GEL CHROMATOGRAPHY

GEL PERMEATION CHROMATOGRAPHY

SIZE EXCLUSION CHROMATOGRAPHY
GEL FILTRATION CHROMATOGRAPHY
MOLECULAR SIEVE CHROMATOGRAPHY
 INTRODUCTION
Gel chromatography is a technique in which the separation of
components is based on the difference in molecular weight or size,
and is one of the effective methods used to isolate and analyze the
bio-macromolecular substances.

It is the most convenient technique for characterizing the complete

molecular weight distribution of a polymer.

It is the preferred method for separating high molecular weight

neutral samples (compared to HPLC which is used for small
molecular weight samples)

In 1963 Jim Waters build the first commercial Gel

Chromatography equipment
 PRINCIPLE
It is a technique that separates dissolved molecules on the
basis of their size by pumping these molecules through
specialized columns containing a microporous packing
material(gel).

Stationary phase is a porous polymer matrix whose pores

are completely filled with the solvent to be used as the
mobile phase.
 PRINCIPLE
The pore size is highly critical, since the basis of the
separation is that molecules above a certain size are totally
excluded from the pores, and the interior of the pores is
accessible, partly or wholly, to smaller molecules.

The flow of mobile phase will cause larger molecules to

pass through the column unhindered, without penetrating
the gel matrix, whereas smaller molecules will be retarded
according to their penetration of the gel.
GPC SEPARATION
 Sample is prepared as a dilute solution in the eluent and injected
into the system

 The GPC column is packed with porous beads of controlled

porosity and particle size

 Large molecules are not able to permeate all of the pores and
have a shorter residence time in the column

 Small molecules permeate deep into the porous matrix and have
a long residence time in the column

 Sample molecules are separated according to molecular size,

eluting largest first, smallest last

 As a result of the GPC separation mechanism, sample molecules

elute from the column in order of size in solution

 Largest elute first, smallest elute last

 The separation is purely a physical partitioning, there is no

interaction or binding

 The separation is isocratic

GPC SEPARATION MECHANISM

Big Ones Come Out First

Assume all molecules are simple spheres
Smaller spheres will spend more time in
stationary phase
Large spheres will be excluded from the pore of
stationary phase
No interaction between sample and stationary
phase
GPC COLUMN

Biggest comes out first

 PRINCIPLE
A GPC column is made up of swollen gel particles and the solvent used
to swell the gel in a suitable tubular container. An equation is given
below:

Vt = V0 + Vi + Vm where,

Vt = the total volume of the column (which can be measured),

V0 = the volume of liquid outside the gel matrix (known also void or dead
volume),

Vi = the volume of liquid inside the matrix,

Vm = the volume of the gel matrix

GPC SYSTEM
 MOBILE PHASE RESERVOIR
CLEAN, INERT CONTAINER LIKE LAB FLASK

USUAL CAPACITY 1-2 LIT WITH CAP ALLOWS TUBING

INLET

 SOLVENTS USED MUST BE OF HIGH PURITY,

PREFERABLY HPLC GRADE AND FILTERED THROUGH
0.45µM FILTER.

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Solvent degassing – remove gases from the mobile phase
used in HPLC. If degassing of solvent is not done it will
lead to formation of bubbles in the HPLC tubing which
may lead to fluctuation in pressure and flow rate

 Helium purging or sparging- helium is bubbled through

the solvent and removes up to 80% of dissolved air
 Vacuum degassing - the solvent is exposed to a vacuum
and the reduced pressure removes more than 60% of the
dissolved air
 Sonication – high frequency vibration is used. These
ultrasonic baths as a stand-alone technique only
removes up to 30% dissolved air

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PUMPING SYSTEM
To ensure the delivery of a precise, reproducible,
constant and pulse free flow of the mobile phase
There are mainly two types of pumps used in GPC process
1. Reciprocating pump
2. Displacement or syringe pump

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RECIPROCATING PUMP
Displacement or syringe pump - The solvent is pumped out of a large
chamber by a plunger. The pump produces a pulse-free flow which is
also independent of column back pressure and solvent viscosity.

But it has a limited solvent capacity (~250 mL) and requires refilling of
solvent chamber for continual use.

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 SAMPLE INJECTION SYSTEM
Autosampler
With commercially available automatic sampling devices, large numbers of
samples can be routinely analyzed by GPC without operator intervention.

Such equipment is popular for the analysis of routine samples particularly

when coupled with automatic data-handling systems.

Most of the autosamplers have a piston metering syringe type pump to suck
the preestablished sample volume (generally 250 μl) into a line and then
transfer it to the relatively large loop (~100 ml) in a standard six-port valve.

The simplest autosamplers utilize the special vials with pressuarization

caps.

A special plunger with a needle, push the cap down to the vial and displace
the sample through the needle into the valve loop.

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SAMPLE INJECTION SYSTEM

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 COLUMN
The separation of sample takes place here. The sample
passes through the column with the mobile phase and
separates in its components when come out of the
column.
GPC columns are straight, have length of around 25-
60cm and inside diameter of around 7.5-8 mm
Smaller column diameter will result in greater sensitivity
because sample dilution within a column decreases as
column i.d. decreases
Narrow bore column of 2-3mm diameter are available.

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 DETECTOR
Detectors are used in the detection of the analyte
(sample) present in the eluent coming from the GPC
column. They are capable of determining the identity
and concentration of eluting compounds in the mobile
phase.
Desirable characteristics of detectors –
• Adequate sensitivity
• Stability and reproducibility
• Wide linear dynamic range
 DETECTOR
Desirable characteristics of detectors –
• Short response time
• Minimum volume for reducing zone broadening
• High reliability and ease of use
• Similarity in response toward all analytes
• Selective response toward one or more classes of analytes
• Non-destructive
 TYPES OF GPC DETECTOR
The detectors used in HPLC can be broadly categorized
into two – Specific detectors and Bulk property detectors
Specific detectors – respond to a specific compound and
their response is not dependent on mobile phase
composition
UV-Visible detectors
Photo Diode Array detectors
Fluorescence detectors
 DETECTOR
UV-Visible detector – it is the most widely used detector in
HPLC due to its specific response to the particular
compounds or functional groups (chromophore) present in
the sample.
A beam of EMR is passed through the detector flow cell,
and due to interaction (some absorption) of EMR with the
sample, EMR experiences a change in intensity.
The measurement of this change in intensity of EMR is the
basis of UV-Visible detector.
They are of three different types – fixed wavelength
(254nm), variable wavelength and diode array detector.
 DETECTOR
Photo diode array detector – a large number of diodes
serving as detector elements make it possible for
simultaneous monitoring of absorbing component at
different wavelength. (i.e., full range from 200-800 nm)
PDA saves a lot of time
PDA reduces cost on expensive solvent
PDA is less sensitive compared to UV-Visible detector
Fluorescent detector – more specific, sensitive and selective
detector than UV-Visible detector.
Very few compounds are naturally fluorescent. Thus,
derivatization is needed.
 TYPES OF GPC DETECTOR
Bulk property detectors – response is dependent on the
collective changes in the composition of the mobile phase
and sample.
Refractive index detector
 DETECTOR
Refractive Index Detector – Here detection depends on the
changes in refractive index of eluting molecules in the
mobile phase.
RI of mobile phase – 1.4
RI of mobile phase and sample eluent – 1.8
Change in RI = 0.4, Detector detect this change
These detectors are used for detecting those compounds
which neither absorb nor fluoresces in the UV region.
The problem with RI detector is that, it is having limited
sensitivity.
Moreover, equilibrium time is also too long.
 APPLICATIONS
GPC can also be successfully applied for the efficient removal
of small compounds from large biomolecules (i.e., the
separation and purification of pharmaceutical compounds,
fatty acids, and peptides from proteins)

The determination of the molecular weight of large

biomolecules (i.e., proteins and nucleic acids) and the
characterization of molecular weight distribution of a polymeric
structure are other application areas of the SEC process.
 APPLICATIONS
It can be used to determine the quaternary structure of
purified proteins

Separation of sugar, proteins, peptides on the basis of their

size