A simple presentation on principle, instrumentation and applications of size exclusion chromatography.

1. SIZE EXCLUSION CHROMATOGRAPHY (SEC)
PRESENTED BY : MR K V NANDA KUMAR
ASSOCIATE PROFESSOR,
DEPARTMENT OF PHARMACEUTICAL ANALYSIS,
KRISHNA TEJA PHARMACY COLLEGE, TIRUPATHI-517506

2. CONTENTS
INTRODUCTION AND
PRINCIPLE
TYPES OF SEC COMPONENTS OF SEC
COLUMN PACKING
ADVANTAGES AND
DISADVANTAGES
PHARMACEUTICAL AND
OTHER APPLICATIONS
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3. Also known as…
Molecular exclusion chromatography
Gel exclusion chromatography
Gel filtration chromatography
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5. INTRODUCTION AND PRINCIPLE
Size exclusion chromatography (SEC) separates molecules based on their size by filtration through a
gel.
The gel consists of spherical beads containing pores of a specific size distribution.
Separation occurs when molecules of different sizes are included or excluded from the pores within
the matrix.
Small molecules diffuse into the pores and their flow through the column is retarded according to
their size, while large molecules do not enter the pores and are eluted in the column's void volume.
Consequently, molecules separate based on their size as they pass through the column and are eluted
in order of decreasing molecular weight (MW).
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6. TYPES OF SEC
There are two basic types of size exclusion chromatography.
One is gel permeation chromatography (GPC), which uses a hydrophobic column
packing material and a non-aqueous mobile phase (organic solvent) to measure the
molecular weight distribution of synthetic polymers.
The other is gel filtration chromatography (GFC), which uses a hydrophilic packing
material and an aqueous mobile phase to separate, fractionate, or measure the
molecular weight distribution of molecules soluble in water, such as polysaccharides
and proteins.
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7. COMPONENTS OF SEC
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8. COMPONENTS OF SEC
1. Pump
Pumps the polymer in solution through the system.
2. Injector
Introduces the polymer solution into the mobile phase.
3. Column Set
Efficiently separates sample components from one another.
4. Detector
Monitors the separation and responds to components as they elute from the column.
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9. COLUMN PACKING
To perform a separation, SEC medium is packed into a column between 300 and 600
mm in height for high-resolution fractionation and up to 100 mm in height for group
separations.
The volume of the packed bed is determined by the sample volumes that will be
applied.
Efficient column packing is essential, particularly for high-resolution fractionation.
The efficiency of a packed column defines its ability to produce narrow symmetrical
peaks during elution.
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10. SEC MEDIUM
Superdex Increase or Superdex are designed for high resolution, short run times, and
high recovery.
Superdex prep grade and Sephacryl are suitable for fast, high-recovery separations at
laboratory and industrial scale.
Sephadex is recommended for rapid group separations such as desalting and buffer
exchange
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11. PURIFICATION BY SEC(WORKING)
To perform a separation, the medium is packed into a column to form a packed bed.
SEC media consist of a porous matrix of spherical particles with chemical and physical stability and
inertness (lack of reactivity and adsorptive properties).
The packed bed is equilibrated with buffer, which fills the pores of the matrix and the space
between the particles.
The liquid inside the pores, or stationary phase, is in equilibrium with the liquid outside the
particles, or mobile phase.
Samples are eluted isocratically so there is no need to use different buffers during the separation.
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12. SEC GENERAL APPLICATIONS
Size-exclusion chromatography has been used with great success in the separation of
the sugars, polypeptides, proteins, liquids, asphalts, butyl rubbers, polyethylene’s,
polystyrenes, silicon polymers, and others.
Size exclusion chromatography has been mainly applied to studies of complex,
biochemical or highly polymerized molecules.
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13. MAJOR APPLICATIONS
Purification
Molecular
weight
determination
Solution
concentration
Desalting
Protein
building
studies
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14. PURIFICATION
The main application of exclusion chromatography is in the purification of biological
macromolecules.
Viruses, proteins, enzymes, hormones, antibodies, nucleic acids, and polysaccharides
have all been separated and purified by the use of appropriate gels or glass granules.
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15. MOLECULAR WEIGHT DETERMINATION
The effluent volumes of globular proteins are largely determined by their molecular
weight.
It has been shown, that over a considerable molecular weight range, the effluent
volume is approximately a linear function of the logarithm of the molecular weight.
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16. SOLUTION CONCENTRATION
Solution of high molecular weight substances can be concentrated by the addition of
dry sephadex G-25 (coarse).
Water and low molecular weight substance remain in solution.
After ten minutes the gel is removed by centrifugation, leaving the high molecular
material in a solution whose concentration has increased but whose pH and ionic
strength are unaltered.
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17. DESALTING
By use of a column of sephadex G-25, solutions of high molecular weight compounds
may be desalted.
The high molecular weight substances move with the void volume while the low
molecular weight components are distributed between the mobile phase and hence
move slowly.
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18. PROTEIN BUILDING STUDIES
Exclusion chromatography is one of a number of methods commonly used to study the
reversible binding of a ligand to a macromolecular such as proteins including receptor
proteins.
A sample of the protein/ligand mixture is applied to a column of a suitable gel (e.g.
G-25) which has previously been equilibrated with a solution of the ligand of the same
concentration as that in the mixture.
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