Size Exclusion Chromatography

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Sumit Khanna
Department of Pharmaceutical Sciences, G. J. University, Hisar-125 001, India. 397, Sec-13, Hisar, Haryana, India GSM: +919416045445 E-mail: sumkhanna@hotmail.com

Size-exclusion chromatography (also known as gel filtration chromatography) is a technique for separating proteins and other biological macromolecules on the basis of molecular size. Originally developed in the 1950s, the technique was developed using cross-linked dextran. Some confusion over nomenclature has been created by the term gel permeation, used to describe separation by the same principle in organic mobile phases using synthetic matrices. It is now generally agreed that the terms gel filtration and gel permeation do not accurately reflect the nature of the separation. Size exclusion chromatography (SEC) has been widely accepted as a universal description of the technique and in line with the IUPAC nomenclature, this term will be adopted. Size-exclusion chromatography is a commonly used technique, because of the diversity of the molecular weights of proteins in biological tissues and extracts. SEC has been employed for many roles, including buffer exchange (desalting), removal of nonprotein contaminants (DNA, viruses), protein aggregate removal, the study of biological interactions, and protein folding. It also has the important advantage of being compatible with physiological conditions. (3)

Key Words: Gel Permeation Chromatography (GPC), Gel Filtration Chromatography (GFC)

Introduction

Size-exclusion chromatography (SEC) being a versatile technique where in the separation is based on differences in the size of sample molecules. The column packing is made from beads of a porous gel. Molecules of various sizes are drawn on the photofilm and show that the large molecules cannot fit at all and would be washed out of the column. The smallest molecular weight molecule that does not enter the gel is called the exclusion limit, and the volume required to elute the large molecule is called the void volume, V. The smaller molecules can permeate the gel; the smaller the molecule, the farther the penetration and greater the retardation. This results in a separation because it requires more solvent to wash the smaller molecule out of the gel. This has a limit. If the molecule becomes too small, then their molecule size compared to the pores is so small that they behave in the same manner and require essentially the same amount of solvent to elute them. (1,14)

      SIZE-EXCLUSION CHROMATOGRAPHY.jpg           

This volume is called the total permeation volume; Vt. Vi is the interstitial volume.

{mospagebreak title=Gel permeation chromatography}

Gel permeation chromatography

When SEC is carried out in non-aqueous mobile phase, it can be called gel permeation chromatography (GPC) (4,6,8,10). Gel permeation chromatography is an accepted technique for characterizing polymers. It does not rely on chemical differences to effect the separation but separates polymer molecules on the basis of their size relative to the pores in the packing particles. Its primary use is in measuring molecular weight and molecular weight distributions. However, closer consideration reveals that the size of polymer molecule also depends on primary and secondary structural factors. The major limitation of conventional GPC using only a concentration detector, such as a refrectrometer, is that it is unable to distinguish between the effects of molecular weight and structural differences on molecular size.

Gel Filtration chromatography

When SEC is carried out in aqueous mobile phase, it is often called gel filtration chromatography (GFC). (6,10) Purifying biological macromolecules entails separating them according to some distinguishing property, and then collecting those you want. Two of the more useful distinguishing properties of macromolecules for purification purpose are their size and shape, and a variety of techniques have been developed to separate macromolecules by size. Liquid gel filtration chromatography has been widely used for this purpose because it is simple, economical, rapid, highly reproducible, and very gentle on the samples.

As on date gel filtration chromatography is used to fractionate a crude protein preparation by size. We will first calibrate the column using markers of known molecular weight. This calibration curve shall then be used to estimate the size and Kd of several enzymes present in the sample.

Method

The basic components of a SEC procedure are the matrix, chromatography column and eluant. The matrix is a stationary phase consisting of spherical gel beads typically made from polyacrylamide, dextran or agarose. The beads contain pores and internal channels that span a defined size range. This range is chosen according to the sizes of molecules within sample to be separated. The chromatography column is a tube in which the matrix is contained, and where the separation takes place. The eluant constitutes the mobile phase of SEC and is usually a moderate buffer or salt solution, which helps to maintain the biological activity of molecules. (7,8,15)

The sample mixture is dissolved in the eluant. This solution is applied to the top of the column and migrates downwards through the matrix. Smaller molecules are able to penetrate the pores in the gel beads, while larger molecules have access to fewer pores and tend to travel around the beads. The large molecule will, therefore, pass through the column more quickly and will elute (separate from the matrix) first. Throughout the process, the column is washed with eluant and molecules are collected in fractions as they exit from the bottom. Since elution volume is inversely proportional to mass, molecules are collected in order of decreasing molecular weight.

Principle

SIZE-EXCLUSION CHROMATOGRAPHY1.jpg
When a sample is injected into the column, the polymer molecules are separated according to their hydrodynamic volumes; hence polymer molecules larger than the pores of the packing material cannot enter the pores and are eluted at the interstitial volume Vi. Note that no fraction of the sample can be eluted before the interstitial volume has passed through the column. Small molecules, however, have an access to the pores and will, therefore, elute as the sum of both the interstitial and pore volume, i.e. Vi + Vp. Molecules which have sizes between the above two extreme values will have access to only a part of the pore volume and will therefore be eluted at an elution volume Ve which is given by the following expression:

SIZE-EXCLUSION CHROMATOGRAPHY2.gif                                                      

where KSEC is the equilibrium constant of a sample in size exclusion  chromatography (0<KSEC<1). (2,11)

Note that to obtain an absolute molecular mass value of a sample, standards with known molecular masses must be used to obtain a suitable calibration curve.

Instrumentation

Modern size-exclusion chromatography requires somewhat different equipment from that used in traditional gel permeation chromatography and radically different apparatus that used in conventional gel filtration chromatography. Although elegant   separations have been carried out in the past with relatively modest apparatus, more   sophistication is featured in modern size-exclusion chromatography, whether in   determining accurate molecular weights of synthetic polymers or analyzing samples with   biologically active macromolecules. Important parts of SEC instrument include pumps   for maintaining constant, pulseless rates of flow, column types for the molecular weight range for interest, and the detector system for quantifying the result.

Components employed in SEC (3,15)

  • Column
  • Stationary phase
  • Pumps
  • Detectors
Applications

  • Determination of the molecular weight
  • Determination of the molecular weight distribution of natural and synthetic polymers
The molecular weight and molecular weight distribution of polymers can be determined easily using size-exclusion chromatography (SEC). The polymer sample is dissolved in an appropriate solvent and injected into the chromatography system. The system contains separation columns, which are filled with polymer gels with cavities of known size. The smaller molecules of low molecular weight diffuse deeper into the cavities of the polymer gel than the bigger molecules of high molecular weight. These molecules move faster through the polymer gel and are eluted earlier than the smaller molecules. The result in a separation of the molecules in the chromatography column caused by their hydrodynamic volume.

After separation of the polymer sample, the molecules are detected with different detector system, differential e.g., a refractrometer or an UV-VIS detector. The detector intensity is recorded as a function of the volume of the eluted solvent. A calibration of the system with polymer samples of known molecular weight allows the evaluation of the molecular weight and molecular weight distribution of the polymer sample. Using a light scattering detector, the molecular weight of polymers can be determined directly and thus, no calibration substances are necessary. (9)

References

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  3. A.F.Buijtenhuijs and C.M.A. van de Riet, “Aqueous Size Exclusion Chromatography” MolMass Proceedings, 1999.
  4. Meehan, “High temperature gel permeation chromatography” MolMass Proceedings, 1999.
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  11. Cohen, Y., S.R. Faibish and M. Rovira-Bru, "Polymer-Modified Silica Resins for Aqueous Size Exclusion Chromatography," In :Interfacial Phenomena in Chromatography, Pefferkorn, E. (ed.), Marcel-Dekker, New York, (1999) 263-310.
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  13. Cohen, Y. and P. Eisenberg, "Polyvinylpyrrolidone-Grafted Silica Resin: A New Promising Material for SEC of Water Soluble Polymers,” in "Polyelectrolyte Gels: Properties, Preparation, and Applications," Harland, R. S. and R. K. Prud'homme, ACS Symposium Series 480, American Chemical Society, Washington, DC (1992) 254-268.
  14. S.Mori & H.G.Barth; Size-exclusion chromatography; Springer, berlin, 1999
  15. E.Meehan, In C.S. WU, ed.; “ Column handbook of SEC”; Academic Press, New York, 1999,349-366
  16. M.Potschka and P.L.Dublin,”Stratigies in Size exclusion chromatography”, American Chemical Society, Washington DC, 1996
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  18. M. Strlic, J. Kolar, M. Zigon, B. Pihlar: "Evaluation of Size-exclusion Chromatography and Viscometry for the Determination of Molecular Masses of Oxidised Cellulose". J. Chromatogr. A, 805 (1998) 93-99.
  19. M. Strlic, J. Kolenc, J. Kolar, B. Pihlar: "Enthalpic interactions in size exclusion chromatography of pullulan and cellulose in LiCl/DMAc", J. Chromatogr. A, 964 (2002) 47-54.
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  21. J. C. Giddings, "Universal Calibration in Size Exclusion Chromatography and Thermal Field-Flow Fractionation," Anal. Chem., 66 (1994) 2783-2787.
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