Chromatography involves separating mixtures into their components based on how they partition between two phases, a stationary phase and a mobile phase. The different affinity of components for the stationary phase causes separation over time as they travel through at different rates. Key types of chromatography include liquid column chromatography using columns packed with particles, gas-liquid chromatography using an inert gas as the mobile phase, and planar chromatography techniques like thin-layer chromatography using coated plates.

2. CHROMATOGRAPHY
(DEMONSTRATION)

3. 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.

4. 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 )

5. 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 solvent
Rf=

6. 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.

7. 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)

8. 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.

9. 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)

11. 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

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

15. 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.

16. 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

17. 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

18. 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)

19. 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

20. PAPER CHROMATOGRAPHY