CHROMATOGRAPHY

(DEMONSTRATION)

Mrs. Chaitali MaitraAssistant ProfessorDept.of BiochemistryPCMS&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.

Definition:

 

Different affinity of these 2 components to stationary phase causes the separation.

Concentration of component in stationary phase

Concentration of component in mobile phase

Distribution Coefficient (Equilibrium Distribution )

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

Distance traveled by solventRf=

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)

HPLCBased 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