A Separation technique.……. Chromatography Chromatography is a technique of separation of different dissolved constituents of a mixture by absorbing them over an appropriate adsorbent material .It is a versatile method of separating different kinds of chemical mixtures .

TYPES OF CHROMATOGRAPHY Paper chromatography Thin Layer Chromatography(TLC) Column Chromatography Adsorption Chromatography Gel Filtration Chromatography Ion Exchange Chromatography Affinity Chromatography

Paper Chromatography The technique in which the filter paper is used as an adsorbent material for the separation of various constituents of a mixture is called paper chromatography. In Paper Chromatography, the sample to be analyzed is applied in the form of a spot near one of the edges of a “Whatman Filter Paper”.

Depending upon the direction of flow of mobile phase , the two commonly used systems are : Ascending & descending paper chromatography In the ascending method, the solvent is kept at the base of the chamber & the edges of the paper where the sample has been applied is immersed in the solvent taking care that the sample spots do not get dipped in the solvent but remain just above the surface of the solvent. The solvent moves up or ascends the paper by capillary action & the separation of different components ocuur on the basis of differences in the partition coefficients. In the descending method, the paper is hung in such a way that the side where the sample has been spotted sips in a trough which is fitted at the top of the chamber & contains the mobile phase & this solvent travels down the paper under the force of gravity.

types of paper chromatography

Identification OF COMPOUNDS The separated compounds are identified on the basis of Rf values which denotes relative to front. The Rf value is calculated as follows: Rf = dist. Travelled by the component from base line dist. Travelled by the solvent from the base line

APPLICATIONS OF PAPER CHROMATOGRAPHY It is used in separation of amino acids , sugar from urine , drugs from the samples of blood and colors from dyes . It is used for the detection of adulterants and contaminants in foods and drinks . It is used for the detection of drugs and dopes in animals & humans . It is used to control the purity of pharmaceuticals .

Thin layer chromatography This technique is similar to paper chromatography and is more convenient & less time consuming. Instead of paper, the supporting material is a glass plate, a plastic sheet or a piece of foil. The layer may be 250µm for analytical separations & 2-5 mm for preparative separations. A agent such as Calcium sulphate or a Gypsum are incorporated into the chromatographic media to facilitate a firm adhesion of adsorbent to plate. Most widely used adsorbent in TLC is Silica Gel which contains 13% CaSO4 .

Thin layer chromatography

APPLICATIONS OF TLC It is used as pilot technique to quickly determine the complexity of a mixture . Thin layer techniques are used to identify drugs, contaminants and adulterants . It has also been used to resolve plant extracts and many other biochemical preparations .

Column chromatography Paper and thin layer chromatography described in proceeding sections use a flat bed or surface for the separation of molecules. In column chromatography, separation is achieved by the passage of sample through vertical fixed tubular glass or polypropylene column which is packed with an chromatography media.

Column chromatography

APPLICATIONS OF COLUMN CHROMATOGRAPHY It is used to separate or purify natural compound mixtures like alkanoids , glycosides . It is used for the removal of impurities . It is used for the isolation of active constituents It is used for the estimation of drugs in formulations .

Adsorption chromatography Adsorption is a phenomenon in which compounds are held on to the surface of a solid adsorbent, having specific adsorption sites, though weak non-ionic interactions such vander waal forces and H-bonding. For good resolution, selection of right type of the adsorbents includes charcoal, silica, alumina etc.

APPLICATIONS OF ADSORPTION CHROMATOGRAPHY It is widely used for biochemical separations that includes amino acids , mono & disaccharides , neutral lipids , phospholipids , cholesterol easters , carotenoids , etc It is used for the clarification of sugar as the sugar is decolorized by treating sugar solution with charcoal powder . The latter adsorbs the undesirable colors present . It is also used in paint industry .

Gel filtration or exclusion column chromatography The chromatographic media used in this technique are porous, polymeric organic compounds with molecular sieving properties. These are cross-linked polymers which swell in water forming a gel of 3-D networks.

Gel filtration

Applications of gel filtration Removal of salts and small molecules from macromolecules can be easily performed by using gel filtration . It is used in the determination of molecular weight of macromolecules . It is used for the purpose of separation of biological molecules leading to their ultimate purification .

Ion exchange chromatography Ion exchange chromatography is a type of adsorption chromatography in which retention of a solute occurs due to its reversible electrostatic interaction with the oppositely charged groups on an ion exchanger. Hence, this technique is useful for separation of compounds which bear a net electric charge such as proteins, amino acids, nucleic acids etc.

Ion exchange chromatography

APPLICATIONS OF ION EXCHANGE CHROMATOGRAPHY The most stimulating use of ion exchange chromatography is in amino acid analysis . It is used to determine the base composition of nucleic acids . It is the fast and effective method of water purification . It is also used for the separation of many vitamins , other biological amines and organic acids .

Affinity chromatography Affinity chromatography is a type of adsorption chromatography in which the substance to be is specificially and reversibly bound to a complementary binding substance immobilized on an insoluble chromatographic bed material (matrix).

Affinity chromatography

Applications of affinity chromatography It is used to purify a large variety of macromolecules such as enzymes , immunoglobins , nucleic acids etc . It is also used to separate cells that include fat cells , T and B lymphocytes , spleen cells , lymph node cells , oocytes and chick embryo neural cells .

Submitted to: Dr.shiffali singla SUBMITTED BY: ISHA DHIMAN BSc.BT (hons.) – I

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