Gel exclusion chromatography, also known as size exclusion chromatography, separates analytes based on their size or hydrodynamic volume. Smaller analytes have a longer retention time as they are able to penetrate the pores in the stationary phase beads, while larger analytes pass through more quickly. The principle allows for separation of a number of molecular weights within each column. Detectors are used to monitor the concentration of analytes as they elute from the column. Though useful for determining relative molecular weights, gel permeation chromatography has limitations including poor resolution of peaks for polymer samples where molecular weights are closely spaced.

1. Gel Exclusion Chromatography
By
Dr. Nidhi Gupta
Assistant Professor
M.M. College of Pharmacy
Maharishi Markandeshwar (Deemed to be University), Mullana,
Ambala, India

2. Introduction
Gel permeation chromatography (GPC) is a form of size exclusion
chromatography (SEC) that uses organic solvents to separate analytes
based on their size. Polymer analysis is a popular application of this
technique. SEC was first developed as a technique by Lathe and Ruthven in
1955. The term gel permeation chromatography was coined by J.C. Moore
of the Dow Chemical Company, who researched the technique in 1964 and
licensed the patented column technology to Waters Corporation, which
commercialized it in 1964.

3. Principle of Gel Exclusion chromatography
•The analytes are separated by GPC based on their size or hydrodynamic volume
(radius of gyration). Other separation methods, on the other hand, rely on
chemical or physical interactions to distinguish analytes. Porous beads packed in
a column are used to separate the particles.
•Since smaller analytes can penetrate pores more quickly, they spend more time
in them and hence have a longer retention time. Since these smaller molecules
spend more time in the column, they elute later. Larger analytes, on the other
hand, spend little or no time in the pores and are easily eluted. A number of
molecular weights can be divided in each column.

4. Analytes that are too large will not be retained; on the other hand,
analytes that are too small will be entirely retained. Analytes that are not
retained are eluted with the free volume outside of the particles (Vo),
while those that are completely retained are eluted with the volume of
solvent held in the pores. The following equation can be used to calculate
the total volume, where Vg is the volume of the polymer gel and Vt is the
total volume:
Vt = Vg + Vi + Vo

5. Methods of Gel Filtration Chromatography
Almost all gel permeation chromatography is done in chromatography columns.
The experimental model is very similar to that of other liquid chromatography
techniques. Samples are dissolved in a suitable solvent, which in the case of GPC
is usually organic, and then filtered before being injected onto a column. The
column is where a multi-component mixture is separated. The use of a pump
ensures a steady supply of fresh eluent to the column. A detector is needed
because most analytes are not visible to the naked eye. To obtain additional
information about the polymer sample, several detectors are often used.
The stationary process for GPC is gel. In order to apply a gel to a specific
separation, the pore size of the gel must be carefully monitored. The absence of
ionizing groups and low affinity for the substances to be separated in a given
solvent are also desirable properties of the gel-forming agent.

6. •Microporous packing material is used to fill the GPC column. The gel is poured
into the column known as the gel filtration column.
•The eluent (mobile phase) should be a good solvent for the polymer, allowing
the polymer to have a high detector response and wetting the packing surface.
Tetrahydrofuran is the most common element in GPC polymers that dissolve at
room temperature (THF).
•Piston or peristaltic pumps are the two types of pumps available for uniform
distribution of relatively small liquid volumes for GPC.
•In GPC, a detector will continuously monitor the polymer concentration by
weight in the eluting solvent. There are several different types of detectors,
which can be classified into two groups. UV absorption, differential
refractometer (DRI) or refractive index (RI) detectors, infrared (IR) absorption,
and density detectors are the first types of concentration-sensitive detectors.

8. Application of Gel Filtration Chromatography
GPC is often used to determine the relative molecular weight of polymer
samples as well as molecular weight distribution. The molecular volume and
shape function, as determined by the intrinsic viscosity, are what GPC truly
measures. This relative data can be used to calculate molecular weights
within 5% precision if comparable criteria are used. To calibrate the GPC,
polystyrene standards with disparities of less than 1.2 are commonly used.

9. Disadvantages of Gel Permeation chromatography
GPC, on the other hand, has several drawbacks. First, the number of peaks that
can be resolved within the short time frame of a GPC run is small. Furthermore,
for a satisfactory resolution of peaks, GPC as a technique needs at least a 10%
difference in molecular weight. When it comes to polymers, the molecular
masses of most of the chains are too close together for the GPC separation to
produce anything other than large peaks. Another downside of GPC for
polymers is that it needs filtration prior to use to prevent dust and other
particulates from destroying the columns and interfering with the detectors.

10. Thank you………..