On several occasions, a researcher requires a specific group of macromolecules separated from a biological extract in order to understand the molecule or the process indepth. Chromatography is one of the techniques to separate biological molecules. There are many types of chromatography based on the physicochemical properties of the molecules.
These different types of chromatography include gel filtration - ion-exchange - adsorption - affinity chromatography and so on, based on the molecular size and shape, ionic nature, molecular topography and biological specificity of the molecule.
Essentially, any chromatography consists of two phases: one is stationary phase which may be a solid, liquid or a solid/liquid mixture and is immobilized while the other, mobile phase, is a fluid which flows through the stationary phase.
Chromatographic separations in practice may take any one of three modes-paper, thin layer or column chromatography.
In column chromatography, the stationary phase is packed in a cylindrical column made of plastic or glass.
The concept of gel filtration chromatography is the different molecules are separated based on the molecular size and shape where the stationary gel matrix serves as a sieve.
The Principle underlying the ion-exchange chromatography is the attraction between the biological compounds and the stationary phase, each with opposite electrical charges, thus attracting each other. The ionic nature of chemical compounds is hence exploited.
Affinity chromatography is based on the attraction of a partially compound specifically to combine with the molecule of our interest. For instance, the inhibitor of an enzyme serves as affinity compound to separate that particular enzyme. However, the inhibitor has to be initially combined with an inert matrix to serve as stationary phase.
Majority of the chromatography is routinely carried out Using the column mode. The apparatus and general techniques used for gel exclusion, ion-exchange, adsorption and affinity chromatography have much in common. Gas-liquid chromatography and high performance liquid chromatography each have their own special apparatus, materials and protocols. The column chromatography nowadays been made sophisticated, easier and faster by combining together pumps, detectors, recorders etc. to the columns.
As a model, column chromatographic separation of proteins based on their molecular size by gel filtration is described below:
The basis of any form of chromatography is the partition or distribution coefficient (Kd) which describes the way in which a compound distributes itself between two immisible phases, such as solid/liquid or gas/liquid.
Chromatography columns are considered to consist of a number of adjacent zones in each of which there is sufficient space for the solute to achieve complete equilibrium between the mobile and stationary phases. Each zone is called a theoretical plate and its length in the column is called the plate height. The more efficient the column is the greater the number of theoretical plates involved.
» Chromatographic column of suitable dimension made up of transparent plastic or glass: Generally, gel filtration is carried out in longer columns (up to 1 m) depending upon the type of gel filtration medium used and the size of the protein to be purified from the bulk
» Stationary phase: (ex) Sephadex G 100
There are different types and grades of gel filtration media available. It is to be chosen on the basis of the size of the protein under study
» Elution buffer
» Fraction collector
» Peristaltic pump
» Marker proteins (a set of highly pure proteins with known molecular weight (e.g.) cytochrome C, 12,400; carbonic anhydrase, bovine 1,50,000; b-amylase, potato 2,00,000; blue dextran 2,00,000 daltons.
A. Packing the column
1. Suspend the gel (for instance, Sephadex G 100) in a large volume of water or preferably in elution buffer until the gel is fully swollen. The swelling can be done by overnight suspension or by heating in a water bath for 2-4 hr (Follow the manufacturer's instructions for this purpose, carefully).
2. Plug the bottom of column tube with glasswool or sintered filter and stand upright the column.
B. Packing the column
1. Gel filtration: Theory & Practice — Pharmacia fine chemicals Hand-Book 97,1974.
2. A Biologists Guide to Principles and Techniques of Practical Biochemistry (eds. Wilson and Goulding) ELBS Publication, 1986.
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