CHROMATOGRAPHY

Nur Kesuma Bt Ishak 2017403804

Siti Zulaikha Bt Zulkefli 201768044
Nur Adrianna Bt Adam 2017441478
Nur Aqilah Ainaa Bt Sahrol 2017644738
Umul Jasrina Bt Sied Abd Jalil 2017635534
GEL FILTRATION CHROMATOGRAPHY
 introduction
Otherwise known as: molecular sieving, gel permeation, size exclusion
chromatography (SEC)

Used to separate mixtures of macromolecules, particularly enzymes,

antibodies and other globular proteins

Based on size to achieve rapid separation of molecules

Mobile phase – liquid

Stationary phase – porous beads or material with a well – defined range

of pore size
 gels
• Majority available in 2 particle size grades:
coarse ( 300 – 100 μm) and fine ( 20 – 80 μm)
• Available in bead form than powder form to
ease column packing
• Consists of an open, cross – linked 3-D
molecular network of pores and channels into
which molecules of less than maximum pore
size may penetrate
 choice of buffer
• Protein purification: desirable to have proteins of interest to be well
separated from unwanted proteins
• Buffer must be compatible with target molecules for gel filtration
• Most common buffer used are sodium phosphate buffer and Tris-
HCl (control pH)
• Buffer composition will not directly influence the resolution unless
it effects the shape or activity of the molecule
• Extreme pH and ionic strength, denaturing agents and detergents
can cause conformational changes, dissociation or association of
protein complexes
• To reduce dissociation, avoid extreme pH changes. Avoid also
chaotropic agents and detergents (can induce conformational
change, inactive biologically active or dissociate proteins into
subunits)
• Choice of column:
• High resolution fractionation: 30-60cm
• Group separation: 10cm

• Choice of gel matrix/media selection:

• Sephadex (a cross-linked dextrans) – ideal for rapid
group separation
• Superdex – High resolution. Short run times, high
recovery
• Polyacrylamide beads
• Agarose
The separation depends on depends on the shape
and MW of the molecules.

Pores within beads are of such sizes that are not

accessible to large molecules, but smaller
molecules can penetrate all pores.

As solvent moves through the column, 3 possible

things can happen to solutes in sample:

• - can run with solvent front

• - be washed out quickly

• - totally adsorbed on to gel matrix

• Molecules are eluted in
order of their decreasing
size.

• Very large molecules that

are too large to enter the
pores move through the
chromatographic bed are
the fastest.

• Smaller molecules moves

slowest as they moves in
and out of the beads and
into the pores as they
travel down the column.

• As liquid exits from column,

results will be taken by
monitoring UV absorbance
at 280 nm.
 AFFINITY
CHROMATOGRAPHY
 A separation technique based on
a specific binding interaction
between an immobilized ligand
INTRODUCTION and its binding partner

Lead to protein properties

changes such as it can be
separated from other proteins

Application of biospecific
interaction
Only protein that can bind to
ligand will retain on column

Other mixtures will pass

through the column

The protein bind to ligand can

be released by eluting it with
soluble version of ligand.
 AFFINITY CHROMATOGRAPHY

• An immunoadsorbent that consists of solid matrix in which the

antigen binds by covalent bond
STEP 1

• The sample which is the serum is passed through the

immunoadsorbent
• Antibodies in the serum sample that are specific for the antigen
will bind and retain at the antigen on the solid matrix
STEP 2 • Antibodies of other specificities and serum proteins just pass
through the immunoadsorbent without any blockage
 AFFINITY CHROMATOGRAPHY
• Elution process occurs when the a reagent is passed into the
column to release the antibodies from the immunoadsorbent
by using buffers containing high concentration of salts or low

STEP 3 pH, denaturing agent like 8M urea and soluble form of the
antigen

• The eluted antibodies is then dialyzed against buffered saline

is a process of dialysis

STEP 4 • To remove the reagent used for elution