curs_hplc - copy

CHROMATOGRAPHY

Brief History and Definition

Liquid chromatography was defined in the early 1900s by the work of the Russian botanist, Mikhail S. Tswett. His pioneering studies focused on separating compounds [leaf pigments], extracted from plants using a solvent, in a column packed with particles.

Tswett coined the name chromatography [from the Greek words chroma, meaning color, and graph, meaning writingliterally, colorwriting] to describe his colorful experiment. [Curiously, the Russian name Tswett means color.] Today, liquid chromatography, in its various forms, has become one of the most powerful tools in analytical chemistry.

Tswett filled an open glass column with particles. Two specific materials that he found useful were powdered chalk [calcium carbonate] and alumina. He poured his sample [solvent extract of homogenized plant leaves] into the column and allowed it to pass into the particle bed. This was followed by pure solvent. As the sample passed down through the column by gravity, different colored bands could be seen separating because some components were moving faster than others. He related these separated, different-colored bands to the different compounds that were originally contained in the sample.

Column Chromatography

Separation TechniquesSeparation processes are used to decrease the complexity of material mixtures. Chromatography and electrophoresis are representative of this field.

Figure: Separation of black ink on a thin layer chromatography plate.

Chromatography (Encyclopedia Britannica):

Technique for separating the components, or solutes, of a mixture on the basis of the relative amounts of each solute distributed between a moving fluid stream, called the mobile phase, and a contiguous stationary phase. The mobile phase may be either a liquid or a gas, while the stationary phase is either a solid or a liquid.

Chromatography(IUPAC Compendium of Chemical Terminology):

A physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase)moves in a definite direction.

CHROMATOGRAPHY

Chromatography basically involves the separation of mixtures due to differences in the distribution coefficient (equilibrium distribution) of sample components between 2 different phases.

One of these phases is a mobile phase and the other is a stationary phase.

Definition:

Different affinity of these 2 components to stationary phase causes the separation.

Concentration of component A in stationary phase

Concentration of component A in mobile phase

Distribution Coefficient (Equilibrium Distribution )

Kinds of Chromatography

1. Liquid Column Chromatography

2. Gas Liquid Chromatography

3. Thin-Layer Chromatography

Schematic Presentation of a Chromatogram

Skoog and Leary: Principals of Instrumental Analysis, 4th ed. Suanders, 1992

The 4 basic liquid chromatography modes are named according to the mechanism involved:

1. Liquid/Solid Chromatography (adsorption chromatography)

A. Normal Phase LSC

B. Reverse Phase LSC

2. Liquid/Liquid Chromatography (partition chromatography)

A. Normal Phase LLC

B. Reverse Phase LLC

3. Ion Exchange Chromatography

4. Gel Permeation Chromatography (exclusion chromatography)

FOUR BASIC LIQUID CHROMATOGRAPHY

Normal phase

In this column type, the retention is governed by the interaction of the polar parts of the stationary phase and solute. For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample

Structure of silica gel

Stationary Phase: Alumina

O

AlO O

AlO

OH

AlO

OH

AlO

OH

Al

OH

O

Acidic: -Al-OH

Neutral: -Al-OH + -Al-O-

Basic: -Al-O-

LIQUID SOLID CHROMATOGRAPHY

30 Si - O - H

+

Normal phase LS Reverse phase LS

Silica Gel

The separation mechanism in LSC is based on the competition of the components of the mixture sample for the active sites on an absorbent such as Silica Gel.

LIQUID SOLID CHROMATOGRAPHY

Si - OH

HEXANE

OH

C-CH3

CH3

CH3- CCH3

CH3

OH

OH

CH3

CH3

Reverse phase

In this column the packing material is relatively nonpolar and the solvent is polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water.

LIQUID-LIQUID CHROMATOGRAPHY

ODPN(oxydipropionylnitrile)

Normal Phase LLC Reverse Phase LLC

NCCH3 CH2 OCH2 CH2 CN(Normal)CH3 (CH2 )16 CH3 (Reverse)

The stationary solid surface is coated with a 2nd liquid (the Stationary Phase) which is immiscible in the solvent (Mobile) phase.

Partitioning of the sample between 2 phases delays or retains some components more than others to effect separation.

Reverse phase chromatographySilica is alkylated with long chain hydrocarbon groups, using 18carbons long. This is usually referred to as C-18 silica.

O

Si

O

O

SiO

OO

O

Si

OO

O

Si

OO

O

SiO O

O

Si

OO

Si

OO

Si

OO

SiO O

OSi

OO

Si

OO

SiO O

O

CH2

CH3

17Si

CH3

CH3

CH2

CH3

17Si

CH3

CH3SiCH3)3

SiCH3)3SiCH3)3

Size exclusion

In size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size. Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores. The large molecules elute before the smaller molecules.

Gel-Permeation Chromatography is a mechanical sorting of molecules based on the size of the molecules in solution. Small molecules are able to permeate more pores and are, therefore, retained longer than large molecules.

GEL-PERMEATION CHROMATOGRAPHY

Ion exchange

In this column type the sample components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase. Separations are made between a polar mobile liquid, usually water containing salts or small amounts of alcohols, and a stationary phase containing either acidic or basic fixed sites.

MECHANISM OF ION-EXCHANGE

CHROMATOGRAPHY OF AMINO ACIDS

SO3-

SO3-

Na+

COO-

H 3N+

Na+

COOHH 3N

+

pH2

pH4.5

Ion-exchange Resin

Mechanism of separation in

different forms of HPLC

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY(HPLC)

HP

igherformance

LiquidChromatography

HP

ighressure


HP

ighriced


HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

High Performance Liquid Chromatography (HPLC) is one of the most widely used techniques for identification, quantification and purification of mixtures of organic compounds.

In HPLC, as in all chromatographic methods, components of a mixture are partitioned between an adsorbent (the stationary phase) and a solvent (the mobile phase).

The stationary phase is made up of very small particles contained in a steel column. Due to the small particle size (3-5 um), pressure is required to force the mobile phase through the stationary phase.

There are a wide variety of stationary phases available for HPLC. In all labs can be used a normal phase (Silica gel), although reverse phase (silica gel in which a 18 carbon hydrocarbon is covalently bound to the surface of the silica) columns are currently one of the most commonly used HPLC stationary phases.

HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector.

Compounds are separated by injecting a sample mixture onto the column. The different component in the mixture pass through the column at differentiates due to differences in their partition behavior between the mobile phase and the stationary phase. The mobile phase must be degassed to eliminate the formation of air bubbles.

http://www.chemistry.nmsu.edu/Instrumentation/Waters_HPLC_MS_TitlePg.html


General Schematic of LC

Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.

Chromatography: HPLCHewlettHewlett--PackardPackard

Series 1100 HPLCSeries 1100 HPLC

solventsolvent

pumppump

injectorinjector

columncolumn

detectordetector

Chromatography: HPLCHPLC ColumnHPLC Column

Chromatography: HPLCHPLC PumpHPLC Pump

Chromatography: HPLCHPLC Autosampler and InjectorHPLC Autosampler and Injector

Chromatography: HPLCHPLC DetectorHPLC Detector

UV/Visible SpectrophotometerUV/Visible Spectrophotometer

Chromatography: HPLCHPLC Waste CollectionHPLC Waste Collection

http://www.labhut.com/education/flash/introduction07.php

TLC vs High Performance Liquid Chromatography (HPLC)HPLC Optimization

Modes of HPLCAdsorption Chromatography is another name for liquid-solid chromatography.

The solid phase is usually silica or alumina, which have highly polar surfaces. The mobile phases are commonly some of the less polar solvents.

Normal-Phase Chromatography is based on a polar liquid phase coated or bonded onto a silica support. For historical reasons, this is called normal phase because the stationary phase is polar and the mobile phase is typically a nonpolar solvent such as hexane or isopropylether. In normal phase, the least polar component is eluted first.

Reversed-Phase Chromatography uses a non-polar stationary phase (also coated or bonded onto silica or another support) and polar solvents such as water, acetonitrile or methanol. In reversed-phase chromatography, the most polar component is eluted first.

Ion-Pair Chromatography is one of the methods used to separate ions. It uses regular reversed phase columns, and can separate acids, bases and neutral compounds during the same chromatographic run.

Ion-Exchange Chromatography is based on the use of ion-exchange resins as the stationary phase.

Ion Chromatography was developed to separate the ions of strong acids and bases. The equipment used is different from that of ion-exchange chromatography.

CH3|

-0-Si- CH3|CH3

+ HClSilica

CH3|

-Si- CH3|CH3

OH + ClSilica

Could be many differentfunctional groups here

Bonding Phases onto the Silica Support

Usually half or more of the silanol groups remain unreacted after bonding with C18. One method used to reduce the effects of these residual silanol groups is a process called endcapping.

End Capping

End Capping

After bonding with C18 use small silane molecules such as trimethylchlorosilane to react some of the remaining OH groups

CH3|CH2|

-0-Si- CH3|CH2|CH3

Silica

Steric Protection

Hydrolysis may degrade the column by breaking off the bonded phase.

Hypersil HyPURITY C18Column: Symmetry C18

Sample: 1. Uracil2. Pyridine 3. Phenol4. Dimethyl phthalate5. N,N-Dimethylaniline6. 4-Butylbenzoic acid7. Toluene

Mobile Phase: 60% CH3CN40% 50mM KH2PO4, pH 3.2

Comparison of Different C18 Columns

Common RP Packings

Other Bonded Phases

Phenyl phases show weak dipole - induced dipole interactions with polar analytes. Usually this type of bonded phase is used for separating closely related groups of molecules.

Other Bonded Phases

The amino-phase is the most polar, and it can also act as weak anion-exchanger, (protonated at low pH). Amino columns are mainly used in normal-phase mode, specially for selectiveretention of aromatic compounds.

Other Bonded Phases

Diol columns are slightly polar and are used for normal-phase separations. Diols are useful for samples containing many compounds with different polarities , and which usually have strong retention on unmodified silica.

Non-Silica SupportsThe most common polymer support material for reversed-phase separation is made of divinylbenzene cross-linked polystyrene

The main advantage of porous polymers is that they can be used in the pH range from 1 to 13. (silica supports tend to dissolve at pH greater than 9).

Because of the surface acidity of silica supports, polymer supports can be a better choice for separating basic compounds.

All C18 Columns Are Not Created Equal

Differences in:

Particle SizePore SizeSurface AreaCarbon LoadEnd-CappingSilica Type*Bonding Density

RP Column Properties

Hydrophobic Surface Particle Size and Shape Particle Size Distribution Porosity, Pore Size and Surface Area

Particle Size

Columns have a distribution of particle sizes

Reported particle diameter is an average Broader distribution ---> broader peaks

RP Mechanism (Simple)

Reversed Phase Mechanisms

Classical measures of retention retention factors partition coefficients Vant Hoff Plots

Give bulk properties only - do not give molecular view of separation process

Proposed RP Mechanisms

Hydrophobic Theory Partition Theory Adsorption Theory

See Journal of Chromatography, volume 656.

Hydrophobic Theory

Chromatography of cavities in solvent created by hydrophobic portion of analyte molecule

Surface Tension Interaction of polar functions with solvent Stationary phase is passive

Partition Theory

Analyte distributes between aqueous mobile phase and organic stationary phase

Correlation between log P and retention organic phase is attached on one end Does not explain shape selectivity effects

Adsorption Theory

Analytes land on surface - do not penetrate

Non-polar interactions between analyte hydrophobic portion and bonded phase

Weak interactions dipole-dipole dipole-induced dipole induced dipole-induced dipole

None of these can completely explain all of theobserved retention in reversed phase HPLC

Important Reversed Phase Parameters

Solvent (mobile phase ) Strength Choice of Solvent Mobile Phase pH Silanol Activity

HPLC Solvents Mobile Phase

LC Mobile Phase Qualities

High purityReasonable cost (and disposal)Boiling point 20-50 C above column temperatureLow viscosityLow reactivityImmiscibile with stationary phaseCompatible with detectorSafety limited flammability and toxicity

SOLVENTS

Polar Solvents

Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile

Non-polar Solvents

N-Decane > N-Hexane > N-Pentane > Cyclohexane

HPLC Solvents Properties

HPLC Solvents Groups

LC Mobile Phase Selection

k of 2-5 for two or three component mixturek of 0.5-20 for multicomponent mixture

Match analyte polarity to stationary phase polarityMobile phase of different polarity

Normal Phase:nonpolar solvent, polar stationary phaseleast polar component elutes firstincreasing mobile phase polarity decreases elution time

Reversed Phase:polar solvent (water, MeOH, ACN), nonpolar stationary phasemost polar component elutes firstincreasing mobile phase polarity increases elution timemost widely used

LC Pumping Systems

General Requirements:Generate pressures up to 6000 psiPulse-free outputFlow rates from 0.1-10 mL/min0.5% or better flow control reproducibilityCorrosion resistant

LC Pumping SystemsReciprocating Pumps Pulsed flow must be damped Small internal volume High output pressures Adaptable for gradient elution Constant flow rates independent of column back-pressure or solvent viscosity

Displacement Pumps Flow independent of viscosity and back-pressure Limited solvent capacity Inconvenient to change solvents

Pneumatic Pumps Inexpensive Pulse free Limited capacity and pressure Dependent on solvent viscosity and backpressure Not good for gradient elution

HPLC Pump Head

Piston HPLC Pump Head

Gradient HPLC

High Pressure

Low Pressure

HPLC Chromatograph injectors

The function of the injector is to place the sample into the high-pressure flow in as narrow volume as possible so that the sample enters the column as a homogeneous, low-volume plug. To minimize spreading of the injected volume during transport to the column, the shortest possible length of tubing should be used from the injector to the column.

When an injection is started, an air actuator rotates the valve:solvent goes directly to the column; and the injector needle is connected to the syringe. The air pressure lifts the needle and the vial is moved into position beneath the needle. Then, the needle is lowered to the vial.

HPLC columns

The column is one of the most important components of the HPLC chromatograph because the separation of the sample components is achieved when those components pass through the column. The high performance liquid chromatography apparatus is made out of stainless steel tubes with a diameter of 3 to 5mm and a length ranging from 10 to 30 cm.

Normally, columns are filled with silica gel because its particle shape, surface properties, and pore structure help to get a good separation. Silica is wetted by nearly every potential mobile phase, is inert to most compounds and has a high surface activity which can be modified easily with water and other agents. Silica can be used to separate a wide variety of chemical compounds, and its chromatographic behavior is generally predictable and reproducible.

Skoog and Leary: Principals of Instrumental Analysis,5th ed. Suanders, 1998

Reverse Phase HPLC

Al

l

t

e

c

h

C

h

r

o

m

a

t

o

g

r

a

p

h

y

S

o

u

r

c

e

b

o

o

k

,

2

0

0

4

-

0

4

c

a

t

a

l

o

g

Alltech Chromatography Sourcebook, 2004-04 catalog

xa

b

c

a b c

c b a

0

0

Time

Time

Normal Phase (SiO2)

Reverse Phase (C18)

Normal Phase (SiO2) TLC

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY(TLC vs Normal Phase and Reverse Phase HPLC)

RP-HPLC Stationary Phase


http://www.chemistry.nmsu.edu/Instrumentation/Waters_HPLC_MS_TitlePg.html


Characteristics of PerformanceThe performance criteria affecting quality of the result include:

Accuracy Precision (repeatability and reproducibility) Sensitivity (LOD and LOQ) Selectivity Linearity Dynamic range Stability

The performance criteria for the economics include:

Cost of purchase, installation and maintenance Analysis time Safety aspects Running costs supplies, gases, consumables Training Sample throughput

HPLC Detectors No universal or versatile detector ...?! Types

General respond to mobil phase bulk properties which vary in the presence of solutes (e.g. refractive index)

Specific respond to some properties of the solute (not possessed by the mobil phase (e.g. UV adsorption)

Hyphenated detector LC-MS

Common LC Detectors

Bulk property detectors

Refractive index detector Conductivity detector Light scattering detector.

Analyte property detectors

9 UV detector9 Fluorescence detector9 Amperometric detectorMass spectrometric detectorCombination detectors

Refractive Index Detector1. The RI detector is one of the few universal detector available in LC 2. Principle:

The RI detectors measure a bulk property of the mobile phase leaving the column: its ability to refract to bend light (i.e., its refractive index). This property changes as the composition of the mobile phase changes, such as when solutes from the column. By detecting this change, the presence of solutes can be detected.

3. Detector Design:

i. One of simplest of RI detectors is the deflection RI detector

ii. In this detector, light is created by a source and passed through flow-cells containing mobile phase eluting from the column (sample stream) and a reference stream (usually mobile phase with no solute in it). The light passing through these flow/cells is passed through a second time using a mirror and passed to a detector where its intensity is measured.

iii. When the refractive index of liquid in the sample and reference flow-cell are the same, little or no bending of light occurs at the interface between the low-cells. This allows the largest amount of light possible to reach the detector.

iv. As solute elute from the column, the refractive index of the liquid in the sample flow-cell will be different that that in the reference flow-cell and light will be bent as it passes between them. This changes the amount of light reaching the detector, producing a response.

4. Applications:

RI detector are universal applicable to the detection of any solute in LC. This makes them useful in preliminary work in LC where the nature or properties of a compound may not be known yet. They also the detector of choice for work with carbonhydrates or in the separation of polymer by size-exclusion chromatography.

Some disadvantages: (1) they do not have very good limits of detection, (2) they can not used with gradient elution, where the composition of the mobile phase is changing with time. (3) The temperature of the system must also be controlled to avoid baseline fluctuations with these detectors.

5. SensitivityThe response of a RI detector is approximately the same for all compounds.

6. Limit of Detection: 10-5 to 10-6 M

7. Linearity/ Dynamic Range: The response of a RI is usually linear over a 104-fold range in concentration.

Refractive Index Detector

Plus:1) Measures a bulk property2) Nearly Universal (different RI than mobile phase)3) Comparable response for different analytes4) Detects species with no chromophores

Minus:1) Temperature dependent2) Poor sensitivity (LOD 100 ng)3) No gradient elution

HPLC Bulk Property Detectors

2. Principle:

i. This detector measures the ability of a solution to conduct a current when placed in an electrical field. This ability depends on the number of ions or ionic compounds present in the solution.

ii. The relationship between the current, electric field and conductivity of the solution is shown as follows:

I = C E

I = CurrentC = conductivityE = electric field strength

Conductivity Detector1. A conductivity detector is an example of a universal detector for ionic

compound.

3. Detector Design

4. Applications: for any compound that is ionic or weakly ionic. It is widely used in ion chromatography.

5. Sensitivity:The response of a conductivity detector depends on the charge and size

of the compound of interest. Small, highly charged compounds tend to produce larger response that large, less charged compound.

6. Limit of Detection: 10-6 M7. Linearity/Dynamic range: 104-fold

Plus:1) Measures a bulk property2) Nearly Universal (must be ionic)3) Common for Ion Exchange LC4) Detects species with no chromophores5) Simple, robust

Minus:1) Fair sensitivity (LOD 1 ng)2) No salts or buffers in mobile phase3) Gradients a challenge


Conductivity Detector

Evaporative Light Scattering Detector

Plus:1) Measures a bulk property2) Nearly Universal (must be non-volatile)3) Does not detect liquids (gradients are ok)4) Detects species with no chromophores

Minus:1) Signal not linear with concentration2) Fair sensitivity (LOD 1 ng)3) No salts or buffers in mobile phase

Evaporative Light Scattering Detector


Absorbance Detector (UV/Vis)1. The absorbance detector is the most common type of detector in LC.

2. Principle:

Absorbance detector measures the ability of solutes to absorb light at a particular wavelength range. This absorbance is described by the Beer-Lambert Law.

A = l cwhere: A = Absorbance of light at a given wavelength

= Molar absorption coefficient of the solutel = path length of the flow-cellc = concentration of solute

3. Detector design:i. There are three types of UV-Vis absorbance detector: fixed wavelength detectors, variable and diode array detector. They are generally based on the following type of design:

ii. In a fixed wavelength detector, absorbance of only one given wavelength is monitored by the system at all time. The wavelength is usually 254 nm.

A fixed wavelength detector is the simplest and cheapest of types of detector, but is limited in terms of it flexibility and the types of compounds it can used to monitor.

iii. In a variable wavelength detector, a single wavelength is monitoredat any given time, but any wavelength in a wide spectral range can be selected.

The wavelengths that can be monitored can vary from 190 nm to 900nm. The ability to use one instrument for more than one wavelength is achieved by adding in more advanced optics to the system.

Diode Array Detector

HPLC UV/VIS Spectrophotometer

5. Sensitivity:

The response of an absorbance detector depends on the molar absorption coefficient. The larger this value is, the larger the response of the detector

6. Limit of detector: 10-8 M

7. Linearity/Dynamic range: 105-fold range

4. Applications:

Absorbance detector can be used to detect any compound absorbing at the wavelength monitored. Absorbance detector can be sued with gradient elution.

HPLC Specific Property Detectors

Multi-wavelength UV-Vis Absorption Detector

Plus:1) Measures a specific property2) Nearly Universal (must have chromophore)3) Most common of all detectors (~75%)4) Potential to provide qualitative info.5) Simple, robust

Minus:1) Fair sensitivity (LOD 1 ng)2) Expensive with PDA, limited with Hg Lamp3) Misses some important analytes

F = I (1-e- l c) = I l c (at low concentration)

F = Fluorescence intensityI = intensity of the excitation light = Fluorescence quantum yield = Molar absorption coefficient of the solutel = path length of the flow-cellc = concentration of solute

2. Principle:

Fluorescence Detector

1. A fluorescence detector is an example of a selective detector, with limits of detection smaller than those by either RI or absorbance monitors.

4. Applications:

It can be used to detect any compound absorbing and emitting lightat the given excitation and emission wavelength.

3. Detector design

5. Sensitivity:

F = I (1- e- l c) = I l c (at low concentration)

6. Limit of detection: 10-10 M

7. Linearity/Dynamic Range: 103 to 104-fold


Plus:1) Measures a specific property2) Highly Selective (must fluoresce)3) Second most common of all detectors (~15%)4) High sensitivity (LOD 0.01 ng)5) Can interrogate very small volumes

Minus:

1) Not Universal2) Limited Applications

Fluorescence Detector

Electrochemical detector1. It can be used to detect an compound which can undergo an electrochemical reaction2. Principle:i. This detector measure the ability of a solute to undergo either oxidation

(i.e., loss of electrons) or reduction (i.e. gain of electrons)

Oxidation: A A+ + e-

Reduction: A + e- A-

ii. One way in which such a reaction can be monitored is by measuring the change in current under a constant electric field. Another way is to measure the change in the electric field produced when a constant current is present.

3. Detector Design

4. Applications:

Electrochemical detectors can be used to detect any solute that can undergo oxidation or reduction.

Detection by reduction: aldehydes, ketones, nitriles, conjugated acids

Detection by oxidation: phenols, peroxides, purines, diols

5. Sensitivity: It depends on the extent of oxidation or reduction that occurs at given potential of the electrode.

6. Limit of detection: 10-11 M

7. Linearity/Dynamic Range: 106-fold

8. Disadvantages: destructive detector


Plus:1) Measures a specific property2) Highly Selective (depends on reduction potential)3) High sensitivity (LOD 0.01 ng)

Minus:

1) Not Universal2) Must have electrolyte in mobile phase3) Mobile phase must be aqueous4) Gradients not possible

Electrochemical Detector

MassMass spectrometryspectrometryMass spectrometry probably is the most versatile and comprehensive analytical technique currently used by chemists and biochemists.

It measures the masses of individual molecules, fragments of molecules and atoms.

It provides ultrahigh detection sensitivity requiring only a few picomoles of a compound to obtain characteristic information regarding the structure and the molecular weight of a compound.

In all cases, energy is transferred to the compound molecules to effect ionization, causing the formation of the molecular ion of the compound.

The molecular ion fragments into a variety of fragment ions and the resulting fragmentation pattern constitutes the mass spectrum.

The mass spectrum of each compound is unique and can be used as chemical fingerprint together with its retention time to characterize the compound.

The first essential step in mass spectrometry analysis to convert the analyte molecules by ionization into gas phase ionic species.

The excess energy transferred to the molecules leads fragmentation.A mass analyzer separates the molecular ion and fragment ions according to their mass/charge (m/z) ratio.

Data are recorded and then converted into a mass spectrum.These steps are carried out under high vacuum [10-2 10-6 Pascal (Pa)].

(1 Pa = 0.0075 torr; 1 atm = 1.013 x 105 Pa)

General concepts General concepts ofof mass mass spectrometryspectrometry

Basic components of a mass spectrometerBasic components of a mass spectrometer

GC or LC

Ionization methods

1. Electron-impact ionization (GC/MS)

2. Chemical ionization (GC/MS)

3. Atmospheric pressure electrospray ionization (LC/MS)

van Deemter Equation

H = A + B/u +Cu

HPLC HPLC -- Column EfficiencyColumn Efficiency


HPLC HPLC -- Column EfficiencyColumn Efficiency

H = H = AA + B/+ B/uu + C+ Cuu

A = 2A = 2ddpp

1.1. depends on particle size distribution, the depends on particle size distribution, the narrower the distribution the smaller the l narrower the distribution the smaller the l

2.2. ddpp = particle size= particle size3.3. Independent of mobile phase flow rateIndependent of mobile phase flow rate4.4. Also known as eddy diffusionAlso known as eddy diffusion


HPLC HPLC -- Column EfficiencyColumn Efficiencyparticle sizeparticle size


HPLC Column EfficiencyHPLC Column Efficiency

Longitudinal Diffusion (B)Longitudinal Diffusion (B)

H = A + H = A + B/B/uu + C+ Cuu

B/u = 2B/u = 2DDMM/u/u

1. = constant depending on quality of packing

2. DM is the mobile phase diffusion coefficient

3. Inversely related to mobile phase flow rate

HPLC Column EfficiencyHPLC Column Efficiency

Mass Transfer Mass Transfer (Cs + Cm)

H = A + B/u + (Cs + Cm)u

CS = fS(k)df2 / DS

CM = fM(k)dp2 / DM

DDMM is the mobile phase diffusion is the mobile phase diffusion coefficientcoefficient

DDSS is the stationary phase is the stationary phase diffusion coefficientdiffusion coefficient

ddff is film thicknessis film thickness ddpp is particle sizeis particle size Directly related to mobile phase Directly related to mobile phase

flow rateflow rate


Flow Chart I: Small Molecules1. m. wt.

2. solubility

1. m. wt.

2. solubility

Flow Chart II: Large Molecules

Uses of HPLC This technique is used for chemistry and biochemistry research

analyzing complex mixtures, purifying chemical compounds, developing processes for synthesizing chemical compounds, isolating natural products, or predicting physical properties. It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product stability and monitor degradation.

In addition, it is used for analyzing air and water pollutants, for monitoring materials that may jeopardize occupational safety or health, and for monitoring pesticide levels in the environment. Federal and state regulatory agencies use HPLC to survey food and drug products, for identifying confiscated narcotics or to check for adherence to label claims.

Area of application:

Separation and purification of substances and Analysis

Chemistry Biomedical and Clinical Pharmaceutics Agriculture and Food Enviromental Veterinary

RP-HPLC - Example


RP-HPLC Gradient Elution


Chromatogram of Organic Compounds from Fermented Cabbage

Chromatogram of Orange Juice Compounds

Liquid chromatograph/mass spectrometerLiquid chromatograph/mass spectrometer

MassSpectrometer

MassSpectrometer

LiquidChromatograph

LiquidChromatograph

Rough pumpRough pump

UVDetector

UVDetector

SamplerInjection port

SamplerInjection port

ColumnColumn

SolventsSolvents

Ion sourceIon source

PumpsPumps InterfaceInterface

ComputerComputer

Separation TechniquesNormal phase Stationary Phase: AluminaReverse phase Reverse phase chromatographySize exclusion GEL-PERMEATION CHROMATOGRAPHY Ion exchange MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS Mechanism of separation in different forms of HPLCHHHCommon RP PackingsRP Column PropertiesParticle SizeRP Mechanism (Simple)Reversed Phase MechanismsProposed RP MechanismsHydrophobic TheoryPartition TheoryAdsorption TheoryImportant Reversed Phase ParametersHPLC Solvents Mobile PhaseHPLC Solvents PropertiesHPLC Solvents GroupsHPLC Pump HeadGradient HPLCHPLC Chromatograph injectors HPLC columnsCharacteristics of PerformanceHPLC DetectorsHPLC UV/VIS SpectrophotometerFlow Chart I: Small MoleculesFlow Chart II: Large MoleculesUses of HPLC

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chromatography chromatography

stationary stationary

contiguous stationary

layer chromatography

normal phase lscb

reverse phase lsc2

normal phase llcb

reverse phase llc3