SIZE EXCLUSION CHROMATOGRAPY

ARKAJYOTI GUPTA1460027

IMTH 5(A)School of Biotechnology

KIIT University

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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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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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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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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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Pictorial representation of the working principle

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Factors affecting SEC• Column LengthAn increase in column length will enhance the resolution.

• Column DiameterIncreasing 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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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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Types of matrix

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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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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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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

Fig : Mol wt. of polymethacrylate ( *) and cellulose trinitrate ( +) as a function of elution volume

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Pros and cons of SECAdvantages

• 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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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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