CONTENTS:-

INVENTION OF CHROMATOGRAPHYABOUT CHROMATOGRAPHYWORKING OF CHROMATOGRAPHYINTRODUCTION OF GAS CHROMATOGRAPHYPRINCIPLETHEORETICAL PLATE INSTRUMENTATIONAPPLICATIONS

Invention of Chromatography

Mikhail TswettRussian Botanist

(1872-1919)

Mikhail Tswett invented chromatography in 1901 during his research on plant pigments.

He used the technique to separate various plant pigments such as chlorophylls, xanthophylls and carotenoids.

Chromatography: (Greek = chroma “color” and graphein “writing” ) Tswett named this new technique chromatography based on the fact that it separated the components of a solution by color.

Common Types of Chromatography

There are now several common chromatographic methods. These include:•Paper Chromatography•Thin Layer Chromatography (TLC)•Liquid Chromatography (LC)•High Pressure Liquid Chromatography (HPLC)•Ion Chromatography•Gas Chromatography (GC)

How Does Chromatography Work?In all chromatographic separations, the sample is transported in a mobile phase. The mobile phase can be a gas, a liquid, or a supercritical fluid.

The mobile phase is then forced through a stationary phase held in a column or on a solid surface. The stationary phase needs to be something that does not react with the mobile phase or the sample.

The sample then has the opportunity to interact with the stationary phase as it moves past it. Samples that interact greatly, then appear to move more slowly. Samples that interact weakly, then appear to move more quickly. Because of this difference in rates, the samples can then be separated into their components.

Good for volatile samples (up to about 250 oC)

0.1-1.0 microliter of liquid or 1-10 ml vapor

Can detect <1 ppm with certain detectors

Can be easily automated for injection and data analysis

Gas ChromatographyIt is based on distribution of gaseous solutes between a gas & liquid or solid phase thus according to the nature of stationary phase they are classified in two categories:-Gas solid chromatography(SP-solid)Gas liquid chromatography(SP-non volatile liquid on solid support)

PROPERTIES:-

Principle

The organic compounds are separated due to differences in their partitioning behavior between the mobile gas phase and the stationary phase in the column.

Theoretical plate is a term coined by Martin & Synge.

Theoretical plates (N) measure how efficiently a column can separate a mixture into its components. This efficiency is based on the retention time of the components and the width of the peaks.

The Theoretical Plate

wb

tR

N = 16( t R

w b

) 2

N = Number of theoretical plates (a measure of efficiency)

tR is the retention time; it is measured from the injection peak (or zero) to the intersection of the tangents.

wb is the width of the base of the triangle; it is measured at the intersection of the tangents with the baseline.

Instrumentation of a GasChromatography

Gas Supply: (usually N2 or He)

Sample Injector: (syringe / septum)

Column: 1/8” or 1/4” x 6-50’ tubing packed with small uniform size, inert support coated with thin film of nonvolatile liquid

Detector: TC - thermal conductivityFID - flame ionization detector

Schematic of a Gas Chromatography

Our GC SystemCarrier Gas : The main purpose of carrier gas is to transport sample components through the column.(H , He , Ar & N are common carrier gases)

Injection Port : It is placed so that the sample is introduced directly into the carrier gas.

Column :columns are of three type:-•Packed column(PC)•Open tubular column(OTC)•Support coated open tubular column(SCOTC)PACKED COLUMN : The column packed by the liquid phase over an inert solid support.OPEN TUBULAR COLUMN : It is about 100-130 fit diameter about 0.01-0.03inch the inside wall of capillaring tube is coated by liquid phase

in the form of thin & uniform film.SUPPORT COATED OPEN TUBULAR COLUMN : columns are made by depositing a micron size porous layer of support material on the inside wall of a capillaries columns & then coated with a thin film. These column are of coarse having higher sample capacity & yeild better resolution .Detector : After resolution of solutes each is vapourised components in turn from column & is carried into the detector mixed with carrier gas.The detectors receive the impulse from the elute of the column in the form of solute. The vapour is sensed by the detectors it converts this impulse into an electric signal proportional to the concentration of the solute in carrier gas . The impulse signal is emplified & recorded as a peak. Various detectors are as follows:-Thermal conductivity detectorFlame ionized detectorFlame photometric detectorElectron capture detectors

Detectors: Flame IonizationDetectors: Flame Ionization

FID uses fuel mixed with carrier and organic analyte. Analyte forms ions in the flame.Cations from the flame are gathered by the negative electrode—produces a current.Advantage:inorganics do not respond (He carrier gas), so the low background signal allows for LOD 100- to 1000-fold lower then TCD. Disadvantage: destructive

Detectors: Electron CaptureDetectors: Electron CaptureDetects electronegative atoms or groups (Cl, -NO2) and also polynuclear aromatics and conjugated carbonyl compoundsThese groups capture e– that are produced from nuclear radiation from 3HCarrier gas hit by these e– can release secondary e–, which will be absorbed by analyteHas good LOD, but a narrow linear range and requires radioactive source

THERMAL CONDUCTIVITY DETECTOR (TCD)

A TCD detector consists of an electrically-heated wire.The temperature of the sensing element depends on the thermal conductivity of the gas flowing around it. Changes inthermal conductivity, such as when organic molecules displace some of the carrier gas, cause a temperature rise in the element which is sensed as a change in resistance. The TCD is not as sensitive as other detectors but it is non-specific and non-destructive.

GAS CHROMATOGRAPHY (INSTRUMENT)

ApplicationsThere are two type of analytical methods of gas chromatography:-•Qualitative Analysis•Quantitative Analysis

QUALITATIVE ANALYSIS:-

This can be used to confirm identity of a suspected compound but it along can not establish the structure of unknown compound.

To supplement identification of the unknown by retention data , suspected compound can be added to the suspected compound & to see if there is a concomitent increase in the peak heights.

QUANTITATIVE ANALYSIS:-

With the help of gas chromatography qualitative analysis of individual component in a mixture.

The method has advantages over titrimetry or spectrophotometry because both evaluations of a mixture can be performed simultaneously.

There are numerous advantages of GAS CHROMATOGRAPHY:-Gas chromatography plays important role in the analysis of pharmaceutical products & drugs.

It is used in quality control.

Analysis of new products.

In monitoring metabolites & biological fluids.

Few of application includes:-•Antibiotics : penicillin•Anti tuberculosis drugs : isoniazid•Anti neoplastics : chlorambucil& florourasil•CNS stimulants : caffine & theophyllin