LAHORE COLLEGE FOR WOMEN
UNIVERSITY
ANALYTICAL CHEMISTRY
ASSIGNMENT
Submitted to: Dr. Mahwish
Submitted by: Azka Asim
Topic: Applications of Gel Filtration Chromatography
INTRODUCTION
Gel filtration chromatography is a technique which is used to separate dissolved molecules on
the basis of their size and shape by pumping them through specialized columns containing a
microscopic packing material (gel). It is also known as molecular sieve chromatography.
The stationary phase consists of a porous polymer matrix whose pores are completely filled
with solvent to be used as mobile phase.
The pore size is highly critical, since the basis of separation is that the molecules above a
certain size are excluded from the pores and the anterior of the pores is accessible, partly or
wholly, to smaller molecules.
The flow of mobile phase will cause the mobile phase will cause the larger molecules to pass
through the column unhindered, without penetrating the gel matrix, whereas, small molecules
will be retarded according to their penetration of the gel.
METHOD
1. Firstly, a column of spherical gel beads is prepared for the gel filtration chromatography.
2. The packed bed is equilibrated with a buffer.
3. Then, the test solution (mobile phase) is eluted through the column.
4. After that, the particles in the test sample will enter into or diffuse out of the porous gel
matrix (stationary phase).
5. The molecules with a small molecular size will enter the gel pores, i.e. small molecules will
cover a longer path or stay longer on the column.
6. Large molecules with large molecular sizes cannot get into the pores of gel beads or easily
pass through the column.
7. Thus, the separation of the particles occurs at different intervals by following isolation and
identification of the components separated.
8. Fractionation and desalting are the methods that facilitate the separation of components in
size exclusion chromatography.
APPLICATIONS
The following are the major applications of gel chromatography;
• Protein-Ligand Binding
• Molecular weight determination
• De-salting
• Purification of enzymes
PROTEIN-LIGAND BINDING
Ligand is a substance, usually a small molecule that is able to bind to a biomolecule. This binding
produces a complex and serve as a biological purpose; activation, inhibition or act as a
neurotransmitter. It binds to a site of the target protein. This binding of the ligand to protein
occurs by intermolecular forces such as ionic bonds, hydrogen bonds or van der waals force.
Because the binding is takes place by intermolecular forces and not with covalent bonding which
is a strong bond, ligand binding is reversible. Ligand binding changes the conformation of the
receptor protein, the three-dimensional shape of the protein.
A protein ligand binds to receptor sites on the surface of a protein. Ligands are involved in a
wide variety of processes, from protein folding to change structure and they provide a function in
immune reactions.
By using the gel filtration chromatography, we use albumin because different ligands can bind
to it easily. We use sephadex as gel in the experiment. Before using it, it is waited in water for 3-4
hours so that it can swell.
During the experiment after collecting 10 drops of the sample from chromatography we add
NaOH and 2 ml water to the cuvette. The reason we add NaOH is because we need to obtain a
coloured solution so that we can measure absorbance values with spectrophotometer. In the acidic
medium samples are yellow. The reason why we add 2 ml water is also to be able to obtain
absorbance values. Approximately, 3 ml sample in the cuvette is needed in order to have results.
DE-SALTING AND BUFFER EXCHANGE
In desalting and buffer exchange, the macromolecular components of a sample are recovered in
the buffer used to pre-equilibrate the gel-filtration matrix through which the sample is processed.
The method is commonly referred to as desalting when the goal is to remove buffer salts from a
sample in exchange for water (with water used to pre-equilibrate the gel-filtration resin). Buffer
exchange is the term used when one set of buffer salt in a sample is exchanged for another set.
Desalting and buffer exchange use gel filtration chromatography (or size-exclusion
chromatography) to separate soluble macromolecules from smaller molecules. When an aqueous
solution is used to transport the sample through the column, the technique is referred to as gel-
filtration chromatography. Desalting and buffer exchange are two of the most widely used gel
filtration chromatography applications, and both can be performed using the same materials.
Experiment is carried out when a sample solution is passed through a column of packed gel
filtration resin, small molecules in the sample (buffer salts, small molecules, etc.) enter the pores
of resin beads that they encounter and are forced to follow a circuitous path before later exiting
the beads. By contrast, large molecules flow around the resin beads, taking a relatively direct path
through the column. In effect, small molecules experience a much larger column volume than
large molecules. (Keep in mind that resin beads are extremely porous; most of their total volume
is water). Thus, the difference in the flow rates of small molecules and excluded molecules allows
the faster-flowing macromolecules to become separated from the slower small molecules as the
sample travels the distance of the resin bed packed in the column.
DE-SALTING
MOLECULAR WEIGHT DETERMINATION
The distribution of an analyte between the stationary and mobile phases depends on its size. It is
described by a parameter called its equilibrium distribution coefficient (Kd). Kd is the ratio of the
concentrations of the component in the stationary and the mobile phases. For component A, Kd =
0, since it cannot access the pores of the stationary matrix. For component B, Kd can be written as:
K_{d} = \frac{ V_{i} }{V_{0}}Kd=V0Vi
where Vi is the total solvent-accessible volume of the pores of the beads.
The smaller the particle, the more it permeates into the pores of the beads, and therefore, the
larger the Kd value. So, Kd depends on particle size. It is essential to determine Kd to estimate
particle size.
Considerations
Note that we must choose the molecular weight standards carefully; take the range of pore sizes in
your matrix beads into account.
Additionally, bear in mind, this assumes that the molecular shapes of the standards and your
samples are the same. Commercially available protein standards are largely globular in shape. If
protein has an elongated shape, then the determined molecular weight is likely to be inaccurate.
In such cases, use analytical ultracentrifugation or gel filtration coupled with multiple angle light
scattering for molecular weight determination.
REFERENCES
• https://www.ukessays.com/essays/biology/using-gel-filtration-to-
study-ligand-protein-interactions-biology-essay.php
• https://www.slideshare.net/shovameghbalika/gel-chromatography
• https://www.wise-geek.com/what-is-a-protein-ligand.htm
• https://www.thermofisher.com/pk/en/home/life-science/protein-
biology/protein-biology-learning-center/protein-biology-resource-
library/pierce-protein-methods/desalting-gel-filtration-
chromatography.html
• https://biologyreader.com/gel-filtration-chromatography.html
• ttps://bitesizebio.com/29685/determine-molecular-weight-gel-
filtration-chromatogram/
• Google Images
