11th Class Biology Tools And Techniques Chromatography

Chromatography

Category : 11th Class

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.

Adsorption or Column chromatography : The stationary phase consists 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 absorbed 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.

Types : (a) Ascending (b) Descending (c) 2-D chromatography.

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 / Gel filtration chromatography (Molecular sieve chromatography) : The stationary phase consists of gel forming hydrophilic beads which contain pores, e.g., sephadex (cross-linked dextran). As the mixture is poured over the gel, larger molecules pass out unimpeded while small molecules are trapped in the pores. The technique is used in separation of proteins. It is also employed in determining their molecular weight by calibrating the column with proteins of known molecular weight.

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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