Size Exclusion

Chromatography
Ayush Nair
2017A5PS1068P
 Principle

• Particles of different sizes elute through a stationary phase at different

rates, resulting in the separation of particles based on size. Particles of the
same size should elute together.
 Types

• 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.
• 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.
Working

• Process is usually performed within a column,

which typically consists of a hollow tube tightly
packed with micron-scale polymer beads
(Sephadex, Sepharose, Sephacryl or BioGel
P).containing pores of different sizes.

• After sample has been applied, molecules

larger than the pores are unable to diffuse into
the beads, so elute first.
Working

• Smaller molecules can penetrate the pores to

varying degrees based on their size.

• Larger molecules therefore flow through the

column more quickly than smaller molecules,
that is, the smaller the molecule, the longer
the retention time.
 Analysis

• Eluent collected in fractions. Particles of similar size eluted in same

fractions.
• Concentration of protein in fractions examined by UV and RI spectroscopy.
• Molar mass and sample fraction size, can be detected by MALS, coupled
with concentration detector.
Molecular Weight Determination

• Calibration curves prepared by using MW markers with

known weights to find the elution volume and plotting the
logarithm of MW versus the elution volume.
• Molecules larger than the pore size pass straight through (are
excluded). This is called the exclusion limit.
• Conversely, molecules below a certain size completely
penetrate the pores and tend to elute almost in the same
position. This is called the permeation limit.
• The calibration curve is valid in the range between this
exclusion limit and permeation limit. Within this range, the
larger the molecule, the sooner it elutes and the smaller the
molecule the later it elutes.
 Applications

• Protein Purification

Source-The separation of substances and estimation of their relative molecular sizes by the
use of colums of starch in water.LATHE GH, RUTHVEN CR
Biochem J. 1956 Apr; 62(4):665-74.
Applications

• Understanding Protein Folding/Unfolding

Applications

• Protein desalting and purification

 Applications

• Analysis of Protein Aggregation

Source-M.D. Bond, et. Al. Evaluation of a dual-wavelength size exclusion HPLC

method with improved sensitivity to detect protein aggregates and its use to better
characterize degradation pathways of an IgG1 monoclonal antibody
J. Pharm. Sci., 99 (2010), pp. 2582-2597
 Limitations

• Polymer interaction with analyte may increase elution time.

• Turbulence in flow of solvent may cause broadening of bands.

 References

• M.D. Bond, et. Al. Evaluation of a dual-wavelength size exclusion HPLC method with
improved sensitivity to detect protein aggregates and its use to better characterize
degradation pathways of an IgG1 monoclonal antibody. J. Pharm. Sci., 99 (2010),
pp. 2582-2597
• The separation of substances and estimation of their relative molecular sizes by the use of
colums of starch in water.LATHE GH, RUTHVEN CR. Biochem J. 1956 Apr; 62(4):665-74.
• https://www.shimadzu.com/an/hplc/support/lib/lctalk/55/55intro.html
• Aline Staub, Davy Guillarme, Julie Schappler, Jean-Luc Veuthey, Serge Rudaz,Intact protein
analysis in the biopharmaceutical field,Journal of Pharmaceutical and Biomedical
Analysis,Volume 55, Issue 4,2011.
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