Biology Doubts

About Chromatography

Discovered by Michael Tswett (1906). This technique is used to separate the molecules of different substances present together. Mixture of molecules is run over an adsorption medium. Chromatography may be following types.

Types of Chromatography

Adsorption or Column chromatography

The stationary phase consist of a column of charcoal, silica, alumina, calcium carbonate or magnesium oxide. The solution is made to percolate through this column when different chemicals get adsorb at various levels. The technique is useful for separation of tissue lipids.

Thin layer chromatography

The stationary phase consists of a thin plate of cellulose powder or alumina. As a few drops of mixture are poured over it, the different chemicals spread to different distances. The method is useful in separation of amino acids, nucleotides and other low molecular weight products.

Paper chromatography

A paste of mixture is applied near one end of a chromatographic paper (or Whatman 1). The lower end below the paste is dipped in a solvent. As the solvent rises in chromatographic paper, the different chemicals of the mixture spread to different distances. The paper can be rotated to obtain two dimensional chromatogram.  

Ion exchange chromatography 

Beads of cellulose and other materials having negative and positive charges are placed in a column. The mixture (mobile phase) is poured over the column. As the mixture passes through the column, its constituents separate according to their charges. The technique is used in purification of insulin, plasma fractionation and separation of proteins.

Gel fractionation chromatography (Molecular sieve chromatography)

Dextran gel sephadex is available  with various pore size. A mixture is poured over a column of sephadex. The various chemicals pass through the pores and come out of the column with heavier larger molecules do so first followed by progressively smaller sized molecules provided  the pores are larger than the size of largest molecules. The technique is used in determining the molecular weight of proteins by calibrating the column with proteins of different molecular weights.

Affinity chromatography

Satationary phase consists of column of ligands (molecules that bind to other specific molecules at particular sites). Mixture is allowed to pass through the column. Chemical linkages are established between ligands and their specific chemicals. Others pass out of the column. The technique is used in separation of enzymes, immunoglobulins, mRNA, etc.

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