Medical brain notes 25

  Chromatography One of the tasks of biochemists is to identify, separate and purify one or more biological components in a mixture of such compounds in a biological sample. One of the most important convenient methods for achieving such separation is the use of chromatographic techniques. The term chromatorgraphy was originally applied by Micheal Tswett, a Russian Botanist, in 1906 to a procedure where a mixture of different coloured pigments (chlorophylls and xanthophylls) was separated from each other. Principle :  The basis of all forms of chromatography is the partition or distribution   co-efficient which describes the way in which a compound distributes itself between two immiscible phases. For a compound distributing itself between equal volumes of two immiscible solvents A and B (Fig. 10.1), the value of distribution co-efficient is a constant at a given temperature and is given by the expression Basically all chromatographic systems consists of two phases. One i...

  Types of chromatography Column chromatography:  In this type, the stationary   phase is packed into a glass or metal columns (wide tubes or cylinders). The mixture to be separated is layered on the top of the column in the form of a solution at particular concentration. After equilibration the components are eluted out of the column one by one using specific mobile phases (Fig.10.2).The solvent used to elute the separated components is known as eluant. Thin layer chromatography:  In this type, the stationary   phase is thinly coated on to a glass, plastic or foil plates. The mixture to be separated is applied on the stationary phase at one end and kept vertical in the petridish containing the mobile phase. When the mobile phase reaches the other end of the plate the plate is removed from the petridish and the compounds separated are identified by using specific staining reagents  Paper chromatography:  In this type, the stationary  column chroma...

  Column chromatography:  In this type, the stationary   phase is packed into a glass or metal columns (wide tubes or cylinders). The mixture to be separated is layered on the top of the column in the form of a solution at particular concentration. After equilibration the components are eluted out of the column one by one using specific mobile phases (Fig.10.2).The solvent used to elute the separated components is known as eluant. Column chromatography : All the major types of chromatography are routinely   carried out using column type (Fig. 10.3). The different types of column chromatography are i. adsorption chromatography ii. partition chromatography iii. ion-exchange chromatography iv. exclusion chromatography v.    affinity chromatography i. Adsorption chromatography Principle: An adsorbent may be described as a solid which has the property of adsorbing molecules at its surface, particularly when it is porous and finely divided. Adsorption c...

  Thin Layer chromatography Principle:  Partition, adsorption, exclusion chromatography can be carried out in a thin layer   mode. In this technique the stationary phase is made in the form of a slurry and applied as a thin coating on the surface of a glass plate.After activating the plate, the sample to be separated is applied at one end of the plate . The plate is kept vertically in a chamber specially designed for this purpose( TLC chamber) and allowed the sample and the mobile phase to raise through the stationary phase by capillary action. The whole procedure consists of. a. Thin layer preparation :  A slurry of the stationary phase, generally applied to a glass,   plastic or foil plate as a uniform thin layer by means of a plate spreader starting from one end of the plate and moving progressively to the other. Calcium phosphate is incorporated into the slurry in order to facilitate the adhesion of the adsorbent to the plate. The plate is heated in an oven ...

  Paper chromatography Principle:  The cellulose fibres of chromatography paper act as the supporting matrix for the   stationary phase. The stationary phase may be water or a non-polar material such as liquid paraffin. The components get separated between the liquid stationary phase and the liquid mobile phase. The procedure consists of a. Paper development:  There are two techniques which may be employed for the   development of paper, ascending and descending methods. In both cases, the solvent is placed in the base of a sealed tank or glass jar to allow the chamber to become saturated with the solvent paper. The sample spots should be in a position just above the surface of the solvent so that as the solvent moves vertically up the paper by capillary action, separation of the sample is achieved. b. Component detection:  The separated components can be detected by (i) examining   the paper under ultraviolet light; (ii) spraying of papers with specif...

  Applications of chromatography ·             Thin layer chromatography is used for the separation of alkaloids, phospholipids and other lipids. ·             Gas liquid chromatography is applied for the separation of fatty acids in a lipid mixture. ·             Ion exchange chromatography can be used for the separation and identification of amino acids in a mixture of protein hydrolysate. This principle is used in auto-analyzer. ·             Exclusion chromatography can be applied for the determination of the molecular weight of the components separated. ·             Affinity chromatography is applied for the purification of a wide range of enzymes and other proteins like immunoglobulins.

  Electrophoresis 1. General principle:  Many important biological molecules such as amino acids,   peptides, proteins, nucleotides and nucleic acids posses ionisable groups and can therefore be made to exists in solution as electrically charged species either as cations (+) or anions (-). When a mixture of these components are subjected to electric field, they migrate differentially and thus can be separated. 2. Types of electrophoresis (i) low voltage thin sheet electrophoresis; (ii) high voltage electrophoresis, (iii) gel electrophoresis – native poly acrylamide gel electrophoresis and sodium dodecyl sulphate (SDS) poly acrylamide gel electrophoresis, (iv) isoelectric focussing and (v) isotachophoresis. Gel electrophoresis The most commonly used electrophoresis is gel electrophoresis. In this technique either agarose or poly acrylamide is used as supporting media. Electrophoresis units are available for running either vertical or horizontal gel systems. Vertical slab g...

  Centrifugation techniques Principle These techniques are based up on the behaviour of particles in an applied centrifugal field such as density, shape or size of the molecules being separated. The particles are normally suspended in a specific liquid medium held in tubes or bottles which are located in rotor. The rotor is positioned centrally on the drive shaft of the centrifuge. Particles which differ in density, shape and size can be separated since they sediment at different rates in the centrifugal field. Each particle sediments at a rate which is proportional to the applied centrifugal field. The rate of sedimentation can be expressed as rpm (revolutions per minute) or as g (gravitational force). The rate of sedimentation is dependent up on the applied centrifugal field (G) which is determined by the square of the angular velocity of the rotor (ω) and the radial distance (r) of the particle from the axis of rotation according to the equation G= ω2r. The sedimentation rate or...