CHROMATOGRAPHY Presented by Mr.Halavath Ramesh M.A,M.sc,B.ED,PGDCAQM,PGDCA,M.Phil,(P.HD)(UoH) University of Madras Dept. of Chemistry Loyola College,Chennai.

INTRODUCTION Chromatography is one of the most useful analytical technique by which closely related molecules in a mixture of biomolecules could be separated,isolated and purified. The separation processes is based on differential distribution of substances between two immiscible phase including a mobile phase and stationary phase,one of which moves relatively to the other. Based on the type of interaction between the substances and the stationary phase, chromatographic techniques can be classified into three major groups 1.Partition Chromatography 2.Permeation or molecular exclusion chromatography 3.Absortion chromatography

Based on the type of Stationary phase used, the partition and adsorption chromatography are further classified into Partition chromatography- Paper chromatography and Thin Layer chromatography Adsorption chromatography-Affinity chromatography(AC) Ion-exchange chromatography -Cation exchange chromatography - Anion exchange chromatography similarly,based on the type of mobile phase used, the chromatography could be classified into three types: 1. Liquid chromatography(LC) 2.Gas chromatography(GC) 3. Gas-Liquid chromatography(GLC) Paper and thin layer chromatography In paper chromatography,the paper (made of cellulose) acts as stationary phase and the solvent system acts as mobile phase. When the mixture of substances moves on a stationary phase(paper) along with the mobile phase (the solvent flowing along the paper), the separation is brought about by continuous partition between the mobile phase and water held in the paper, where paper together with water acts as an adsorbent which results in retardation of substances at certain levels of flow on the paper. Thus the paper chromatographic separation is achieved as a resultant of propelling (mobile) and retardation (stationary) forces.

Similarly,in thin layer chromatography silica gel coated plates are being used instead of paper. Here again the separation is achieved by the result partition between the mobile phase and an inert stationary phase (silica).This is being commonly used in several laboratories to separate hydrophobic non-polar soluble substances such as lipids. In both these techniques, one should consider certain properties of solvents as mobile phases which include 1.Solvent should be stable in air and when mixed with small quantities acid or alkaline vapor. 2.Components of the mobile phase should be relatively non-volatile or their volatilability should be similar to the closed apparatus. 3.The solvent should be removed rapidly and completely from the stationary phase after the chromatogram has been completed. 4.The solvent should remain homogenous throughout the range of temperature experienced in laboratory. 5.The solvent should not react with any of the substances to be separated or with stationary phase. Identification of compounds separated An important characteristics used in both paper and thin layer chromatography for identification of compounds of substances that are separated is identified after using certain specific staining techniques such as ninhydrin for amino acid,silver nitrate for reducing sugars and for lipids. After staining,Rf values of each spot could be determined.

This value (Rf) can be calculated by measuring the distances travelled by the substance(separate) and the solvent( mobile) from their point of origin. Permeation of Molecular exclusion or Gel filtration chromatography G el filtration chromatography is one of the most commonly employed tools for separating biomolecules on the basis of their molecular weight. Thence it is other wise called as molecular exclusion or molecular sieve chromatography. This system involves a stationary phase (usually a preswollen gel beads) and a mobiles phase(buffer system). Principle: In this technique, the mixture of biomolecules dissolved in a suitable buffer is allowed to flow by gravity down a column packed with beads of an inert highly hydrated polymeric material that has previously been washed and equilibrated with the buffer. During this process, biomolecules of different molecular size penetrate into the internal pores of the beads to different degrees and thus travel down the column at different rates. Very large biomolecules cannot enter the pores of the beads,hence excluded and remain in the exclusion volume of the column. On the otherhand,small biomolecules which can enter the gel pores, move more slowly through the column, sinces they spend a proportation of the their time in stationary phase. Biomolecules of intermediate size will be excluded from

the beads to a degree that depends on their size. The biomolecules are therefore eluted in a order of decreasing molecular size and thus a separation of molecules is achieved. The column materials that are commonly used include: 1.Sephadex G-type(G-10,G-25,G-50,G-75,G-100,& G-200) 2.Sepharose CL-series(2B,4B,6B). 3. Sephacryl(S-100,S-300). 4.Bio-gel 5.Silica gel Applications: The gel filtration chromatography is routinely used in several bio-medical and research laboratories 1.To fractionate proteins,carbohydrates,nucleic acids and enzymes. 2. to fractionate cell organelles,viruses etc., 3.To remove co-factors, inhibitors etc., from enzymes 4.To desalt the biomolecules before lyophilization or concentration or further purification. 5.To remove free and low moleculres weight labels such as 125I,FITC from the solution of labelled proteins.

Adsorption chromatography: Ion-exchange chromatography This techniques involves separation of biomolecules depending on their net charges. In gel filtration techniques, a mixture of biomolecules are separated based on molecular size, but the different biomolecules having identical or similar molecules weight or size cannot be effectively separated. Such possibilities are significant because many unrelated biomolecules may be identical in their molecules weight or size. To solve this kind of problems,other characteristics of biomolecules such as net charge iso-electric point or their specific binding ability are being utilized to separate the unrelated biomolecules having identical or similar molecular weight. APPLICATIONS: The ion-exchange chromatography in mostly used in certain bio-medical and research laboratories to 1. Separated many blood product such as albumin,IgG,plasma b-endorphins. 2. Separate products of recombinant DNA technology. 3.Separate T3 and T4. 4. Separate renal proteinuria in urine.

Affinity Chromatography Affinity chromatography is a type of adsorption chromatography in which the biomolecules or specific type of cells to be purified is specifically and reversibly adsorbed by a complementary binding substances (Ligand) which is immobilized on a insoluble support (matrix). It occupies a unique place in separation technology since it is the only techniques which enables purification of almost any biomolecules on the basis of its biological function or individual chemical structure.In addition purification of a biomolecules is often in the order of several thousand fold and recovery of active materials are generally very high and being a single step purification it is an immense time saver over the other multi-stage purification procedures. Applications: 1.To purify enzyme using substrate as ligands. 2. To purify hormones using cell menbrance receptors. 3.To purify specific antibodies using antigens. 4.To purify mRNA using complementary DNA. 5.To purify interferons, viral RNA,etc.