Size exclusion/ molecular seive chromatography.

1. SIZE EXCLUSION CHROMATOGRAPY
ARKAJYOTI GUPTA
1460027
IMTH 5(A)
School of Biotechnology
KIIT University

2. What is Chromatography?
• Russian scientist Tswett (1906) was the first to use the term
“chromatography”
• Derived from Greek words "Chroma" meaning color and "graphein"
meaning to write.
• IUPAC definition: Physical method of separation in which the components to
be separated are distributed between two phases, one of which is stationary
while the other moves in a definite direction.
• Stationary phase - solid, or a liquid supported on a solid or gel
• Mobile phase - either a gas or a liquid.
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3. Size Exclusion Chromatography
• Also known as Gel Filtration Chromatography or Molecular Sieve
Chromatography
• Separates molecules according to their size, shape & molecular
weight.
• It is usually applied to large molecules or macromolecular complexes
such as proteins and industrial polymers.
• Basic representation:
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4. History
• Invented by Grant Henry Lathe and Colin R
Ruthven
• They later received the John Scott Award for
this invention.
• While Lathe and Ruthven used starch gels as
the matrix, Jerker Porath and Per Flodin
later introduced dextran gels.
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5. Basic Principle
• Works by trapping smaller molecules in
the pores of the adsorbent materials
adsorption ("stationary phases").
• The larger molecules simply pass by the
pores because those molecules are too
large to enter the pores.
• Larger molecules therefore flow through
the column more quickly than smaller
molecules
• In short : the smaller the molecule, the
longer the retention time.
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6. Pictorial representation of the working
principle
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7. Factors affecting SEC
• Column Length
An increase in column length will enhance the resolution.
• Column Diameter
Increasing the column diameter increases the capacity of the column.
• Proper Column Packing : Column packing should be optimum
It is important to maximize resolution as
An over packed column can collapse the pores in the beads, resulting in a
loss of resolution.
An under packed column can reduce the relative surface area of the
stationary phase accessible to smaller species, resulting in those species
spending less time trapped in pores.
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8. Factors considered while designing the system
• Choice of matrix : Compatibility & inertness w.r.t the molecules being
separated.
• Effect of flow rate : flow rate and resolution are inversely related.
• Sample size and conc. : With increase in sample conc. w.r.t to the
eluent, viscosity increases. A high viscosity will result in irregular
sample migration through the column.
• Column parameters : Long columns maximize resolution.
• Choice of eluent : High ionic strength of the eluent minimizes
protein-matrix & protein-protein associations by electrostatic or van
der Waals interactions.
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9. Types of matrix
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10. Applications
• Used in the purification of thousands of proteins and peptides from
various sources. These range from therapeutic proteins to enzymes
and proteins for the brewing, food-processing and diagnostics
industries
• Separation of cells and virus particles. Cells of different sizes can be
efficiently separated from one another using SEC . Example
separation of erythrocytes and platelets from blood
• Molecular mass estimation : Gel-filtration chromatography is an
excellent alternative to SDS-PAGE for the determination of relative
molecular masses of proteins, since the elution volume of a globular
protein is linearly related to the logarithm of its molecular weight
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11. Purification and separation of proteins
• Column is composed of porous beads made from polyacrylamide, dextran
or agarose.
• Proteins flow around the spherical beads in gel filtration chromatography.
• The surface of the beads is punctured by large holes, and proteins will
spend some time within these holes.
• Because smaller proteins can penetrate into the beads more easily than
larger proteins, they travel through a gel filtration column more slowly than
larger proteins
• The total volume of liquid required to elute a protein from the column
depends on its mass: the smaller the mass, the greater the elution volume
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12. Molecular mass estimation of biomolecules
• Elution time : the time between the start of the separation (the time
at which the solute enters the column) and the time at which the
solute elutes
• Elution volume : the volume of eluent required to cause elution.
• log M = f (Ve) .
Where, M = Molecular weight ; Ve = Elution volume
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Fig : Mol wt. of polymethacrylate ( *) and cellulose trinitrate
( +) as a function of elution volume

13. Pros and cons of SEC
Advantages
• Simplicity ,reliability &
versatility
• Ease of scale up
• Low chances of sample loss
• Less time of analysis
Disadvantages
• large size of columns need large
volumes of eluent often creating
excessive running costs
• When separating proteins by SEC
proteolysis increasing problem
• Low resolution compared to other
chromatographic techniques
• Low sample handling capacity
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14. References
• https://en.wikipedia.org/wiki/Size-exclusion_chromatography
• Gel-Filtration Chromatography Ciarán Ó’Fágáin1, Philip M. Cummins
and Brendan F. O’Connor1
• Lathe, GH and Ruthven, CR (1955) The separation of substances on
the basis of their molecular weights, using columns of starch and
water.
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