3 Main Types of Chromatography | Microbiology

The following points highlight the three main types of chromatography. The types are: 1. Adsorption Column Chromatography 2. Thin Layer Chromatography 3. Gas Chromatography.

Type # 1. Adsorption Column Chromatography:

The general chromatography method was introduced into biology by Tswett in 1906 and various adsorbents such as starch and aluminium oxide are poured as a slurry into a long glass tube. The name column chromatography was given to it because column of materials was used for adsorption of different substances.

An unknown compound is poured on the open top of the tube and identified by slowly trickling down an appropriate solvent through the long tube commonly spoken as a column.

The rate at which any compound migrates through the column depends upon the balance between its affinity for the solvent and its adsorption on the particles in the column. By column chromatog­raphy the different coloured components of a mixture are separated and the separated components travel down the column at different rates.

By changing receivers the different fractions are collected separately as they leave the bottom of the column. Colourless substances are frequently separated by collecting many small fractions in succession from the column, testing each fraction chemically or by other means, and combining all fractions containing single component.

Various adsorbents which are in common use are usually inert compounds like charcoal, alumina, CaCO₃, Ca(PO₄)₂, Cellulose, silica, glass kaolin (clay material). Silica gel and Kiesulguhr are most commonly used now a days. The individual adsorbent particles should be of uniform size and sufficiently hard for adsorption during the packing of the column.

Adsorption chromatography is widely used in the separation of aminoacids, lipids, steroids, sugars, and similar other compounds of relatively low molecular weight. A molecule that is adsorbed more strongly will migrate very slowly in comparison to another molecule which is poorly adsorbed.

Type # 2. Thin Layer Chromatography:

(i) Principle:

The separation of compounds on a thin layer is similar in many ways to paper chromatography but has the added advantage that a variety of supporting media can be used so that separation can be done by adsorption, ion exchange, partition chromatography or gel Alteration depending on the nature of the medium employed.

The method is very quick and separations can be completed in a hour. Compounds can be detected at a lower concentration than on paper as the spots are very compact. Furthermore, separated compounds can be detected by corrosive sprays and elevated temperature with some thin materials, which of course is not possible with paper.

(ii) Preparation of Thin Layer:

The Rf value is affected by the thickness of the layer below 200 µm and a depth of 250 µm is suitable for most separations. There are several good spreaders on the market which, when carefully used, can produce even layer of required thickness.

The slurry (5ml) of alumina (made for TLC) following the directions given as per manufacturer. Take the glass slides and keep them flat, pipette out 1-2 ml of the slurry into them. By tilting the slides, spread the slurry evenly on the surface.

Lining the edges with vaseline will be of help. Spot a mixture of methylene blue and cresol red, and dip the slide in a beaker jar containing Na₂CO₃ solution saturated with butanol. The slide must be handled with care. There are now a number of prepared thin layer plates that are commercially available and these may be more convenient to use than drying to prepare plates in the lab.

(iii) Solvents:

Electrostatic attraction play a big role in the adsorption phenomena, and therefore, the polar nature of the solvent used influences adsorption considerably. Generally, adsorption is maximal in nonpolar solvents and decreases as the polarity is increased.

(iv) Development:

It is essential to make sure that the atmosphere of the separation chamber is fully saturated; otherwise Rf values will vary widely from tank to tank. This can be ensured by using as small tank as possible. Development of the plate is usually by the ascending technique is very rapid. The spots are evaluated in the same way as in paper chromatography.

(v) Precautions:

(a) The coated glass plates should be clean and dried before making a thin layer over it.

(b) Plates should be activated before use in drying over at 105°C.

(c) Samples should be spotted carefully.

Type # 3. Gas Chromatography (GC):

The technique of gas chromatography was used for the first time by A.T. James and P. Martin (1952) to separate long chain fatty acids. The phenomenon of differential adsorption can be used to separate gases and vaporizable substances as well.

This technique is called Gas Liquid chromatography. In this an inert solid base is coated with a liquid like paraffin oil or silicone oils, and packed in long tubes. This column is kept in an oven and generally kept around 150-200°C.

An inert gas like N₂ is passed through as carrier. The mixtures of gases to be separated are now injected into one end of the column and as they are carried through by the carrier gas, they get absorbed, released and reabsorbed.

The gas with least absorptivity emerges first out of the column. There are several methods of detection of the emerging material; primary among them are flame ionization detector, electrical resistance detector, and electron capture device.

The principle of the gas chromatography is also the same as any other type of chromatography. In this case, the substance to be analysed is partitioned between mobile phase and a stationary phase. The mobile phase in GC is a gas instead of a liquid.

The stationary phase consists of a liquid material coated on an inert solid support. The equipment consists of supply of carrier gas, an injector, temperature regulated oven, column, detector and recorder. Most commonly used carrier gases are helium, argon, nitrogen or hydrogen. The material to be analysed can be injected in the form of solid, liquid or gases.

The injector helps the column temperature in order to vaporise the sample and to prevent its condensation in an injector system. The column in a GC made up of glass or metal measuring about 1.8-3.6 m long with 3-6 mm diameter.

The column has an inert solid support coated with a liquid phase. Column is heated to the desired temperature by an oven around the column. The detectors in the gas chromatography are situated at a short distance from the column. Recorder is a multi voltmeter which converts the signal of the detector on graph.