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CHROMATOGRAPHY (DEMONSTRATION) Mrs. Chaitali Maitra Assistant Professor Dept.of Biochemistry PCMS&RC CHROMATOGRAPHY Chromatography basically involves the separation of mixtures due to differences in the distribution coefficient (equilibrium distribution) of sample components between 2 different phases which are immiscible. One of these phases is a mobile phase and the other is a stationary phase. porous medium through which the mobile phase migrates is called the support. Distribution Coefficient (Equilibrium Distribution ) Definition: Concentration of component in stationary phase Concentration of component in mobile phase Different affinity of these 2 components to stationary phase causes the separation. DUE DIFFERNCE IN DISTRIBUTION COEFFICIENT OF VARIOUS COMPONENT OF MIXTURE, THERE MOBILITY IN TWO PHASES DIFFER HENCE, SEPARATION MEASURE OF THIS MOBILITY IS CALLED RF VALUE I.E. RELATIVE FRACTION VALUE Distance traveled by substance Rf= Distance traveled by solvent STATIONARY PHASE: IN SOME CASES SOLID SUPPORT IT SELF FORM THE STATIONARY PHASE , WHILE IN OTHERS , IT ABSORBS LIQUID PHASE WHICH IN TURN ACTS AS STATIONARY PHASE. IT IS SOLID OR LIQUID. MOBILE PHASE: PHASE WHICH MOVE OVER OR THROUGH STATIONARY PHASE AND CARRIES SAMPLE ALONG WITH IT, THUS RESULTING IN THE SEPARATION OF ITS COMPONENT. IT IS EITHER LIQUID OR GAS. Kinds of Chromatography 1. Liquid Column Chromatography gel filteration ,ion exchange, affinity, adsorption,reverse phase,metal binding.(column) 2. Gas Liquid Chromatography(column) 3. Thin-layer Chromatography & paper(planar) LIQUID COLUMN CHROMATOGRAPHY A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid. With the proper solvents, packing conditions, some components in the sample will travel the column more slowly than others resulting in the desired separation. ION EXCHANGE • protein interact with stationary phase by charge-charge interaction •Positively charged proteins adhere to negatively charged functional groups(carboxylates,sulfates:cation exchanger) •Tertiary or quaternary amines: anion exchanger •Sequential elution, change of pH •Diethyl aminoethyl(DEAE)cellulose(anion exchanger) •Carboxymethyl(CM)cellulose(cation exchanger) SIZE EXCLUTION/GEL FILTERATION/GEL PERMEATION POROUS BEADS AS STATIONARY PHASE STROKE RADIUS;FUNCTION OF MOLECULAR MASS AND SHAPE GREATER THE STROKE RADIUS FASTER WLII BE THE ELUTION GEL IS MADE OF DEXTRAN AGAROSE OR POLYACRYLAMIDE AFFINITY SENSITIVITY OF MOST PROTEINS TOWARDS LIGANDS USE OF RESINS TO ATTACH LIGANDS ELUTION BY COMPETITION WITH SOLUBLE LIGAND OR BY DISRUPTION OF INTERATION. Purification of recombinant protein METAL BINDING Poly his-tag of c-terminal,binding with divalent metal ion nickel, cobalt eluted with imidazole Purification of recombinant protein linked with glutathione s-transferase by glutathione matrix. REVERSE PHASE & HYDROPHOBIC INTERACTION separation of hydrophobic proteins stationary phase is non polar liquid immobilized on inert solid polar mobile phase polarity of mobile phase reduced to dislodge proteins. hydrophobic interaction chromatography phenyl sepharose and octyl sepharose is used with weak inetraction to prevent denaturation ADSORPTION molecules are absorbed by stationary phase, insoluble substance like alumina,silicon,powdered sucrose is used elution by pure solvents (chloroform, hexane ,ethyl ether) HPLC Based on other chromatographic technique employment of incompressible matrix of silica or alumina micro beads thin layer of stationary phase mobile phase is forced through the column at a pressure of upto 5000psi high resolution,reduced analysis time Gas liquid chromatography ~separation of volatile mixture ~stationary phase is non-volatile liquid, coated on a inert solid ~mobile phase is a inert gas. argon) Planar chromatography 1. Thin layer chromatography solid support: glass stationary phase : silica gel , alumina solvent system : mixture of organic solvents(chloroform,methanol,acetic acid) ~separation of phospholipids and pigments ~separation based on differential adsorption PAPER CHROMATOGRAPHY