1. CHROMATOGRAPHY (DEMONSTRATION) Mrs. Chaitali Maitra
Assistant Professor
Dept.of Biochemistry
PCMS&RC

2. 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.

3. Distribution Coefficient (Equilibrium Distribution )
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

4. 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

5. 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.

6. Kinds of Chromatography
Liquid Column Chromatography
gel filteration ,ion exchange, affinity, adsorption,reverse phase,metal binding.(column)
2. Gas Liquid Chromatography(column)
Thin-layer Chromatography & paper(planar)

7. 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.

8. 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)

10. 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

12. AFFINITY
SENSITIVITY OF MOST PROTEINS TOWARDS LIGANDS
USE OF RESINS TO ATTACH LIGANDS
ELUTION BY COMPETITION WITH SOLUBLE LIGAND OR BY DISRUPTION OF INTERATION.

14. 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.

15. 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

16. 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

17. 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)

18. Planar chromatography
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

19. PAPER CHROMATOGRAPHY