gas chromatography, gc ppt
DESCRIPTION
GAS CHROMATOGRAPHYTRANSCRIPT
Gas Chromatography
Gas Chromatography Presented By -
Mr Shaise Jacob
Faculty
Nirmala College of Pharmacy
Muvattupuzha Kerala
India
Email ndash jacobshaisegmailcom
What is Gas ChromatographyWhat is Gas Chromatography
bull It is also known ashellipIt is also known ashellipndash Gas-Liquid Chromatography (GLC)Gas-Liquid Chromatography (GLC)
GAS CHROMATOGRAPHY
Separation of gaseous amp volatile substances Simple amp efficient in regard to separation
GC consists of GSC (gas solid chromatography) GLC (gas liquid chromatography
Gas rarr MP
Solid Liquid rarr SP
GSC not used because of limited no of SP
GSC principle is ADSORPTION
GLC principle is PARTITION
Sample to be separated is converted into vapour
And mixed with gaseous MP
Component more soluble in the SP rarr travels slower
Component less soluble in the SP rarr travels faster
Components are separated according to their Partition Co-efficient
Criteria for compounds to be analyzed by GC 1VOLATILITY
2THERMOSTABILITY
What is Gas ChromatographyWhat is Gas Chromatography
bull The father of The father of modern gas modern gas chromatography is chromatography is Nobel Prize winner Nobel Prize winner John Porter MartinJohn Porter Martin who also developed who also developed the first liquid-gas the first liquid-gas chromatograph chromatograph (1950)(1950)
The Next Generation in Gas Chromatography
How a Gas Chromatography Machine How a Gas Chromatography Machine WorksWorks
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
Chromatographic SeparationChromatographic Separation
ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid
within a columnwithin a column
Chromatographic SeparationChromatographic Separation
bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are
uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times
ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn
ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence
Chromatographic Separation Chromatographic Separation (continued)(continued)
ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture
ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time
Chromatographic AnalysisChromatographic Analysis
ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks
ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks
ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times
Peaks and DataPeaks and Data
PRACTICAL REQUIREMENTS
bull Carrier gas
bull Flow regulators amp Flow meters
bull Injection devices
bull Columns
bull Temperature control devices
bull Detectors
bull Recorders amp Integrators
CARRIER GAS
raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated
compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive
Requirements of a carrier gas
InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance
Flow regulators amp Flow meters deliver the gas with uniform pressureflow
rate flow meters- Rota meter amp Soap bubble
flow meter
Rota meterplaced before column inlet
it has a glass tube with a float held on to a spring
the level of the float is determined by the flow rate of carrier gas
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Gas Chromatography Presented By -
Mr Shaise Jacob
Faculty
Nirmala College of Pharmacy
Muvattupuzha Kerala
India
Email ndash jacobshaisegmailcom
What is Gas ChromatographyWhat is Gas Chromatography
bull It is also known ashellipIt is also known ashellipndash Gas-Liquid Chromatography (GLC)Gas-Liquid Chromatography (GLC)
GAS CHROMATOGRAPHY
Separation of gaseous amp volatile substances Simple amp efficient in regard to separation
GC consists of GSC (gas solid chromatography) GLC (gas liquid chromatography
Gas rarr MP
Solid Liquid rarr SP
GSC not used because of limited no of SP
GSC principle is ADSORPTION
GLC principle is PARTITION
Sample to be separated is converted into vapour
And mixed with gaseous MP
Component more soluble in the SP rarr travels slower
Component less soluble in the SP rarr travels faster
Components are separated according to their Partition Co-efficient
Criteria for compounds to be analyzed by GC 1VOLATILITY
2THERMOSTABILITY
What is Gas ChromatographyWhat is Gas Chromatography
bull The father of The father of modern gas modern gas chromatography is chromatography is Nobel Prize winner Nobel Prize winner John Porter MartinJohn Porter Martin who also developed who also developed the first liquid-gas the first liquid-gas chromatograph chromatograph (1950)(1950)
The Next Generation in Gas Chromatography
How a Gas Chromatography Machine How a Gas Chromatography Machine WorksWorks
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
Chromatographic SeparationChromatographic Separation
ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid
within a columnwithin a column
Chromatographic SeparationChromatographic Separation
bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are
uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times
ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn
ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence
Chromatographic Separation Chromatographic Separation (continued)(continued)
ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture
ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time
Chromatographic AnalysisChromatographic Analysis
ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks
ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks
ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times
Peaks and DataPeaks and Data
PRACTICAL REQUIREMENTS
bull Carrier gas
bull Flow regulators amp Flow meters
bull Injection devices
bull Columns
bull Temperature control devices
bull Detectors
bull Recorders amp Integrators
CARRIER GAS
raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated
compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive
Requirements of a carrier gas
InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance
Flow regulators amp Flow meters deliver the gas with uniform pressureflow
rate flow meters- Rota meter amp Soap bubble
flow meter
Rota meterplaced before column inlet
it has a glass tube with a float held on to a spring
the level of the float is determined by the flow rate of carrier gas
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
What is Gas ChromatographyWhat is Gas Chromatography
bull It is also known ashellipIt is also known ashellipndash Gas-Liquid Chromatography (GLC)Gas-Liquid Chromatography (GLC)
GAS CHROMATOGRAPHY
Separation of gaseous amp volatile substances Simple amp efficient in regard to separation
GC consists of GSC (gas solid chromatography) GLC (gas liquid chromatography
Gas rarr MP
Solid Liquid rarr SP
GSC not used because of limited no of SP
GSC principle is ADSORPTION
GLC principle is PARTITION
Sample to be separated is converted into vapour
And mixed with gaseous MP
Component more soluble in the SP rarr travels slower
Component less soluble in the SP rarr travels faster
Components are separated according to their Partition Co-efficient
Criteria for compounds to be analyzed by GC 1VOLATILITY
2THERMOSTABILITY
What is Gas ChromatographyWhat is Gas Chromatography
bull The father of The father of modern gas modern gas chromatography is chromatography is Nobel Prize winner Nobel Prize winner John Porter MartinJohn Porter Martin who also developed who also developed the first liquid-gas the first liquid-gas chromatograph chromatograph (1950)(1950)
The Next Generation in Gas Chromatography
How a Gas Chromatography Machine How a Gas Chromatography Machine WorksWorks
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
Chromatographic SeparationChromatographic Separation
ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid
within a columnwithin a column
Chromatographic SeparationChromatographic Separation
bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are
uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times
ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn
ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence
Chromatographic Separation Chromatographic Separation (continued)(continued)
ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture
ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time
Chromatographic AnalysisChromatographic Analysis
ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks
ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks
ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times
Peaks and DataPeaks and Data
PRACTICAL REQUIREMENTS
bull Carrier gas
bull Flow regulators amp Flow meters
bull Injection devices
bull Columns
bull Temperature control devices
bull Detectors
bull Recorders amp Integrators
CARRIER GAS
raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated
compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive
Requirements of a carrier gas
InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance
Flow regulators amp Flow meters deliver the gas with uniform pressureflow
rate flow meters- Rota meter amp Soap bubble
flow meter
Rota meterplaced before column inlet
it has a glass tube with a float held on to a spring
the level of the float is determined by the flow rate of carrier gas
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
GAS CHROMATOGRAPHY
Separation of gaseous amp volatile substances Simple amp efficient in regard to separation
GC consists of GSC (gas solid chromatography) GLC (gas liquid chromatography
Gas rarr MP
Solid Liquid rarr SP
GSC not used because of limited no of SP
GSC principle is ADSORPTION
GLC principle is PARTITION
Sample to be separated is converted into vapour
And mixed with gaseous MP
Component more soluble in the SP rarr travels slower
Component less soluble in the SP rarr travels faster
Components are separated according to their Partition Co-efficient
Criteria for compounds to be analyzed by GC 1VOLATILITY
2THERMOSTABILITY
What is Gas ChromatographyWhat is Gas Chromatography
bull The father of The father of modern gas modern gas chromatography is chromatography is Nobel Prize winner Nobel Prize winner John Porter MartinJohn Porter Martin who also developed who also developed the first liquid-gas the first liquid-gas chromatograph chromatograph (1950)(1950)
The Next Generation in Gas Chromatography
How a Gas Chromatography Machine How a Gas Chromatography Machine WorksWorks
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
Chromatographic SeparationChromatographic Separation
ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid
within a columnwithin a column
Chromatographic SeparationChromatographic Separation
bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are
uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times
ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn
ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence
Chromatographic Separation Chromatographic Separation (continued)(continued)
ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture
ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time
Chromatographic AnalysisChromatographic Analysis
ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks
ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks
ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times
Peaks and DataPeaks and Data
PRACTICAL REQUIREMENTS
bull Carrier gas
bull Flow regulators amp Flow meters
bull Injection devices
bull Columns
bull Temperature control devices
bull Detectors
bull Recorders amp Integrators
CARRIER GAS
raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated
compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive
Requirements of a carrier gas
InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance
Flow regulators amp Flow meters deliver the gas with uniform pressureflow
rate flow meters- Rota meter amp Soap bubble
flow meter
Rota meterplaced before column inlet
it has a glass tube with a float held on to a spring
the level of the float is determined by the flow rate of carrier gas
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Sample to be separated is converted into vapour
And mixed with gaseous MP
Component more soluble in the SP rarr travels slower
Component less soluble in the SP rarr travels faster
Components are separated according to their Partition Co-efficient
Criteria for compounds to be analyzed by GC 1VOLATILITY
2THERMOSTABILITY
What is Gas ChromatographyWhat is Gas Chromatography
bull The father of The father of modern gas modern gas chromatography is chromatography is Nobel Prize winner Nobel Prize winner John Porter MartinJohn Porter Martin who also developed who also developed the first liquid-gas the first liquid-gas chromatograph chromatograph (1950)(1950)
The Next Generation in Gas Chromatography
How a Gas Chromatography Machine How a Gas Chromatography Machine WorksWorks
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
Chromatographic SeparationChromatographic Separation
ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid
within a columnwithin a column
Chromatographic SeparationChromatographic Separation
bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are
uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times
ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn
ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence
Chromatographic Separation Chromatographic Separation (continued)(continued)
ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture
ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time
Chromatographic AnalysisChromatographic Analysis
ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks
ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks
ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times
Peaks and DataPeaks and Data
PRACTICAL REQUIREMENTS
bull Carrier gas
bull Flow regulators amp Flow meters
bull Injection devices
bull Columns
bull Temperature control devices
bull Detectors
bull Recorders amp Integrators
CARRIER GAS
raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated
compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive
Requirements of a carrier gas
InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance
Flow regulators amp Flow meters deliver the gas with uniform pressureflow
rate flow meters- Rota meter amp Soap bubble
flow meter
Rota meterplaced before column inlet
it has a glass tube with a float held on to a spring
the level of the float is determined by the flow rate of carrier gas
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
What is Gas ChromatographyWhat is Gas Chromatography
bull The father of The father of modern gas modern gas chromatography is chromatography is Nobel Prize winner Nobel Prize winner John Porter MartinJohn Porter Martin who also developed who also developed the first liquid-gas the first liquid-gas chromatograph chromatograph (1950)(1950)
The Next Generation in Gas Chromatography
How a Gas Chromatography Machine How a Gas Chromatography Machine WorksWorks
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
Chromatographic SeparationChromatographic Separation
ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid
within a columnwithin a column
Chromatographic SeparationChromatographic Separation
bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are
uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times
ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn
ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence
Chromatographic Separation Chromatographic Separation (continued)(continued)
ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture
ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time
Chromatographic AnalysisChromatographic Analysis
ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks
ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks
ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times
Peaks and DataPeaks and Data
PRACTICAL REQUIREMENTS
bull Carrier gas
bull Flow regulators amp Flow meters
bull Injection devices
bull Columns
bull Temperature control devices
bull Detectors
bull Recorders amp Integrators
CARRIER GAS
raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated
compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive
Requirements of a carrier gas
InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance
Flow regulators amp Flow meters deliver the gas with uniform pressureflow
rate flow meters- Rota meter amp Soap bubble
flow meter
Rota meterplaced before column inlet
it has a glass tube with a float held on to a spring
the level of the float is determined by the flow rate of carrier gas
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
The Next Generation in Gas Chromatography
How a Gas Chromatography Machine How a Gas Chromatography Machine WorksWorks
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
Chromatographic SeparationChromatographic Separation
ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid
within a columnwithin a column
Chromatographic SeparationChromatographic Separation
bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are
uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times
ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn
ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence
Chromatographic Separation Chromatographic Separation (continued)(continued)
ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture
ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time
Chromatographic AnalysisChromatographic Analysis
ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks
ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks
ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times
Peaks and DataPeaks and Data
PRACTICAL REQUIREMENTS
bull Carrier gas
bull Flow regulators amp Flow meters
bull Injection devices
bull Columns
bull Temperature control devices
bull Detectors
bull Recorders amp Integrators
CARRIER GAS
raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated
compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive
Requirements of a carrier gas
InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance
Flow regulators amp Flow meters deliver the gas with uniform pressureflow
rate flow meters- Rota meter amp Soap bubble
flow meter
Rota meterplaced before column inlet
it has a glass tube with a float held on to a spring
the level of the float is determined by the flow rate of carrier gas
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
How a Gas Chromatography Machine How a Gas Chromatography Machine WorksWorks
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
Chromatographic SeparationChromatographic Separation
ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid
within a columnwithin a column
Chromatographic SeparationChromatographic Separation
bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are
uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times
ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn
ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence
Chromatographic Separation Chromatographic Separation (continued)(continued)
ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture
ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time
Chromatographic AnalysisChromatographic Analysis
ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks
ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks
ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times
Peaks and DataPeaks and Data
PRACTICAL REQUIREMENTS
bull Carrier gas
bull Flow regulators amp Flow meters
bull Injection devices
bull Columns
bull Temperature control devices
bull Detectors
bull Recorders amp Integrators
CARRIER GAS
raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated
compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive
Requirements of a carrier gas
InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance
Flow regulators amp Flow meters deliver the gas with uniform pressureflow
rate flow meters- Rota meter amp Soap bubble
flow meter
Rota meterplaced before column inlet
it has a glass tube with a float held on to a spring
the level of the float is determined by the flow rate of carrier gas
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Chromatographic SeparationChromatographic Separation
ndash Deals with both the Deals with both the stationary phase stationary phase and and the the mobile phasemobile phase bull Mobile Mobile ndash inert gas used as carrierndash inert gas used as carrierbull StationaryStationary ndash liquid coated on a solid or a solid ndash liquid coated on a solid or a solid
within a columnwithin a column
Chromatographic SeparationChromatographic Separation
bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are
uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times
ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn
ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence
Chromatographic Separation Chromatographic Separation (continued)(continued)
ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture
ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time
Chromatographic AnalysisChromatographic Analysis
ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks
ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks
ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times
Peaks and DataPeaks and Data
PRACTICAL REQUIREMENTS
bull Carrier gas
bull Flow regulators amp Flow meters
bull Injection devices
bull Columns
bull Temperature control devices
bull Detectors
bull Recorders amp Integrators
CARRIER GAS
raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated
compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive
Requirements of a carrier gas
InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance
Flow regulators amp Flow meters deliver the gas with uniform pressureflow
rate flow meters- Rota meter amp Soap bubble
flow meter
Rota meterplaced before column inlet
it has a glass tube with a float held on to a spring
the level of the float is determined by the flow rate of carrier gas
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Chromatographic SeparationChromatographic Separation
bull Chromatographic SeparationChromatographic Separationndash In the mobile phase components of the sample are In the mobile phase components of the sample are
uniquely drawn to the stationary phase and thus uniquely drawn to the stationary phase and thus enter this phase at different times enter this phase at different times
ndash The parts of the sample are separated within the The parts of the sample are separated within the columncolumn
ndash Compounds used at the stationary phase reach the Compounds used at the stationary phase reach the detector at unique times and produce a series of detector at unique times and produce a series of peaks along a time sequence peaks along a time sequence
Chromatographic Separation Chromatographic Separation (continued)(continued)
ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture
ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time
Chromatographic AnalysisChromatographic Analysis
ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks
ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks
ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times
Peaks and DataPeaks and Data
PRACTICAL REQUIREMENTS
bull Carrier gas
bull Flow regulators amp Flow meters
bull Injection devices
bull Columns
bull Temperature control devices
bull Detectors
bull Recorders amp Integrators
CARRIER GAS
raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated
compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive
Requirements of a carrier gas
InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance
Flow regulators amp Flow meters deliver the gas with uniform pressureflow
rate flow meters- Rota meter amp Soap bubble
flow meter
Rota meterplaced before column inlet
it has a glass tube with a float held on to a spring
the level of the float is determined by the flow rate of carrier gas
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Chromatographic Separation Chromatographic Separation (continued)(continued)
ndash The peaks can then be read and analyzed by a The peaks can then be read and analyzed by a forensic scientist to determine the exact forensic scientist to determine the exact components of the mixturecomponents of the mixture
ndash Retention time is determined by each component Retention time is determined by each component reaching the detector at a characteristic timereaching the detector at a characteristic time
Chromatographic AnalysisChromatographic Analysis
ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks
ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks
ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times
Peaks and DataPeaks and Data
PRACTICAL REQUIREMENTS
bull Carrier gas
bull Flow regulators amp Flow meters
bull Injection devices
bull Columns
bull Temperature control devices
bull Detectors
bull Recorders amp Integrators
CARRIER GAS
raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated
compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive
Requirements of a carrier gas
InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance
Flow regulators amp Flow meters deliver the gas with uniform pressureflow
rate flow meters- Rota meter amp Soap bubble
flow meter
Rota meterplaced before column inlet
it has a glass tube with a float held on to a spring
the level of the float is determined by the flow rate of carrier gas
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Chromatographic AnalysisChromatographic Analysis
ndash The number of components in a sample is The number of components in a sample is determined by the number of peaksdetermined by the number of peaks
ndash The amount of a given component in a The amount of a given component in a sample is determined by the area under sample is determined by the area under the peaks the peaks
ndash The identity of components can be The identity of components can be determined by the given retention timesdetermined by the given retention times
Peaks and DataPeaks and Data
PRACTICAL REQUIREMENTS
bull Carrier gas
bull Flow regulators amp Flow meters
bull Injection devices
bull Columns
bull Temperature control devices
bull Detectors
bull Recorders amp Integrators
CARRIER GAS
raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated
compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive
Requirements of a carrier gas
InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance
Flow regulators amp Flow meters deliver the gas with uniform pressureflow
rate flow meters- Rota meter amp Soap bubble
flow meter
Rota meterplaced before column inlet
it has a glass tube with a float held on to a spring
the level of the float is determined by the flow rate of carrier gas
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Peaks and DataPeaks and Data
PRACTICAL REQUIREMENTS
bull Carrier gas
bull Flow regulators amp Flow meters
bull Injection devices
bull Columns
bull Temperature control devices
bull Detectors
bull Recorders amp Integrators
CARRIER GAS
raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated
compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive
Requirements of a carrier gas
InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance
Flow regulators amp Flow meters deliver the gas with uniform pressureflow
rate flow meters- Rota meter amp Soap bubble
flow meter
Rota meterplaced before column inlet
it has a glass tube with a float held on to a spring
the level of the float is determined by the flow rate of carrier gas
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
PRACTICAL REQUIREMENTS
bull Carrier gas
bull Flow regulators amp Flow meters
bull Injection devices
bull Columns
bull Temperature control devices
bull Detectors
bull Recorders amp Integrators
CARRIER GAS
raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated
compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive
Requirements of a carrier gas
InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance
Flow regulators amp Flow meters deliver the gas with uniform pressureflow
rate flow meters- Rota meter amp Soap bubble
flow meter
Rota meterplaced before column inlet
it has a glass tube with a float held on to a spring
the level of the float is determined by the flow rate of carrier gas
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
CARRIER GAS
raquo Hydrogen better thermal conductivity disadvantage it reacts with unsaturated
compounds amp inflammableraquo Helium excellent thermal conductivity it is expensiveraquo Nitrogen reduced sensitivity it is inexpensive
Requirements of a carrier gas
InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance
Flow regulators amp Flow meters deliver the gas with uniform pressureflow
rate flow meters- Rota meter amp Soap bubble
flow meter
Rota meterplaced before column inlet
it has a glass tube with a float held on to a spring
the level of the float is determined by the flow rate of carrier gas
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Requirements of a carrier gas
InertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance
Flow regulators amp Flow meters deliver the gas with uniform pressureflow
rate flow meters- Rota meter amp Soap bubble
flow meter
Rota meterplaced before column inlet
it has a glass tube with a float held on to a spring
the level of the float is determined by the flow rate of carrier gas
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Flow regulators amp Flow meters deliver the gas with uniform pressureflow
rate flow meters- Rota meter amp Soap bubble
flow meter
Rota meterplaced before column inlet
it has a glass tube with a float held on to a spring
the level of the float is determined by the flow rate of carrier gas
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Soap Bubble Meter
loz Similar to Rota meter amp instead of a float soap bubble formed indicates the flow rate
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Injection Devices
Gases can be introduced into the column by valve devices
liquids can be injected through loop or septum devices
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
COLUMNSbull Important part of GCbull Made up of glass or stainless steelbull Glass column- inert highly fragile
COLUMNS can be classified Depending on its use
1 Analytical column
1-15 meters length amp 3-6 mm dm
2 Preparative column
3-6 meters length 6-9mm dm
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Depending on its nature
1Packed column columns are available in a packed manner
SP for GLC polyethylene glycol esters amides hydrocarbons polysiloxaneshellip
2Open tubular or Capillary column or Golay column
Long capillary tubing 30-90 M in length Uniform amp narrow dm of 0025 - 0075 cm Made up of stainless steel amp form of a coil Disadvantage more sample cannot loaded
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
3SCOT columns (Support coated open tubular column
Improved version of Golay Capillary columns have small sample capacity
Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column
Then coated with a thin film of liquid phase
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Columns
bull Packed
bull Capillary
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Equilibration of the column
Before introduction of the sample Column is attached to instrument amp
desired flow rate by flow regulators Set desired temp Conditioning is achieved by passing
carrier gas for 24 hours
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Temperature Control DevicesPreheaters convert sample into its vapour
form present along with injecting devices
Thermostatically controlled oven
temperature maintenance in a column is highly essential for efficient separation
Two types of operationsIsothermal programming-Linear programming- this method is
efficient for separation of complex mixtures
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Temperature Control
bull Isothermal bull Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Tem
p (
deg
C)
Instrumentation - Oven
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
DETECTORSHeart of the apparatus
The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by
temperature flow ratehellip Non destructive Simple amp inexpensive
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Measures the changes of thermal conductivity due to the sample (g) Sample can be recovered
1Thermal Conductivity Detector(Katharometer Hot Wire Detector)
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Thermal Conductivity Basics
When the carrier gas is contaminated by sample the cooling effect of the gas changes The difference in cooling is used to generate the detector signal
The TCD is a nondestructive concentration sensing detector A heated filament is cooled by the flow of carrier gas
Flo
w
Flo
w
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
When a separated compound elutes from the column the thermal conductivity of the mixture of carrier gas and compound gas is lowered The filament in the sample column becomes hotter than the control column
The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded
Thermal Conductivity Detector
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
1048698 Measures heat loss from a hot filament ndash
1048698 filament heated to const Tbull when only carrier gas flows heat loss to
metal block is constant filament T remains constant
bull when an analyte species flows past the filament generally thermal conductivity goes
down T of filament will rise (resistance of the filament will rise)
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Relative Thermal Conductivity
CompoundRelative Thermal
Conductivity
Carbon Tetrachloride 005
Benzene 011
Hexane 012
Argon 012
Methanol 013
Nitrogen 017
Helium 100
Hydrogen 128
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple amp inexpensive
Disadvantages Low sensitivityAffected by fluctuations in temperature and
flow rateBiological samples cannot be analyzed
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Flame Ionization Detector
Destructive detector The effluent from the column is mixed with H
amp air and ignited Organic compounds burning in the flame
produce ions and electrons which can conduct electricity through the flame
A large electrical potential is applied at the burner tip
The ions collected on collector or electrode and were recorded on recorder due to electric current
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
FIDs are mass sensitive rather than conc sensitive
ADVANTAGESbull microg quantities of the solute can be
detectedbull Stablebull Responds to most of the organic
compoundsbull Linearity is excellent
bull DA destroy the sample
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
FID
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Argon ionization detector Depends on the excitation of argon atoms to a
metastable state by using radioactive energy
Argonrarr irradiation Argon + e- rarrcollision Metastable
Argonrarr collision of sub rarr Ionization rarruarrCurrent
ADVANTAGES
1Responds to organic compounds
2High sensitivity
DISADVANTAGES
1Response is not absolute
2Linearity is poor
3 Sensitivity is affected by water
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
ELECTRON CAPTURE DETECTOR The detector consists of a cavity
that contains two electrodes and a radiation source that emits - radiation (eg63Ni 3H)
The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma containing electrons and positive ions
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
bull If a compound is present that contains electronegative atoms those electrons are captured and negative ions are formed and rate of electron collection decreases
bull The detector selective for compounds with atoms of high electron affinity
bull This detector is frequently used in the analysis of chlorinated compounds
bull eg ndash pesticides polychlorinated biphenyls
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
ADVANTAGEHighly sensitive
DISADVANTAGEUsed only for compounds with electron
affinity
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
RECORDERS amp INTEGRATORS
Record the baseline and all the peaks obtained
INTEGRATORSRecord the individual peaks with Rt heighthellip
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Derivatisation of sample
Treat sample to improve the process of separation by column or detection by detector
They are 2 types Precolumn derivatisation
Components are converted to volatile amp thermo stable derivative
Conditions - Pre column derivatisationComponent darr volatileCompounds are thermo labiledarr tailing amp improve separation
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Post column derivatisation
Improve response shown by detector Components ionization affinity towards
electrons is increased
Pretreatment of solid supportTo overcome tailing Generally doing separation of non polar
components like esters ethershellip
Techniques 1 use more polar liquid SP
2 Increasing amt of liquid phase
3Pretreatment of solid support to remove active sites
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Parameters used in GC
Retention time (Rt)
It is the difference in time bw the point of injection amp appearance of peak maxima Rt measured in minutes or seconds
(or) It is the time required for 50 of a component to be eluted from a column
Retention volume (Vr)
It is the volume of carrier gas which is required to elute 50 of the component from the column
Retention volume = Retention time ˣ Flow rate
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Separation factor (S)
Ratio of partition co-efficient of the two components to be separated
If more difference in partition co-efficient bw two compounds the peaks are far apart amp S
Is more If partition co-efficient of two compounds are similar then peaks are closer
Resolution (R)
The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
It is the measure of both column amp solvent efficiencies
R= 2d
W1+W2
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Retention time
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Separation factor
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Resolution
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Resolution
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
THEORETICAL PLATE
An imaginary unit of the column where equilibrium has been established between SP amp MP
It can also be called as a functional unit of the column
HETP ndash Height Equivalent to a Theoretical Plate
Efficiency of a column is expressed by the number of theoretical plates in the column or HETP
If HETP is less the column is uarr efficient If HETP is more the column is darr efficient
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
HETP= L (length of the column)
N (no of theoretical plates)
HETP is given by Van Deemter equation
HETP= A + B +Cu
u
A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column
B = Molecular diffusion depends on flow rate
C = Effect of mass transferdepends on flow rate
u = Flow rate
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Efficiency ( No of Theoretical plates)
It can be determined by using the formula
n = 16 Rt2
w2
N = no of theoretical plates
Rt = retention time
W = peak width at baseThe no of theoretical plates is high the
column is highly efficientFor GC the value of 600 meter
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Asymmetry Factor
Chromatographic peak should be symmetrical about its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of SP amp can be avoided by using less quantity of sample
TAILING
Due to more active adsorption sites amp can be eliminated by support pretreatment
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Asymmetry factor (095-105) can be calculated by using the formula AF=ba
b amp a calculated at 5 or 10 of the peak height
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
ADVANTAGES OF GC
Very high resolution power complex mixtures can be resolved into its components by this method
Very high sensitivity with TCD detect down to 100 ppm
It is a micro method small sample size is required
Fast analysis is possible gas as moving phase- rapid equilibrium
Relatively good precision amp accuracyQualitative amp quantitative analysis is possible
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Gas Chromatography vials caps
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Chromatographic AnalysisChromatographic Analysis
ndash The The number of componentsnumber of components in a sample is in a sample is determined by the determined by the number of peaksnumber of peaks
ndash The The amountamount of a given component in a of a given component in a sample is determined by the sample is determined by the area under area under the peaks the peaks
ndash The The identity identity of components can be of components can be determined by the given determined by the given retention timesretention times
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Applications of GC
bull GC is capable of separating detecting amp partially characterizing the organic compounds particularly when present in small quantities
1 Qualitative analysis
Rt amp RV are used for the identification amp separation
2 Checking the purity of a compound
Compare the chromatogram of the std amp that of the sample
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
3 Quantitative analysis
It is necessary to measure the peak area or peak height of each component
4 used for analysis of drugs amp their metabolites
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Semi-Quantitative Analysis of Fatty Acids
C
C
C
Dete
ctor
Resp
onse
Retention Time
14
16
18
Pea
k A
rea
Sample Concentration (mgml)
2
4
6
8
10
05 10 15 20 25 30
The content of C fatty acids =C
C + C + C
= the content of C fatty acids14
14
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Tentative Identification of Unknown Compounds
Res
pons
e
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane16 min = RT
Res
pons
e
Unknown compound may be Hexane
16 min = RT
Retention Time on Carbowax-20 (min)
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Res
p on s
e
GC Retention Time on SE-30
Unknown compound
RT= 4 min on SE-30
Res
pons
e
GC Retention Time on SE-30
HexaneRT= 40 min on SE-30
Retention TimesRetention Times
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
Advantages of Gas Chromatography
bull Very good separation
bull Time (analysis is short)
bull Small sample is needed - l
bull Good detection system
bull Quantitatively analyzed
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
How a Gas Chromatography How a Gas Chromatography Machine WorksMachine Works
ndash FirstFirst a vaporized sample is injected onto the a vaporized sample is injected onto the chromatographic columnchromatographic column
ndash SecondSecond the sample moves through the the sample moves through the column through the flow of inert gascolumn through the flow of inert gas
ndash ThirdThird the components are recorded as a the components are recorded as a sequence of peaks as they leave the columnsequence of peaks as they leave the column
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
-
- Gas Chromatography
- Slide 2
- What is Gas Chromatography
- GAS CHROMATOGRAPHY
- Slide 5
- Slide 6
- Slide 7
- The Next Generation in Gas Chromatography
- How a Gas Chromatography Machine Works
- Chromatographic Separation
- Chromatographic Separation
- Chromatographic Separation (continued)
- Chromatographic Analysis
- Peaks and Data
- Slide 15
- Slide 16
- Slide 17
- Slide 18
- Slide 19
- Slide 20
- PRACTICAL REQUIREMENTS
- CARRIER GAS
- Requirements of a carrier gas
- Flow regulators amp Flow meters
- Slide 25
- Soap Bubble Meter
- Injection Devices
- COLUMNS
- Depending on its nature
- 3SCOT columns (Support coated open tubular column
- Columns
- Slide 32
- Slide 33
- Equilibration of the column
- Temperature Control Devices
- Temperature Control
- DETECTORS
- 1Thermal Conductivity Detector (Katharometer Hot Wire Detector)
- Thermal Conductivity Basics
- Slide 40
- Slide 41
- Slide 42
- Relative Thermal Conductivity
- Advantages of Katharometer
- Flame Ionization Detector
- Slide 46
- FID
- Slide 48
- Argon ionization detector
- Slide 50
- Slide 51
- Slide 52
- Slide 53
- Slide 54
- Slide 55
- RECORDERS amp INTEGRATORS
- Derivatisation of sample
- Post column derivatisation
- Parameters used in GC
- Slide 60
- Retention time
- Slide 62
- Separation factor
- Resolution
- Slide 65
- THEORETICAL PLATE
- Slide 67
- Efficiency ( No of Theoretical plates)
- Slide 69
- Slide 70
- Asymmetry Factor
- Slide 72
- Slide 73
- Slide 74
- ADVANTAGES OF GC
- Gas Chromatography vials caps
- Slide 77
- Applications of GC
- Slide 79
- Slide 80
- Slide 81
- Slide 84
- Slide 85
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