Chromatography

CHROMATOGRAPHY

 

Introduction

Chromatography is the techniques used for the separation and identification of biomolecules. Most effective technique for isolating and purifying all types of biomolecules. It is widely used as an analytical tool to measure quantitative properties.

Principle

Chromatography are based on very simple principle. The sample to be examined is called the solute, is allowed to interact with two physically distinct entities – a mobile phase and a stationary phase. The mobile phase, which may be a gas or liquid, moves the sample through a region containing the solid or liquid stationary phase called the sorbent. The stationary phase varies from one chromatographic method to another. Considered as having the ability to “bind” some types of solutes. The sample, which may contain one or many molecular components comes into contact with the stationary phase. The components distribute themselves between the mobile and stationary phase. If some of the sample components are preferentially bounds by the stationary phase, they spend more time in the stationary phase and hence, are retarded in their movement through the chromatography system. Molecules that show weak affinity for the stationary phase spend more time with the mobile phase and are more rapidly removed or eluted from the system. The main interaction that occur between solute molecule and stationary phase bring about a separation of molecules because of different affinities for the stationary phase. The general process of moving a solute mixture through a chromatographic system is called development.

The mobile phase can be collected as a function of time at the end of the chromatographic system. The mobile phase, now called as effluent, contains the solute molecules. Process has been effective, fractions or “cuts” that are collected at different time will contain the different components of the original sample. Molecules are separated because they differ in the extent to which they are distributed between the mobile phase and the stationary phase. Preparative or analytical, or both. A preparative procedure is one that can be applied to the purification of a relatively large amount of biological material. The purpose of such an experiment would be to obtain purified material for further characterization and study. Analytical procedures are used more often to determine the purity of the biological samples; however, they may be used to evaluate any physical, chemical, or biological characteristic of a biological system.

Partition vs adsorption chromatography

Chromatographic methods are divided into two types according to how solute bind to or interact with the stationary phase. Partition chromatography is the determination of a solute between two liquid phase. This may involve direct extraction using two liquids, or it may use a liquid immobilized on the solid support as in the case of paper, thin-layer, gas-liquid chromatography. For partition chromatography, the stationary phase consists of inert solid particles coated with liquid adsorbent. The distribution of solutes between the two phases is based primarily on solubility differences. The distribution may be quantified by using the partition coefficient, k_D

 

            Concentration of solute in stationary phase

K_D =------------------------------------------------------------

           Concentration of solute in mobile phase

 

Adsorption chromatography refers to the use of a stationary phase or support, such as an ion-exchange resin, that has a finite number of relatively specific binding sites for solute molecules. Paper, thin-layer, and gas chromatography are separated based primarily on nonspecific solubility factors. Adsorption chromatography relies on relatively specific interactions between the solute molecules and binding sites on the surface of the stationary phase. The attractive force between solute and support may be ionic, hydrogen bonding, or hydrophobic interactions. Binding of solute is, of course, reversible.

Partition processes, are they may be applied to different separation of small molecules, especially those in homologous series. Used for the separation and identification of amino acid, carbohydrate, and fatty acids. Adsorption technique, represented by ion-exchange chromatography, are most effective when applied to the separation of macromolecules including proteins and nucleic acids.

PLANAR CHROMATOGRAPHY (paper and thin-layer)

Example of partition chromatography are paper chromatography, the cellulose support is extensively hydrated, so distribution of the solutes occurs between the immobilized water (stationary phase) and the mobile developing solvent. Initial stationary liquid phase in thin-layer chromatography (TLC) is the solvent used to prepare the thin-layer of adsorbent. As developing solvent molecules move through the stationary phase, polar solvent molecules may bind to the immobilized support and becomes the stationary phase.

Preparation of the stationary support

The support medium may be a sheet of cellulose or a glass or plastic plate covered with a thin coating of silica gel, alumina, or cellulose. Large sheet of cellulose chromatography paper are available in different porosities. May be cut to the appropriate size and used without further treatment. It should never be handle with bare finger. Thin-layer plates can be easily prepared, ready-made plates are available in a variety of size, materials, and thicknesses of stationary support. They are relatively inexpensive and have a more uniform support thickness than hand-made plates.

The samples to be analyzed is usually dissolved in a volatile solvent. Very small drop of solution is spotted onto the plate with a disposable microcapillary pipet and allowed to dry; then the spotting process is repeated by superimposing more drops on the original spot. The exact amount of sample applied is critical. There must be enough sample so the developed spots can be detected, but over loading will lead to “tailing” and lack of resolution. Finding the proper sample size is a matter of trial and error. It is usually recommended that two or three spots of different concentrations be applied for each sample tested. Spots should be applied along a very faint line drawn with a pencil and ruler. TLC plates should not be heavily scratched or marked. Identifying marks may be made on the top of the chromatogram.

Solvent development of the support

If a new solvent system must be developed, a primary analysis must be done on the sample with a series of solvents. Solvents can be rapidly screened by developing several small chromatograms (2 x 6 cm) in small sealed bottles containing the solvents. Actually analysis, the sample should be run on a larger plate with appropriate standards in a development chamber. The chamber must be airtight and saturated with solvent vapors. Filter paper on two sides of the chamber, enhances vaporization of the solvent.

Paper chromatograms may be developed in either of two types of arrangements-ascending or descending solvent flow. Descending solvent flow leads to faster development because of assistance by gravity, and it can offer better resolution for compounds with small R_f values. Two-dimensional chromatography is used for especially difficult separations. The chromatogram is developed in one direction by a solvent system, air dried, turned 90⁰, and developed in a second solvent system.