CHROMATOGRAPHY

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CHROMATOGRAPHY (DEMONSTRATION)
Mrs. Chaitali Maitra Assistant Professor Dept.of
Biochemistry PCMSRC
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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.
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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
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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
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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.
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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)

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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.
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• ION EXCHANGE
• protein interact with stationary phase by
  charge-charge interaction
• Positively charged proteins adhere to negatively
  charged functional groups(carboxylates,sulfatesca
  tion exchanger)
• Tertiary or quaternary amines anion exchanger
• Sequential elution, change of pH
• Diethyl aminoethyl(DEAE)cellulose(anion
  exchanger)
• Carboxymethyl(CM)cellulose(cation exchanger)

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SIZE EXCLUTION/GEL FILTERATION/GEL
PERMEATION POROUS BEADS AS STATIONARY
PHASE STROKE RADIUSFUNCTION OF MOLECULAR MASS
AND SHAPE GREATER THE STROKE RADIUS FASTER WLII
BE THE ELUTION GEL IS MADE OF DEXTRAN AGAROSE OR
POLYACRYLAMIDE
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AFFINITY SENSITIVITY OF MOST PROTEINS TOWARDS
LIGANDS USE OF RESINS TO ATTACH
LIGANDS ELUTION BY COMPETITION WITH SOLUBLE
LIGAND OR BY DISRUPTION OF INTERATION.
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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.
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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
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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
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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)
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• 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

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PAPER CHROMATOGRAPHY
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