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# Types of Chromatography

Chromatography is considered one of the popular techniques utilised in the field of biochemistry and could be employed even in case of normal lab process. It is widely utilized to identify and isolate very small quantities of substance both qualitatively as well as quantitatively. It is utilized as the equipment’s used are very simple and yet very effective. The actual skill to carry out these are very simple and no specific or special skill is necessary. The results that we usually get after the analysis is reproducible and hence is ideal.

Chromatography basically is the technical procedure of analysis by liquid percolation through a body of porous rigid material and carried out irrespective of physiochemical process that lead to the separation of substance. The various physiochemical factors that are involved in this are adsorption, partition, ion exchange along with molecular sieving.

In all types of chromatography there are two phases, a stationary phase and the other a mobile phase. In case the mobile phase is a liquid moves along the stationary phase like the column of a solid or paper and the separation of the substance takes place by one or more physiochemical factors operation.

The mobile phase has a large specific surface area and the combination of both stationary and mobile make sure a very fast mass transfer between phases and rapid local equilibrium. The components of the analysed mixture is ideally be soluble in mobile phase and a physiochemical process which would cause the components of the analysed mixture to have some moderate affinity for stationary phase and maintain an equilibrium between the mobile and stationary phase.

## Gas Chromatography

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Around 1952 A J P Martin Nobel laureate suggested that it is possible to use vapour as the mobile phase and some years later along with James used ethyl acetate vapour to desorb a mixture of fatty acids that are affixed to an adsorbent. The vapour stream eluting from the test tube was directed to an automated titration apparatus which showed a graph showing a series of steps that reflected the sequence of base addition neutralizing the eluted acid through automated titration.

Gas chromatography is now a major method of quantitative analysis of complex mixtures. This is adopted mainly because it is fast, accurate and relatively inexpensive. It has wide range of application as well. Gas chromatography uses computer technology along with data processing and digital electronics as the integration of these into chromatograph makes it a very sophisticated equipment.

The process involves the carrier gas which is injected into the injector, through which the column and finally into the detector. After the detection is over the data is collected and analysed.

The method follows the following steps:

Carrier gas (at constant flow rate or pressure) $\rightarrow$ sample introduced into the carrier gas stream $\rightarrow$ chromatograph column $\rightarrow$ detection system $\rightarrow$ temperature controlled oven $\rightarrow$ data handling.
• Carrier gas supply unit delivers a steady stream of carrier gas with a flow rate controller which is kept constant. The number of moles of gas passing through is kept constant.
• The sampling system which helps in injecting the sample into the stream of gas is modulated in order to vaporize the sample in short time and introduce into column as cylindrical plug of vapour diluted by carrier gas
• The column which is placed inside a temperature controlled oven is kept in at 350 C
• The linear detector which helps in delivering the signal function of carrier gas composition is kept at zero when pure carrier gas is proportional to the concentration of any compound other than carrier gas.

## Liquid Chromatography

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The liquid chromatography separation is based on the selective distribution of analytes between a liquid based mobile phase and a stationary phase. The sample is injected by means of an injection port into the mobile phase stream delivered by high pressure pump and transported through the column where the separation takes place. The separation method is monitored by a flow through detector.

The classification of liquid chromatography is based on various types of distribution mechanism applied in the separation techniques. In practice liquid chromatography in most cases are the result of mixed mechanism.

Most of the liquid chromatography applications are done with reversed phase liquid chromatography or a non-polar stationary phase and polar mobile phase. This is mainly because the non-polar bonded chemical materials are much easier to use and reversed phase liquid chromatography are ideally suited to analyse polar and ionic analytes.

## Ion Exchange Chromatography

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In ion exchange chromatography, the chromatographic support contains ions which are capable of being exchanged with ionic solutes in the mobile phase. The ion exchange chromatography normally make use of a bonded quaternary ammonium group for the anion separation and a bonded sulphonic acid group for the separation of cations. The ion exchange chromatography was utilized almost a quarter of century back to separate nucleic acid components, the amino acids, sugar molecules etc.

In ion exchange chromatography there are two important variables which could be altered to achieve the separation between two or more components. This could be carried out by increasing the ionic strength (µ) of the eluent or buffer solution which makes the process of solute elution faster when the ion exchange is taking place.

## Paper Chromatography

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The paper chromatography consist of applying a small drop of solution containing the substance to be separated to a strip of filter paper at a short distance from one end. The drop is allowed to dry and the end of paper close to the dried spot is placed in the developing solution without immersing the spot in solution.

The solvent starts flowing up due to capillary action and caries the components of the mixture along at different rates. Once the solvent reaches the top end of the paper, it is removed, dried and the separated zones are located and observed. The paper chromatography in contemporary world is very important and is widely used as an analytical tool.

## Thin-layer Chromatography

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Compared to paper chromatography which is basically a good example of plane chromatography, the TLC or thin layer chromatography is mostly utilised in analytical laboratories. The process applied is same as paper chromatography but the only difference is that in paper chromatography we make use of paper fibre while in thin layer chromatography the silica or alumina is used for the stationary phase.

The term thin layer chromatography is used due the thin layer of stationary phase that has been spread out upon a glass plate or even on microscopic slide as a slurry which is then allowed to dry to produce the thin layer stationary phase. The silica gel used as stationary phase contains SiOH groups that form the hydrogen bonds with other polar groups like alcohol, amines, and carboxylic acids.

The retardation factor in thin layer chromatography is a ratio between the distance travelled by the analyte $(d_R)$ from its original spot and the distance travelled by solvent (dM)

Retardation factor = $\frac{distance \ travelled \ by \ analyte \ from \ origin}{distance \ travelled \ by \ solvent \ front}$

Retardation factor = $\left ( \frac{d_R}{d_M}\right )$.

## Affinity Chromatography

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In affinity chromatography the isolation of alpha amylase by means of insoluble substrate such as starch was described long back in 1910 by Starkenstein.

The affinity chromatography is originated on the basis of biologically active substances which locates and bind in specific regions and also reversibly other substances which are better known as ligands or affinity ligands or may be simply affinants. The whole basis of affinity chromatography using affinants chemically bonded to a solid matrix in a covalent manner has been known for more than two decades to isolate anti bodies of cellulose column attached with covalently bonded antigen.

If insoluble affinants is prepared, the covalent coupling to a solid support and a solution containing biologically active product needs to be isolated is passed through a column of affinant then all the compounds will have no affinity for the affinant and will pass through un-retarded. The use of affinity chromatography is not limited to only isolation of biologically active substance but could be used for determining apparent molecular weight of dehydrogenase based on exclusion from gel filtration. The affinity chromatography is ideal for study of interactive biological process where all sorts of protein interactions with nucleic acids etc. could be studied in great detail.
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