Size
Exclusion Chromatography and its
Application for Polymer Analysis
Size
Exclusion Chromatography (SEC), which is also known as Exclusion Chromatography
is a technique used for separating molecules based on their effective
shapes and sizes in a solution.
Introduction and Basic Concepts
Size Exclusion Chromatography
(SEC), which is also known as Exclusion Chromatography is a technique
used for separating molecules based on their effective shapes and
sizes in a solution. The technique is called Gel Permeation Chromatography
(GPC) when used with organic solvents.
The stationary phases used in Exclusion Chromatography are porous
particles with closely controlled pore sizes. Usually, unlike other
chromatographic techniques, there is least interaction between solute
and the surface of the stationary phase in exclusion chromatography.
Depending on their sizes and shapes, solute molecules are able to
enter pores of the stationary phase particles. However, even in SEC,
retention may not be exclusively controlled by sizes of the solute
molecules.
It will still be controlled by molecular interactions between the
solute and the two phases, viz. stationary and mobile phases. Only
if the magnitude of the forces between the solute and both the phases
are same, in that case the retention and selectivity of the chromatographic
system will depend totally upon the pore size distribution of the
stationary phase.
The term “Exclusion Chromatography” therefore, is specially
confined to those separations where retention is almost completely
controlled by size exclusion, molecular interaction of the solute
molecules with the two phases being almost identical. Under these
circumstances, the larger molecules, being partially or completely
excluded, will elute first and the smaller molecules will elute last
and the intermediate ones in between depending on the sizes and shapes,
the solute molecules will be able to enter the pores of the stationary
phase particles. Molecules comparable in size with the mobile phase
molecules will be able to diffuse and enter throughout the porous
network. The larger molecules will not be able to enter and hence
will be excluded from the fine porous structure and will be able to
move freely in the wider passages. The larger the solute molecules,
the fewer small chanvents nels in the porous structure of the stationary
phase it can get into. There may be solute molecules which are so
large that they are completely excluded from the pores. These excluded
molecules can travel only through relatively wide channels between
the stationary phase particles and so are eluted rapidly from the
column. The smaller the molecule, the more easily it will penetrate
the small channels of the stationary phase particles; and naturally
longer it will be retained in the column. This process is shown in
the Fig. 1. GPC Instrumentation The essential components of GPC instrumentation
include:
Solvent Reservoir
High Pressure Pumps
Injector
S.S. Columns
Detectors
Data Handling System/Software
Solvent Reservoir
Solvent reservoirs are typically made up of stainless steel or
glass and the sol-chanvents used are needed to be thoroughly degassed.
This can be achieved by evacuation using vacuum pump or by purging
helium. Pumps
The main function of the pump is to deliver a constant, pulseless
and reproducible flow rate of the solvent/ mobile phase through the
system. Modern pumps based on reciprocating piston design provide
lesser pressure fluctuations are commonly used.
Injector
The injector is used to introduce a sharp plug and exact amount
of sample solution into the column with minimum band broadening. The
commonly used device is the micro sampling injector valve.
Columns
Like any chromatographic technique, column and packing material
(stationary phase) is like the heart of the GPC system and their main
function is to separate the components based on hydrodynamic volume
or molecular size. GPC columns are made up of stainless steel 30 cm
long and of 7.8 mm diameter. The column material has to be selected
for the molecular weight range of interest. There are mainly two types
of stationary phases commonly used in SEC are Silica Gel and micro-reticulated
cross linked polystyrene gels, with spherical beads usually of 5-30m
size. The gel beads are hard and prepared in different porosities.
Depending on the molecular weights and molecular sizes of the analyte
molecules, columns with suitable porosities are selected. The bead
size, pore size, pore distribution, shape, rigidity etc. play an important
role in the separation process.
....contd.
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