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INTRODUCTION TO CHROMATOGRAPHY AND ITS APPLICATIONSKalsoom Saleem

CMS # 8107

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Introduction

Chromatography, literally "color writing", was first employed by Russian-Italian scientist Mikhail Tsvet in 1900, primarily for the separation of plant pigments such as chlorophyll, carotenes, and xanthophylls.

The word chromatography is derived from two Greek words

Chroma ….……..colorGraphos ………..writing

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Definition

‘A technique by which a mixture is separated into its components on the basis of relative ability of each component to be moved along/through a stationary phase by mobile phase’

The technique of chromatography is based on the differences in the rate at which the components of a mixture move through a porous medium (called stationary phase) under the influence of some solvent or gas (called moving/mobile phase).

Chromatography is a nondestructive procedure

Applied both for both qualitative and quantitative studies

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Principle

Like-dissolve-like or like-prefer-like. The basis-partition or distribution coefficient ‘K’ which describes the way in which a compound distributes itself between two immiscible phases.

Defined as the molar concentration of analyte in the stationary phase divided by the molar concentration of the analyte in the mobile phase

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Commonly Used Terms

Analyte

The substance to be separated during chromatography

Analytical chromatography

Determines the existence and the concentration of analyte(s) in a sample

Chromatogram

Visual output of the chromatograph

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Chromatogram

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Commonly Used Terms

Chromatograph

An equipment that enables the separation e.g. gas chromatographic or liquid chromatographic separation.

Eluate

Mobile phase

Eluent

The components obtained after the process.

Eluotropic series

List of solvents ranked according to their eluting power

Elution

Process of extracting a substance that is adsorbed on another surface by moving it with solvent.

Commonly used terms

Immobilized phase

Stationary phase that is immobilized on the support particles, or on the inner wall of the column

Mobile phase

Phase that moves over the stationary phase. It may be a liquid (LC) or a gas (GC).

Retention time

Time required for the mobile phase to sweep a component from the stationary phase.

Sample

Matter analyzed in chromatography- single component or multiple components

Solute

Sample components in partition chromatography.

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Commonly Used Terms

Solvent

Any substance capable of solubilizing another substance, and especially the liquid mobile phase in liquid chromatography

Stationary phase

Substance fixed in place for the chromatography procedure-solid, gel or a liquid. e.g ; silica, alumina, cellulose

Detector

Instrument used for qualitative and quantitative detection of analytes after separation

Rf value or Retention factor (Rf)

The ratio of the distance traveled by the center of a spot (solute) to the distance traveled by the solvent front (solvent)

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Classification

Analytical• To determine the

chemical composition of sample

Preparative• Used to purify and

collect one or more components of a sample

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Types

Adsorption

Partition

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1. Adsorption Chromatography

It uses a mobile phase or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phase accounts for the separation of different solutes. Following are the chromatographic techniques that are included in this category:

Techniques

Gas Solid Chromatogra

phy (GSC)

Gel Permeation

Chromatography

High Performance (pressure)

Liquid Chromatography (HPLC)

Ion Exchange Chromatogra

phy

Column Chromatogra

phy

Thin Layer Chromatogra

phy (TLC)

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Principle

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Thin Layer Chromatography

Stationary phase is a thin layer of a solid such as alumina or silica supported on an inert base such as glass, aluminum foil or insoluble plastic.

The mixture is ‘spotted’ at the bottom of the TLC plate and allowed to dry. The plate is placed in a closed vessel containing solvent (the mobile phase) so that the liquid level is below the spot.

TLC is also called as drop, strip, spread layer, surface chromatography and open column chromatography.

TLC

TLC PAPER

Column Chromatography

A solvent acts as the mobile phase while a finely divided solid surface acts as the stationary phase. The stationary phase will adsorb the components of the mixture to varying degrees. As the solution containing the mixture passes over the adsorbent, the components are distributed between the solvent and adsorbent surface. This process may be described by three-way equilibrium between the sample, the solvent and the adsorbent. The solvent and sample compete for positions on the solid adsorbent, the solvent displacing the sample reversibly and continuously in the direction of the solvent flow. Consequently, a weakly adsorbed compound will spend more time in the solvent, and will therefore be eluted first.

Ion Exchange Chromatography

Ion exchange chromatography is used to remove ions of one type from a mixture and replace them by ions of another type.

The column is packed with porous beads of a resin that will exchange either cations or anions. There is one type of ion on the surface of the resin and these are released when other ions are bound in their place – e.g. a basic anion exchange resin might remove nitrate ions (NO3–) from a solution and replace them with hydroxide ions (OH–).

Principle

High Performance (pressure) Liquid Chromatography (HPLC)

HPLC involves a liquid sample being passed over a solid adsorbent material packed into a column using a flow of liquid solvent under pressure. 

Normal Phase HPLC

• A non-polar, non-aqueous mobile phase (e.g. Chloroform), the analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increased analyte polarity.

Reversed Phase

HPLC

• A non-polar stationary phase and an aqueous, moderately polar mobile phase. With such stationary phases, retention time is longer for molecules which are less polar, while polar molecules elute more readily (early in the analysis).

HPLC Working

Gel Filtration or Gel Permeation Chromatography

The separation of large molecules, often in biochemical situations, can be achieved in a column which works on the basis of molecular exclusion. The mixture of solutes is carried through the column by a solvent. The stationary phase (the gel) typically consists of particles of a cross-linked polyamide which contains pores. Separation occurs according to molecular size – the larger molecules passing through the column fastest. Different gels are available that allow the separation of proteins with relative masses. The greatest resolution is achieved by using very small gel particles, but the flow rate through the column then becomes much slower.

2. Partition Chromatography

This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid.

Paper Chromatography

Gas Liquid Chromatography (GLC)

Principle

Gas Chromatography

Gas-liquid chromatography (GLC): In GLC the mobile phase is a gas and stationary phase is a thin layer of a non-volatile liquid bound to a solid support thus a partition process occurs. In such case small inert particles such as

Diatomaceous earth is coated with the liquid so that a large surface area exists for the solute to equilibrate with.

Gas-solid chromatography (GSC): GSC utilizes a solid adsorbent as the stationary phase while gas as a mobile phase and an adsorption process takes place.

The separation method can be affected by the polarity of stationary phase, temperature, carrier gas flow, length of column, material amount etc.

Principle

Working

Paper Chromatography

Cellulose filter paper is often used as stationary phase in paper chromatography. Since it is hydrophilic, it is usually covered with a thin film of water. That’s why it is often called as liquid-liquid chromatography.

Principle: The substances are distributed between liquid phases. One phase is the water which present in pores of filter paper and other phase is mobile phase which moves on the paper. The separation of mixture is due to different attraction force towards stationary phase (water) and mobile phase (solvents). 

Principle

Affinity chromatography

Detectors

The components elute from the column can be quantified by a detector and/or collected for further analysis. An analytical instrument can be combined with a separation method for on-line analysis. Examples of such "hyphenated techniques" include gas and liquid chromatography with mass spectrometry (GC-MS and LC-MS), Fourier-transform infrared spectroscopy (GC-FTIR), and diode-array UV-VIS absorption spectroscopy (HPLC-UV-VIS)

Visualization of Chromatogram

It can be done by following method,

Use of fluorescent material like manganese with activated zinc silicate

Spray of Iodine vaporsUV exposureNinhydrin for identification of amino acids

Applications

It is commonly used technique for the separation of molecule. For example, it is used to remove pesticides and insecticides like DDT in the water and poly chlorinated biphenyls.

It is widely used to determine the purity of a substance.

In pharmaceutical companies, it is used for producing pure materials for medicines and also for checking the contamination presence in medicines.

It is used in pharmacy for detecting the chiral compounds (Enantiomers and optical isomers).

In the food industry, this technique is very useful for analyzing and the separation of additives, proteins and amino acids etc.

It used in forensic science for detecting the presence of drugs.

Chromatography has been extensively used in the isolation, characterization and determination of the 60 or so carotenoids at present known.

References

M. Younus, Ilmi Kitab Khana, 2011. Organic Spectroscopy And Chromatography, Chromatography, pg. 269, ed. 3rd.

Scott, R. P. W. Introduction to Analytical Gas Chromatography; 2nd ed.; Marcel Dekker, 1998.

Jennings, W. G.; Mittlefehldt, E.; Stremple, P. Analytical Gas Chromatography; 2nd ed.; Academic Press, 1997.

McNair, H. M.; Miller, J. M. Basic Gas Chromatography; Wiley, 1997. Grant, D. W. Capillary Gas Chromatography; Wiley, 1996. Fowlis, I. Gas Chromatography; 2nd ed.; Wiley, 1995. Scott, R. P. W. Techniques and Practices of Chromatography; 2nd ed.; Marcel

Dekker, 1995. Grob, R. L. Modern Practice of Gas Chromatography; 3rd ed.; Wiley, 1995. Baugh, P. E. Gas Chromatography: A Practical Approach; Oxford, 1994. Hinshaw, J. V.; Ettre, L. S. Introduction to Open Tubular Column Gas

Chromatography; Advanstar, 1994. Grob, K. Split and Splitless Injection in Capillary Gas Chromatography; 3rd

ed.; Hüthig, 1993. Hill, H. H.; McMinn, D. G. Detectors for Capillary Chromatography; Wiley,

1992. Grob, K. On-Column Injection in Capillary Gas Chromatography; 2nd ed.;

Hüthig, 1991. Poole, C. F.; Poole, S. K. Chromatography Today; Elsevier, 1991. Baars, B.; Schaller, H. Fehlersuche in der Gaschromatographie; VCH, 1994. Kolb, B. Gaschromatographie in Bildern; Wiley-VCH, New York, 1999. Kenndler, E.; Huber, J. F. K. In Analytiker Taschenbuch; Springer, 1989