Bio and chemistry

  High performance liquid chromatography (HPLC)    In general, the chromatographic techniques are time taking and slow. Therefore, the separation of interest can be improved by using high pressure in the range of 5,000-10,000 psi(pounds per square inch), so this technique is commonly known as high pressure liquid chromatography.  This tecnique requires the use of non-compressible resin materials and strong metal columns. At last the eluants of the column are detected by using methods such as fluorescence and UV absorption. High liquid chromatography is commonly used for the detection and estimation of vitamins (e.g. vitamin A, calcitriol, etc.), drugs (e.g. phenytoin, phenobarbitones, LSD, AZT, etc.) hormones (e.g. epinephrine, norepinephrine, ACTH, etc.), and metabolites (e.g. metanephrines) in clinical laboratories

  Gas-liquid chromatography (GLC)   This is the method of choice for the separation of volatile substances or the volatile derivatives of certain nonvolatile substances. In GLC, the stationary phase is an inert solid material (diatomaceous earth or powdered firebrick), impregnated with a non-volatile liquid (silicon or polyethylene glycol). Gas chromatography is a method of choice used to separate a mixture of compounds that are volatile or can be made volatile. The components in the sample are purified on the basis of partition between a gaseous mobile phase and a liquid stationary phase. Commonly used gas is inert (nitrogen, helium or argon) and simply carries the molecules through the column, it is called a carrier gas. The column packed of a nonvolatile liquid coated on an inert solid support (Diatomaceous earth or powdered firebrick). The mixture of substances is injected into the column along with the mobile phase,which is an inert gas. The partition of the volatile mixt...

  Gel filtration chromatography : In this technique of chromatography, separation of molecules is accomplished, on the base of their size, shape and molecular weight. Gel filtration chromatography is also known as molecular sieve or molecular exclusion chromatography. In this type of chromatography a packed column consists of a sponge like gel beads (usually cross-linked polysaccharides) containing pores.  The pores on the surface of gel allows small molecules to penetrate inside, whereas larger molecules are left outside and eluted with a buffer, hence the name exclusion chromatography. The gels are commercially available in different types (e.g. Sephadex G-25, G-50, G-250, etc.) depending upon the pore size and hence, suitable for different set of molecules depending on their molecular weight.

  Ion-exchange chromatography : Ion exchange chromatography is the process In which separation of molecules is carried out by on the basis of their electrical charges. For this purpose Ion-exchange resins, cation exchangers and anion exchangers are employed. In this method of separation an anion exchanger (R+A) donates its anion (A) with another anion (B) in solution R+A+ B ⇌R+B+ A Likewise, a cation exchanger (H+R) donates its cation (H+) with another cation (C+) in solution. H+R + C⇌ C+R + H Therefore, ions in solution are reversibly replaced by ion exchange resins in ion exchange chromatography. This depends on their binding abilities of ions bearing positive or negative charges are highly pH dependent, since the net charge on the surface of molecules varies with pH. This principle is helpful in the ion-exchange chromatography for the separation of molecules. ion-exchange chromatography Conveniently mixture of proteins or amino acids can be separated by ion exchange chromatogra...

  Adsorption column chromatography :  In this technique adsorbents such as silica gel, charcoal powder, alumina and calcium hydroxyapatite are packed into a column in a glass tube (it is serves as the stationary phase). The sample containing a mixture of components to be separated in a solvent are applied on this column. The components individually get adsorbed on to the adsorbent and then elution is carried out by a buffer system (mobile phase). The Compounds come one by one out of the column at different speed, which may be separately collected and analyzed. For example amino acids can be determined by ninhydrin colorimetric method.

  Thin layer chromatography (TLC) The working principle of thin layer chromatography is similar to paper chromatography. In TLC in place of a paper, an inert substance, such as cellulose, is Placed as supporting material.  Different grades of cellulose are spread on the glass or plastic plates which a re then activated at high temperatures. Comparatively the chromatographic separation carried out  in TLC is rapid. In TLC adsorption, such alumina, kieselguhr, and activated silica gel a re used.

Types of chromatography This technique is mostly used for the separation of amino acids, sugars, sugars derivatives and also for the separation of chemicals by this type of chromatography.  Stationary phase in paper chromatography is Whatman No. 1 or Whatman No. 3 filter papers while Mobile phase is the mixture of water, organic solvents and of various additives. In paper chromatography mixture of compounds to be separate is allowed to rise up and apply on the upward position (called ascending chromatography) at end of filter paper  (usually 2 cm above the paper) by a capillary action or move down by a capillary action on the paper (called descending chromatography). Solution of mixture to be separated applied on filter paper and migration of molecules present in solution occur and after some time paper is dried and dipped in solvent to visualize spots on paper with specific coloring reagents.  Since the speed of migrating molecules is depends on their relative solubility...

The word ‘chromatography’ is derived from Greek word ‘Chromo’ meaning colour and ‘graphy’ meaning to measure; initially it was used to separate coloured compounds from the mixture.   In another words It is an analytical technique used for the separation of closely related compounds from a mixture. These compounds are proteins, peptides, lipids ,amino acids, carbohydrates, vitamins and drugs. Chromatography was discovered by Russian Botanist Tswest in 1903. He used it for separation of plant pigments for the first time. The early methods used for the separation and purification of compounds from mixtures were labourious, and s  s low.  With passage of time and advancement in the separation techniques over years, the chromatographic technique has become to be known as used for the separation of different compounds from mixture and for their identification. Principle of Chromatography Chromatography is usually  consists of two phases first a mobile phase and secondary i...