GPC PPT MSC analytical chemistry JNTUUCEH

CONTENTS
 Introduction
 Objective of separation
 Principle
 Instrumentation
 Advantages
 Disadvantages
 Applications

Introduction
 Size-exculsion chromatography (SEC), uses porous
particles to separate molecules of different sizes.
 It is generally used to separate biological molecules,
and to determine molecular weights and molecular
weight distributions of polymers
 It is usually applied to large molecules or
macromolecular complexes such as proteins and
industrial polymers
 Types
gel-filtration chromatography (GFC),
gel-permeation chromatography (GPC),

 When an aqueous solution is used to transport the
sample through the column, the technique is known
as Gel-filtration chromatography .
 When an organic solvent is used as a mobile phase,
the technique is known as Gel-permeation
chromatography.
 Size of pores in beads determines the exclusion limit
(what goes through the beads and what goes around
the beads)

Objective of separation
 In protein study-proteins are extracted from
animal/ plant source
 When it is in need of specific protein study like
enzyme, hormone, antibody they need to
separate from the mixture.
 One method of separating proteins, gel filtration
relies on the fact that proteins differ in molecular
weight.

PRINCIPLE
 A mixture of molecules dissolved in liquid (the
mobile phase) is applied to a chromatography
column which contains a solid support in the form
of microscopic spheres, or “beads” (the stationary
phase).
 The mass of beads within the column is often
referred to as the column bed.
 The beads act as “traps” or “sieves” and function to
filter small molecules which become temporarily
trapped within the pores.

 Large proteins which cannot enter these pores pass
around the outside of the beads. Therefore, the
volume of the column appears smaller to a large
molecule
 Smaller proteins which can enter the pores of the
beads have a larger volume that they can explore,
thus the volume of the column appear larger to a
small molecule.
 Both large and small molecules experience the same
flow rate of mobile phase (i.e. L/min).Thus, a sample
of proteins passing through a gel filtration column will
separate based on molecular size: the big ones will
elute first and the smallest ones will elute last (and
"middle" sized proteins will elute in the middle).

Mechanism of Size Exclusion
Chromatography:
 Size exclusion (also known as gel filtration
chromatography) is a case of liquid-liquid
partition chromatography, in which the solute
molecules are get distributed in between two
liquid phases, (i) liquid in the gel pores and
(ii) liquid outside the gel.
 The total volume (Vt) of a column packed
with a gel that has been swelled by solvent
is given by
Vt = Vg + VI + Vo

 There are two extremes in the separation profile of
a gel filtration column. There is a critical molecular
mass (large mass) which will be completely
excluded from the gel filtration beads. All
solutes in the sample which are equal to, or
larger, than this critical size will behave
identically: they will all eluted in the excluded
volume of the column.
 There is a critical molecular mass (small mass)
which will be completely included within the
pores of the gel filtration beads. All solutes in
the sample which are equal to, or smaller, than
this critical size will behave identically: they will
all eluted in the included volume of the column

 Solutes between these two ranges of
molecular mass will elute between the
excluded and included volumes

 Where Vg is the volume occupied by the solid
matrix of gel,
 Vi is the volume of solvent held in the pores
or interstices
 Vo is the free volume outside the gel
particles.

Vo = “void volume”
Vt = “bed volume”
Ve = “elution volume”
Vi = Vt - Vo

 For molecules, which can enter the beads, there is an
linear logarithmic relationship between the size of the
molecule and the volume eluted from the column.
Finally, can use a standard curve to estimate the
molecular weight

COMPONENTS OF A SEC
1. Stationary Phase
2. The Mobile Phase
3. The Columns
4. The Pump
5. Detectors

STATIONARY PHASE
 Stationary Phase Semi-permeable, porous
beads with well-defined range of pore sizes .
 Beads are cross linked polymers
 Degree of cross linking is controlled
carefully to yield different pore sizes.
 Smaller pore sizes are used for rapid desalting
of proteins or for protein purification.
 Intermediate pore sizes are used to separate
relatively small proteins.

 Very large pore sizes are used for purification of
biological complexes.
 Stationary phase used for gel exclusion
chromatography include dextran (Sephadex™),
polyacrylamide and dextran-polyacrylamide
(Sephacryl™).
 Each is available with a variety of different
ranges of pore size in the beads, permitting
separation of macromolecules of different size

Selection of Gel type
 SEC analysis of synthetic organic polymers have been
made with crossed linked semi-rigid polystyrine gel
packings, small, rigid, inorganic particles (e.g. Silica).
 How ever have recently shown several significant
advantages over organic gels.
1. Rigid particles are relatively easly packed into
homogenous columns that are machanically
stable over long periods
2. As much wide range of mobile phases can be
used for greater versatility and increased
convenience.

3. Rapid equilibrium occurs with new solvents, allowing
rapid solvent change over.
4. Rigid packing's are stable at high temperatures.
5. Rigid particles can also be used in aqueous systems
for separating high molecular weight water soluble
solutes by GFC.
6. A potential disadvantage of the rigid inorganic
particles is retention of solutes by adsorption or
degradation of certain materials (e.g. Denaturing of
proteins). How ever silica particles can be surface
modified with appropriate organic functional groups
to eliminate this possible disadvantages for most
applications

 Soft gel e.g.- dextran(Sephadex),
Polyacrylamide gels
Separation of proteins.
 Semi-rigid gel e.g.- bio beads
Separation of non-polar polymers in non-polar
solvents.
 Highly rigid gels and glasses
Separation of polar systems.

Mobile phase
 The choice of mobile phase to be used in
any separation will depend on the type of
separation to be achieved and component to
be separated.
 The most common eluents in for polymers
that dissolve at room temperature. e.g.-
Tetrahydrofuran,Chloroform,
Dimethyl formamide.

material solvent
Synthetic elastomers
( polybutadiene ,
polyisoprene )
Toluene
PS, PVC, Styrene-
Butadiene Rubber ,
Epoxy resins
Tetrahydrofuran
Polyolefins Tri- chloro -benzene
Polyurethane Di- methylformamide
Proteins,
polysaccharides
Water / Buffers

SOLVENT SELECTION
The solvents used for mobile phase of SEC are limited
to those follows following criteria:
 The solvent must dissolve the sample completely.
 The solvent has different properties with solute in the
eluent: typically with solvent refractive index (RI) .
 solvent must not degrade the sample during use
Otherwise, the viscosity of eluent will gradually
increase over times.
 The solvent is not corrosive to any components of the
equipment

Commercially Available Columns
 analytical column- 7.5–8mm
diameters.
 Preparative columns-22–25mm for.
 Usual column lengths-25, 30, 50, and
60 cm.
 Recently, narrow bore columns- 2–
3mm diameter have been introduced,
which save time and solve

Selecting SEC column
 Shorter columns save time and solvent.
 Small particles (typically 5 mm) provide a better
resolution.
 On the other hand, 5 mm (or even 3 mm) packings
are more sensitive towards contamination by
samples containing impurities.
 Particles as large as 20 mm have been recommended
for very high-molecular-weight polymers.
 Columns with different porosity or mixed-bed
columns, provide a better separation.

Handling SEC Columns
 A column set in SEC should be always run in the
same mobile phase.(isocratic)
 SEC columns should never be operated in a
backward direction.
 Care should also be taken in connecting columns or
in sample injection.
 Replacing a clogged inlet frit is a dangerous
operation which can reduce column performance.
 A damaged or dirty check valve of pump, can also
reduce column life.

PUMP
 A highly constant flow rate has to be maintained
during the entire chromatogram. This is very
important in SEC.
 A change of the flow rate of only 0.1% can cause
an error in molar mass of up to 10%.
 Most pumps can only reproduce the flow rate to
0.2–0.3%.
 In-line filters in the solvent reservoir may prevent
particles from coming into the pump heads, which
might damage the check valves or the pump seals.

DETECTOR
Concentration sensitive detectors
Bulk Property Detectors- Refractive Index
(RI) Detector
Solute Property Detectors- Ultraviolet (UV)
Absorption Detector
Evaporative Detectors- Evaporative Light
Scattering Detector (ELSD)

ADVANTAGES
 Well defined separation.
 Narrow bands and good sensitivity.
 There is no sample loss.
 It can be carried out at room temperature

DISADVANTAGES
 Limited number of peaks that can be resolved within
the short time scale of the GPC run.
 Filtrations must be performed before using the
instrument to prevent dust and other particulates from
ruining the columns and interfering with the
detectors.
 The molecular masses of most of the chains will be
too close for the GPC separation to show anything
more than broad peaks

APPLICATION
 Proteins fractionation
 Purification
 Molecular weight determination.
 Separation of sugar, proteins, peptides,
rubbers and others on the basis of their size.
 This technique can be determine the
quaternary structure of purified proteins.

 SEC is a widely used technique for the
purification and analysis of synthetic and
biological polymers, such as protein,
polysaccharides and nucleic acid.
 Various species of RNA and viruses have been
purified using agarose gels.
 For Desalting ( faster & more efficient than
dialysis )
 For copolymerisation studies

GPC PPT MSC analytical chemistry JNTUUCEH

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GPC PPT MSC analytical chemistry JNTUUCEH