gas chromatograp
TRANSCRIPT
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By
K Rakesh gupta
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INTRODUCTION
PRINCIPLE
THEORIES
PARAMETERS
INSTRUMENTATION
APPLICATIONS
REFERENCE
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CHROMATOGRAPHY: SEPARATION TECHNIQUE
: TWO PHASES are used
: MIKHAIL TSWETT invented
GAS CHROMATOGRAPHY: MARTIN AND JAMES:GAS as M.phase always
: solid or liquid S.Phase
Choice for THERMALLY STABLEand VOLATILEcompounds
TWO TYPES BASED ON STATIONARY PHASE
GSC:Stationary phase isSOLID
GLC:Stationary phase isLIQUID
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GSC:ADSORPTION
:RELATIVE AFFINITY
:More affinity towards S.P travels slowly-slow elution
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GLC
:PARTITION
: SOLUBILITY
:more partition coefficient travels slowly slow elution
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Mainly three theories are involved in GC
1. PLATE THEORY2. BAND BROADENING THEORY3. RATE THEORY
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Concept compared counter-current distribution
Plates are hypothetical lines where equilibration occur
Plates analogous to tubes in CCD (Catalytic Combustion
Detector)system
In plate theory two main terms are used as quantitativemeasures of chromaographic column efficiency
PLATE HEIGHT(HETP)PLATE COUNT or no. of PLATES(N)
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CALCULATION OF THE DISTRIBUTION
THROUGH 4 TRANSFERS:
Transfer non
Tube no.r = 0
Tube nor=1
Tube no:r=2
Tube no:r=4
Tube no:r=3
n=0 B
A
0/1
p/qn=1 B
A0/qPq/q2
p/0p2 /pq
n=2 BA
0/q2Pq2 /q3
pq/pq2p2 q/2pq2
p2/0p3 /p2 q
n=3 BA
0/q3Pq3 /q4
pq2 /2pq23p2q/3p2 q3
2p2 q/p2 q3p3q/3p2 q2
p3 /0p4 /p3 q
n=4 B 0/q4 Pq3 /3pq3 3p2q2/3p2q2 3p3q/p3 q p/0
Total after
4 transfers
q4 4pq3 6p2q2 4p3q p4
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The expansion of the function (p+q)n is laboriousfor large n, and an easier calculation is available.
The binomial expansion may be written
(p+q)n = qn + nqn-1p + n(n-1) qn-2 q2++ pn
2
which can be expressed
(p+q)n = nr=0 n! prq(n-r)
r!(n-r)!
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Binominal distribution expression for the fraction of totalsolute in rth plate after n mobile phase volumes havepassed into the column
pr q(n-r)r! (n-r)!
n = no.of mobile phase volumes passed into the column
r = plate number( 0,1,2,3,..r)
p = 1/(KU+1) = fraction of solute per plate in M.P at equilibrium
q = KU/(KU+1)= fraction of solute per plate in S.P at equilibria
Tnr = n! pr q(n-r)r! (n-r)!
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When n is large and p is not small (as in CCDS), binomial
distribution approaches normal (Gaussian) distribution
According to statistics MEAN is given as
Standard deviation
= np
= np
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ELUTION CHROMATOGRAM
Concentration
| Tr |time | width |
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CALCULATION FOR NO.OF PLATES(efficiency)
Length= velocity. time
= Rvtr Standard deviation = Rv
Where is zone-standard deviation Combining above equations
= L
tr
tr2 = L 2/ H since: 2 = HL
tr = r since:
=L/H
r = 16(tr/w)2
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Random walk
Reflects loss of efficiency
Rate process controls zone width
From plate theory : 2
= HLH is a measure of zone spreading and columnefficiency
( height equivalent to theoretical plates)
HETP = Length of the columnno.of theoretical plates
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1. LONGITUDINAL MOLECULARDIFFUSION
2. MASS TRANSFER(SORPTION-DESORPTION KINETICS)
3. EDDY DIFFUSION
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1.LONGITUDINAL MOLECULARDIFFUSION
L= vt
=2Dm t
=2Dm L/v
Hdiff =2Dm /v is empirical factor of value 0.6
Hdiff = 2Dm + 2s DS (1-R)/R
v
This is in the form of Hdiff = B/v
Where B is the function of molecular and chromatographic
properties
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2.MASS TRANSFER(SORPTION-DESORPTION KINETICS)
No.of random steps n = 2L/vta
True step length L =vta - Rvta or (1-R)vta According to random walk theory =Ln
=2(1-R)vta mm
Hs-d = Cv
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3.EDDY DIFFUSION
Flow and diffusion mechanism are coupled
Plate height contribution through flow and diffusion is notadditive ,but found to be
Heddy = 11/HF + 1/HD
H is independent of velocity and H is dependent onaverage velocity
Heddy = 11/A + 1/Ev
Where A and E include the partical diameter
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CALCULATION FOR HETP
Total plate height contribution from 3 process
H = Heddy + Hdiff + H(s-d)
Van Deemeter equation
H = A + B/v + Cv
H = 1 + B + Cv1/A + 1/Ev v
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Random walk Fronting tailing Quantitative measure
detector
signal
time
Fronting- saturation of S.P
Tailing-more active adsorption
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RETENTION TIME(Rt)
Retention time is the difference in the time between thepoint of injection and appearance of peak maxima
Rt is the time required for 50% of a component to beeluted from the column
Unites : min or sec
Retention time is also proportional to the distance movedon a chart paper, which can be measured in cm or mm
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RETENTION VOLUMN(VR)
Retention volume is the volume of carrier gasrequired to elute 50% of the component from thecolumn
Corrected retention volume
Where j is pressure drop correction factor
Pi and Po are inlet and outlet pressures
Retention volume = Retention time flow rate
VR0 = j VR
j = 3 . ( Pi / Po )2 - 1
2 . (Pi / Po )3 - 1
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ADJUSTED RETENTIONVOLUMN(VR!)
Adjusted retention volume is calculated as
Where VM is DEAD VOLUME of mobile phase Applying pressure drop correction to VR
! Gives
net retention volume
VR = VR - VM
VN = j VR
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SELECTIVITY( )
A way of improving resolution is to change the selectivityof the column by changing stationary and mobilephases
Selectivity is the ratio of partition coefficients
Selectivity term can be evaluated from the chromatogram
= VR,2 VM (or) = tr2 - tmV
R,1 V
Mtr1
- tm
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RESOLUTION(Rs)
The degree of disengagement of two bands is resolution.
In terms of width and diameter
In terms of time and width
In terms of zone of migration
In terms of , k ,Nwhere k is capacity factor
k=nS/nM
RS = dAdBW
RS = 2 Rt 1 -Rt2wA + wB
RS = L . R16H R
RS = N k -14 k+1
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EFFICIANCY;NO.OF PLATES(n)
Efficiency of column is expressed by the no. of theoreticalplates
If the no.of theoretical plates is high, the column is said tobe highly efficient
If the no.of theoretical plates is low , the column is said tobe less efficient
For GC columns, a value of 600/meter is sufficient
But for HPLC , high values like 40,000 to 70,000/meterare recommended
No.of theoretical plates efficiency
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HETP(H)
Decides the efficiency of separation
If HETP is less, the column is more efficient
If HETP is more, the column is less efficient
HETP = Length of column
no.of theoretical plates
HETP calculated by using Van Deemeter equationHETP = A + B + Cv
v
HETP 1EFFICIENCY
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INSTRUMENTATION
Carrier Gas
Flow regulators and meters
Sample injection system Columns & ovens
Detectors
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SCHEMATICDIAGRAMOF GAS CHROMATOGRAPH
G C
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Gas ChromatographComponents
FlameIonization
Detector
Column
Oven
Injection Port
top view
front view
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Carrier gas
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The mobile phase gas is called the carrier gas and
must be chemically inert.Sample component column detector
mobile phase gas
Helium ,argon ,nitrogen , carbon dioxide andhydrogen also used.
Selection of the best carrier gas very important ,because it effects both the column separation and
detector performance . The ratio of viscosity of diffusion coefficient
should be minimum for rapid analysis thats whyH, He are prepared for a carrier gas .
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Impurities in the carrier gas such as air water vapour
and trace gaseous hydrocarbons can cause sample
reaction, column character and affect the detector
performance.
The carrier gas system should contains a molecular
sieve to remove water and other impurities.
These gases are available in pressurized tanks.pressure regulators and flow meters are required to
control the flow rate of the gas.
The gases are supplied from the high pressure gas
cylinder , being stored at pressure up to 300psi
carrier gas should be better then 99.99%and 99.999%
is often used33
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Air inlet (detector))
H2 inlet (detector)N2 inlet(make-up gas)
He inlet
(carrier gas)
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Process Flow Schematic
Carrier gas(nitrogen orhelium)
Sample injection
Long Column (30 m)
Detector (flameionizationdetector or FID)
HydrogenAir
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Carrier Gas(mobile phase)
Requirements:
It should be inert and available at low cost High purity Easily available Less risk of explosion or fire hazards Pressure:
-Inlet 10 to 50 psi-packed column 25 to 150 mL/min.- capillary column 1 to 25 mL/min.
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Flow regulators & meters
Flow regulators are used to deliver the gaswith uniform pressure or flow rate
Flow rates of carrier gas:
Linear flow rate (cm/s): u = L/tr Volumetric flow rate (mL/min): u ( r2)L is length of column, it is retention time, r is the internal radius of column
Flow rate depends on type of column Packed column: 25-100 mL/min
Capillary column: 1 to 25 mL/min Flow rate will decreaseas column T increases
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FLOW REGULATORS
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Soap bubble flow meter
Aqueoussolution ofsoap ordetergent
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soap bubbles formedindicates the flow rate.
Glass tube with a inlet tubeat the bottom.
Rubber bulb-----store soapsolution
When the bulb is gently
pressed of soap solution isconverted into a bubble bythe pressure of a carrier gas&travel up.
Soap bubble meter
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inlet tube
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INJECTOR
Sample injection port
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Sample injection port Calibrated Micro syringes are used to
inject liquid sample
Purge :volatile components are
removed from sample by gentleheating
Rubber or silicone diaphragm(septum)
Sample port Temp: 50C
Packed Column: sample sizes-1 to 20
L
Capillary Column : 10 to 30 mL
splitter is used to deliver a fraction
of injection(1:50 to 1:500)
Avoid over loading
Slow injection & oversized samples
cause band spreading & poor
resolution
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Micro syringe
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1. Wash a syringe with acetone by filling
the syringe completely and ejecting thewaste acetone onto a papertowel. Wash 2-3 times.
2. Remove air bubbles in the syringe byrapidly moving the plunger up and downwhile the needle is in the sample.
3.Usually 1-2 mL of sample is injectedinto the GC.
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COLUMN OVENS
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Column temperature is very important in GC
The column is ordinarily housed in athermostatic oven.
they are usually formed as coils having
diameters of 10 to 30 cm. The optimum column temperature depends
upon the boiling point of the sample and thedegree of separation required.
Roughly, a temperature equal to or slightlyabove the average boiling point of a sampleresults in a reasonable elution time (2 to 30min).
Column ovens
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COLUMNS
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Columns
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Two types of columns are used in gaschromatography, packed and open
tubular or capillary.
Packed column length from less than 2m to 5 m
Capillary columns from few m to 100 m
They are constructed of stainless steel,glass, fused silica, or Teflon.
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Column
Types of columns
1.packed columns
2. Open tubular or capillary.
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Capillary column- 30mPacked column-3m
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Packed columns
Packed columns are fabricated from glass,metal (stainless steel, copper, aluminum), orteflon tubes that typically have
Lengths------ 2m to 3 m
Internal diameters ------- 2 to 4 mm. These tubes are densely packed with a
uniform, finely divided packing material, orsolid support, that is coated with a thin layer
(0.05 m) of the stationary liquid phase. In order to fit in a thermostatic oven, the tubes
are formed as coils having diameters ofroughly 15 cm. 51
Capillary (or)Open tubular
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Capillary (or)Open tubularColumns
1.Wall-coated open tubular (WCOT)
Capillary tubes coated with a thin layer ofstationary phase
Old: stainless steel, Al, Cu, plastic, glass.
2.Support-coated open tubular (SCOT)
Inner surface of the capillary is lined with a thinfilm (~30m) of a support materials, likediatomaceous earth
Lower efficiency than WCOT, higher than packed
column3.Fused-silica open tubular column (FSOT):Physical strength, low reactivity, flexibility. 0.32 to
0.25 mm
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Column Stationary Phases:
Packed liquid coated silica particles (
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The Stationary Phase
requirements for stationary phase
Low vapor pressure
Thermal stability
Low viscosity (for fast mass transfer)
High selectivity for compounds of interest
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DETECTOR
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DETECTORS Ideal characters of
detectorHigh sensitivity to even
small concentrtion linerity, ie, less response
to low concentration&proportional responseto high concentration
Large linear dynamicrange
Useful at a range oftemperatures
Good stability andreproducibility
Rapid response time
Easy to use
Stable, Predictableresponse
Inexpensive
operation from RT to 400
oC
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Types of detectors1. Thermal Conductivity Detector(TCD)2. Flame Ionization Detector(FID)3. Atomic Emission Detector(AED)4. Electron Capture Detector(ECD)
5. Nitrogen Phosphoroes Detector(NPD)6. Photo Ionization Detector(PID)7. Flame Photometric detector(FPD)8. Electrolytic conductivity detector (Hall
detector)9. Absolute Mass Detector(AMD)10. Thermionic Detector(TD)
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ame on za onDetector(FID)
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Detector(FID)Most widely used, Air-H2 flame
Number of ions depends onnumber of reduced (methylene)
carbons in a moleculeThe positive ions will be attracted tothe cylindrical cathode.
Negative ions and electrons will beattracted to the jet anode.
Organic compounds Producesions and electrons pyrolyzed(tempof flame) burner tip andelectrode.(fhv power)Ions &electrons move toward thecollector
less sensitive for non-hydrocarbongroups
Insensitive to noncombustiblegases(CO2, SO2, NO2 and H2O)Insensitive to functional group
(carbonyl, alcohol, halogen and amine)
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Thermal Conductivity Detector(TCD)
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Thermal Conductivity Detector(TCD)
Thermal conductivity detector cell
Arrangement of the twin detectors
Element(platinum, gold or tungsten wire)is electrically heated at constant power
Temperature depends on thermalconductivity (He & H)of surrounding gas.
Hydrogen and helium have higherthermal conductivity and carrier gas
provide best sensitivity
Six times greater than the Organiccompounds
Poorer sensitivity than FID, but more
universal
Advantages: simplicity, large range,inexpensive, linearity is excellent.organic & inorganic species
DA: low sensitivity ng/mL 60
Electron Capture Detectors (ECD)
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Electron Capture Detectors (ECD)
The sample elute from a column ispassed over a radioactive -
emitter(nickel-63) Selectively to halogen-containingorganic sample ,like pesticides and,polychlorinated biphenyls
Ni-63: radioactive -emitter-- electron --
ionization of carrier gas (N2)High electronegative group (halogen,peroxide, quinones and nitro group) in thesample capture the electronHighly selective and sensitive,
nondestructiveInsensitive to amines, alcohols andhydrocarbons
AD: High sensitivity, analyse thepolyhalogenated organic compounds
Small linear range61
Th i i d t t ( it h h d t t )
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Thermionic detector(nitrogen phosphorus detector)
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N or P containing organic compounds
phosphorus atom is approximately tentimes greater than to a nitrogen atom and104 to 106 larger than a carbon atom.
Compared with the FID , the thermionicdetector is approximately 500 times more
sensitive to phosphorus-containingcompounds and 50 times more sensitive tonitrogen bearing species.
Column effluant + H2 +air(hotgas)electrically hearted Rb2SiO4
(rubidium silicate)bead at 180 V plasma(600 800C ) ions to determinecompounds
useful for detecting and determining themany phosphorus-containing pesticides.
ATOMIC EMISSION DETECTOR
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Eluent(column) helium(carrier) water cooled microwave cavity helium plasma(high temp) characteristic atomic emissionspectra grating diode array optical emission spectrometer detect the element .
ATOMICEMISSIONDETECTOR
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Six elements detect simultaneously .Determine the hetero atoms(H,P,S,O),silicon , heavymetals(Pb , Hg),tin, arsenic ,copper ,iron.
PHOTO IONIZATION
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UV light (10.2 eV H2or 11.7 eV Ar lamp)photo ionization ofmolecular currentto flow betweenbased electrodes
Most sensitive forAromatic and S, Peasily
photoionizedmolecules
Linear range is high
PHOTO IONIZATIONDETECTOR(PID)
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Flame photometric detector
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p(FPD):
S and P compounds photomultiplier to view light of 394
nm for sulphur (H2 + air S2)measurement or 526 nm for
phosphorus (H2 + air HPOspecies) Filteres are used to isolate the
appropriate bands
Intensity is recordedphotometrically X-, N-, Sn , Cr, Se and Ge
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filteres
photomultiplier
H2 + air
Column effluent
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APPLICATIONS
Q lit ti l i
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Qualitative analysis:
Retention time data should be useful foridentification of mixtures
Comparing the retention time of the
sample as well as the standard Checking the purity of a compound:
compar the standard and sample
Additional peaks areobtained..impurities are
present.compound is not pure68
Quantitative
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analysis:
Direct comparison method:-comparing the area of the peak, peak height,
width of peak.
Calibration curves:-standards of varying concentration are used
determine peak areas .o Internal standard method:
-A known concentration of the internal standard isadded separately to the standard solution
-The peak area ratio of sample and internal
standard.unknown concentration is easilydetermined .
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Elemental analysis
Determination of C,H ,O ,S and N .
Determination of mixture of drugs
Isolation and identification of drugs
Isolation and identification of mixture ofcomponents(amino acids ,plant extracts,volatile oils)
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Instrumental Analysis by Douglas A.Skoog , F.JamesHoller & Stanley R.Crouch.
Text book of Pharmaceutical Analysis by KennethA.Connor
www.google .com
Text book of Pharmaceutical analysis by Dr.S.Ravi Sankar
Introduction to instrumental analysis by Robert D.Braun
Chromatograhic methods by Smith
http://www.google/http://www.google/ -
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