Techniques of chromatography

Part 2

Techniques of chromatography

A.OPEN-COLUMN CHROMATOGRAPHYB. PAPER CHROMATOGRAPHY (PC)C.THIN-LAYER CHROMATOGRAPHY (TLC)D.Gas chromatographyE.HIGH-PRESSURE LIQUID CHROMATOGRAPHY F.SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)G.ELECTROPHORESIS (Electrochromatography)H.High Performance Capillary Electrophoresis (HPCE):

A. OPEN-COLUMN CHROMATOGRAPHY

stationary phase (silica or alumina) is packed in glass tubes The stationary phase particle size is large (250 jam) to allow the

passage of solvent. Disadvantage is the long time needed for the separation of

complex mixtures (up to one week or more).

B) PAPER CHROMATOGRAPHY (PC)

The adsorbent is a sheet of paper of suitable texture and thickness

Development may be ascending in which case the solvent is carried up the paper by capillary forces, or descending, in which case the solvent flow is also assisted by gravitational force.

C- Thin-layer chromatography

Separation is based on migration of the sample spotted on a coated (stationary phase) plate with one edge dipped in a mixture of solvents (mobile phase).

However, it is not usually as accurate or sensitive as liquid chromatography.

D- Gas chromatography

Principle:

A pressured gas flows through heated tube coated with liquid stationery phase or packed stationery on a solid support.

The analyte loaded on the head of the column via heated injection port, where it is evaporated.

The separation of a mixture occurs according the relative time spent in the stationary phase

Instrumentation

1. Injection of the simples manually or using autosampler usually size of 0.5 – 2 ul injection volume

2. The sample is evaporated and condensed at the head of the column

3. The column either capillary or packed column, the mobile phase is a gas to carry the sample through the column which is Helium or nitrogen gases.

4. The oven to heat the column up to 400 oC.5. The detector usually flame ionization detector FID

WWU -- ChemistryWWU -- Chemistry

The Gas Chromatograph

Gas Chromatography

Capillary column

Injection site

Control panel

Gas Chromatography Plotter

WWU -- ChemistryWWU -- Chemistry

Gas Chromatography: Separation of a Mixture

Stationery phase for GCTypes of columns

1. Packed columns:Usually glass columns silanised to remove Si….OH The column mobile phase used is nitrogen at flow rate of 20 ml/min. Limitation, can not be used above 280 oC because of the evaporation of the stationary

phase

2. Capillary column The inner surface is coated with orange silicon polymers which are chemically

bonded to silanol groups The mobile phase used usually Helium at low flow of 0.5 to 2 ml/min

Columns

• Packed

• Capillary

Factors governing the retention of compounds in capillary GC;

1. Carrier gas type and flow: Nitrogen and helium

2. Column temperature: increase of column temperature, decreases resolution between two compounds,

3. Column length: increase the column length increases the resolution

4. Film thickness phase loading: the greater the volume of the stationary phase the more solutes will be retained

5. The column internal diameter: the smaller the diameter the more efficient

Gas Chromatogram

Lowestb.p.

Highestb.p. Retention

time

Chromatograms - 551.1

Detectors

1. Flame ionization detector FIDCompounds burned at the detector produced ions Detects carbon – hydrogen compounds till 10 ng Wide application range up to 10-6

2. Electron capture detector ECDHighly halogenated compounds can be detected at 50 fg – 1 pg Wide application for drugs determination in biological fluids. Have wide application in

environmental analysis such as chlorofluorocarbons in the air 3. Nitrogen phosphate collectorsUsed for compounds containing nitrogen and phosphors such as drugs and metabolities in body

tissues and fluids High selective

4. Thermal conductivity detectors TCDResponding to cooling effect of the analyte passing over filament Insensitive, used for determination of water vapour such as in peptides

Application of GC1. Detection of impurities in drug formulation

2. used for quantification of drug substances in formulation specially for drugs lack of chromophore

3. characterization of some row material used for drug synthesis

4. measurements of drugs and their metabolites in biological fluids

Limitation of GC

1. only thermostable compounds can be analysed

2. the sample may require derivatisation to be volatile

3. quantitative sample introduction is more difficult due to the small volume of sample injected

Derivatization: GC

The technique is extended by the preparation of volatile derivatives of the non volatile compounds or of the compounds, which undergo decomposition.

used also for improvement of peak shape, relocation of an interfering peak, improvement of sensitivity or improvement of separation of closely related compounds.

An example of derivatization is silylation by addition of trimethylsilyl group to carboxylic acids, amines, imines, alcohols, phenols and thiols by treatment with hexamethyldisilazane.

ColumnInjector Detector

HPLC

Data Processing

25

Chromatographic Column

The system consists of main parts:1- Mobile phase or solvent reservoir.2- A high pressure pump.3- A sample inlet port.4- Column5- Detector6- Recorder

Schematic diagram of an HPLC unit(1) Solvent reservoirs, (2) Solvent degasser, (3) Gradient valve, (4) Mixing vessel for delivery of the mobile phase, (5) High-pressure pump, (6) Switching valve in "inject position", (6') Switching valve in "load position",

(7) Sample injection loop, (8) Pre-column (guard column), (9) Analytical column, (10) Detector (i.e. IR, UV), (11) Data acquisition, (12) Waste or fraction collector.

The pump, capable of maintaining high pressures draws the solvent (mobile liquid phase) from the reservoir and pushes it through the column.

The sample is injected through a port into the high pressure liquid carrier steam between the pump and the column.

The separation takes place on the columns, which vary, from 25-100 cm length and 2-5 mm in internal diameter. Typical flow rates are 1-2 ml/min with pressures up to several thousand psi.

The column effluent passes through a non-destructive detector where a property such as :

UV absorbance, Rl ormolecular fluorescence

To increase the efficiency of separation, the mobile phase may be altered by changing its polarity, pH or ionic strength. HPLC offers the advantages of speed, resolution and sensitivity.

There are two types of HPLC procedures:

1.LLC: the column consists of an inert support usually silica gel on which the stationary partitioning phase is adsorbed.

In the normal phase mode, the stationary phase is polar (e.g. methanol, acetonitrile or water) while the mobile phase is less polar (e.g. iso-octane, chloroform or n-hexane). This mode is usually used for the separation of polar components.

In the reverse phase LLC, the stationary phase is less polar and the mobile phase is polar. It is usually used for the separation of non-polar components.

2.LSC: The packing may be silica (polar packing) or octadecylsilica, ODS (C18-silica, non-polar packing). Adsorption mechanism is involved here.

In the normal phase LSC, the packing is polar (silica) and the mobile phase is less polar (e.g. n-hexane).

In the reverse phase LSC, the packing is non-polar (eg. ODS) and the mobile phase is polar (e.g. acetonitrile-water or methanol-water).

Again, as under LLC, normal phase LSC is used for polar solutes while reverse phase LSC is used for separation of non-polar compounds.

Elution Approaches

• Isocratic - constant mobile phase composition• Gradient - variable mobile phase composition

– step - change accomplished sharply at a defined point in time

– continuous - change accomplished gradually over time

HPLC Columns

• Analytical columns Made of stainless stele Internal diameter 2.1 – 4.6 mm column long 30 – 300 mm Particle size 3 – 10 micrometer

Gourd columnsShorter column 7.5 mm Used to prevent the adsorption of substances on the analytical column

Stationary phase in HPLC

Chemically inert

Non-soluble in any imaginable mobile phase

Thermal and chemical stability

Appropriate physical sorption of analyte

Shape: Uniform spherical particles

Stationary phase in HPLC1. unmodified silica stationary phasesSpherical and regular Particle size 3-10 uM;Polar surface due to the silanol groupsUsesFor the separation and retention of non polar and moderately polar

compounds such as poly aromatics fats, oil, isomers

2. Modified silica stationary phasesSpherical and regular Particle size 3-10 uM: Non-polar due to the modification of silanol groups by chloroaloxy silane

produces stable stationary phases, Ex ODS Octadecylsilane the most used stationary phase in reversed phase chromatography

UsesFor the separation and retention of wider range of polar and moderately

polar substances such as drugs and amino acids

Si OH+

Cl Si

CH3

CH3

R-HCl

Si O R

CH3

CH3

Si

Si OH

Si OH

+ Cl2SiR2

-2HClSi O

SiR2

OSi

NH2

CN

O OH

OH

octadecyl

octyl

phenyl

amino

cyano

diolPolar phase

Nonpolar phase

F) SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC)

A supercritical fluid: is a substance above its critical temperature and pressure.Critical temperature (Tc): is that above which it is impossible to liquefy a gas, no matter how great a pressure is applied. Critical pressure (Pc): is the minimum pressure necessary to bring about liquefaction at Tc.Critical volume (Vc): is the volume occupied by one mole of gas or liquid at the critical temperature and pressure.

SFC , is a column chromatographic technique in which a supercritical fluid is used as a mobile phase .The used mobile phase is frequently cooled to be maintained in a liquid state for easier pumping to the column. Carbon dioxide is the most frequently used mobile phase .Other mobile phases include ammonia, nitrous oxide , and xenon .SFC ,is an intermediate between GC and HPLC and offers the advantages of both .

Compound Tc, °C Pc, atm.

Carbon dioxide 31.05 72.9

Nitrous oxide 36.4 71.5

Ammonia 132.4 111.3

2-Propanol 235.1 47.6

Methanol 239.4 79.9

Acetonitrile 274.8 47.0

Water 374.1 217.6

Advantage of supercritical fluids as mobile phases in chromatography compared with liquid chromatography is that

1.solutes generally have much higher diffusion coefficient in them than in liquids. This leads to enhanced speed of separation and possibly greater resolution with complex mixtures, especially for large molecules.

2.SFC possesses also advantages over GC in that solutes do not have to be volatile or thermally stable.

C) ELECTROPHORESIS (Electrochromatography)

Introduction:

Electrophoresis is a technique in which solutes are separated by their different rates of travel through an electric field.

- commonly used in biological analysis, particularly in the separations of proteins, peptides and nucleic acids

The rate of migration (electrophoretic mobility) of each species is a function of its charge, shape and size.

Gel electropherograms

– another type of zone electrophoresis – It involves high voltage electrophoresis in narrow bore fused-silica capillary tubes and

on-line detectors similar to those used in HPLC. - On passing through the detector, they produce response profiles that are sharper than

chromatographic peaks.

High Performance Capillary Electrophoresis (HPCE):