INSTRUMENTAL METHODS OF ANALYSIS, B.PHARM 7TH SEM. AND FOR BSC,MSC CHEMISTRY.

1. Gel-filtration chromatography
Presented by : Mr. G.P Kashyap ( Asst. Prof ) M.Pharm
(Q.A)
Siddhi Vinayaka Inst. of Technology & Sciences
(Bilaspur, C.G)

2. Gel-filtration chromatography
• Gel-filtration chromatography is a form of partition chromatography
used to separate molecules of different molecular sizes.
• This technique has also frequently been referred to by various other
names, including gel-permeation, gel-exclusion, size- exclusion, and
molecular- sieve chromatography.
• The basic principle of gel filtration is quite straightforward. Molecules
are partitioned between a mobile phase and a stationary phase
(comprising a porous matrix of defined porosity) as a function of their
relative sizes.
• Biomolecules are purified using different techniques that separate
them according to the differences in their specific properties such as
size, hydrophobicity, biorecognition, charge, etc.

3. Working Principal
• In gel filtration, or as it is sometimes referred to as size
exclusion, chromatography the resin are porous (see figure to
the left).
• Some molecules (blue here) can enter the resin and as the lines
try to indicate it is not a straight path through; thus it takes
longer for small molecules to traverse the column than large
molecules which travel around the outside of the resin.
• This is highlighted in the figure to the right where big molecules
(blue) come off first and smaller molecules (red) later.

6. Ion Exchange
• Unlike gel filtration chromatography, here proteins directly interact
with the resin.
• So generally the column is equilibrated in a buffer solution to
establish a constant pH in the column, then the protein mixture is
loaded where all or some of the proteins interact with the resin
depending upon their own charge.
• Buffer is continued to be applied until all proteins not interacting with
the resin have been washed off.
• At that point usually a gradient of increasing salt concentration
(disrupts ionic and hydrogen binding) in the buffer is applied to
column allowing the most weakly interacting proteins to release first
followed by the more strongly and finally the most strongly
interacting.
• This can also be accomplished by changing the pH of the buffer
being applied to the column.

7. Cont….
• Anion Exchanger
• Anion exchanger means that it removes anions from protein mixture
so that means the resin must be decorated with positively charged
moieties. Before elution begins all positively and uncharged proteins
will fall through the column. When you start eluting, first you will
knock off the weakly negative proteins (e.g. -1 charge), followed by
those with a stronger negative charge (-2), and finally the most
negatively charged proteins (-3).
• Cation Exchange
• It is exactly oppose with a cation exchanger -- here cations are
removed from the protein solution so the resin must be negatively
charged. Again before elution begins all negatively and uncharged
proteins will fall through the column. When you start eluting, first you
will knock off the weakly positive proteins (e.g. +1 charge), followed
by those with a stronger positive charge (+2), and finally the most
positively charged proteins (+3).

9. Types of Gel Filtration Chromatography
Group Separations
• The components of a sample are separated into two major groups
according to the size range.
• A group separation can be used to remove high or low molecular
weight contaminants (such as phenol red from culture fluids) or to
desalt and exchange buffers.
• High-resolution fractionation of biomolecules
• The components of a sample are separated according to differences
in their molecular size.
• High-resolution fractionation can be used to isolate one or more
components, to separate monomers from aggregates, to determine
the molecular weight or to perform a molecular weight distribution
analysis.

11. Steps in Gel Filtration Chromatography
1.Spherical particles of gel filtration medium are packed into a column.
2.The sample is applied to the column.
3.Buffer (mobile phase) and sample move through the column.
4.Molecules diffuse in and out of the pores of the matrix (also described as the
partitioning of the sample between the mobile phase and the stationary phase).
5.Smaller molecules move further into the matrix and so stay longer on the
column.
6.As buffer passes continuously through the column, molecules that are larger
than the pores of the matrix are unable to diffuse into the pores and pass
through the column.
7.Smaller molecules diffuse into the pores and are delayed in their passage down
the column.
8.Separation occurs at different intervals which are followed by detection of
components.

12. Applications of Gel Filtration Chromatography
• Gel filtration plays a key role in the purification of enzymes,
polysaccharides, nucleic acids, proteins, and other biological
macromolecules.
• Gel filtration can also be used to facilitate the refolding of denatured
proteins by careful control of changing buffer conditions.
• It is used in protein fractionation experiments.
• Gel filtration technique is also used in molecular weight
determination.
• Separation of sugar, proteins, peptides, rubbers, and others on the
basis of their size.
• Can be used to determine the quaternary structure of purified
proteins.

13. Advantages of Gel Filtration Chromatography
• Gel filtration is a robust technique that is well suited to handling
biomolecules that are sensitive to changes in pH, the concentration of
metal ions or co-factors and harsh environmental conditions.
• A significant advantage of gel filtration is that conditions can be varied
to suit the type of sample or the requirements for further
purification, analysis or storage without altering the separation.
• Separations can be performed in the presence of essential ions or
cofactors, detergents, urea, guanidine hydrochloride, at high or low
ionic strength, at 37 °C or in the cold room according to the
requirements of the experiment.
• Unlike ion exchange or affinity chromatography, molecules do not
bind to the chromatography medium so buffer composition does not
directly affect resolution (the degree of separation between peaks).

14. •Short analysis time.
•Well defined separation.
•Narrow bands and good sensitivity.
•There is no sample loss.
•The small amount of mobile phase
required.
•The flow rate can be set.