Gel‐Filtration Chromatography

Lars Hagel1

1 Amersham Pharmacia Biotech AB, Uppsala, Sweden
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.9
DOI:  10.1002/0471142727.mb1009s44
Online Posting Date:  May, 2001
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Gel filtration (GF) chromatography separates proteins solely on the basis of molecular size. Separation is achieved using a porous matrix to which the molecules, for steric reasons, have different degrees of access‐‐i.e., smaller molecules have greater access and larger molecules are excluded from the matrix. Hence, proteins are eluted from the GF column in decreasing order of size. This unit describes the experimental theory behind gel filtration and contains many useful tables listing the properties and characteristics of currently available matrices.

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Table of Contents

  • Section IV: Purification of Proteins by Conventional Chromatography
  • Strategic Planning
  • Basic Protocol 1: Desalting (Group Separation)
  • Basic Protocol 2: Protein Fractionation
  • Basic Protocol 3: Determination of Molecular Size
  • Support Protocol 1: Column Calibration
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Desalting (Group Separation)

  • GF matrix (Table 10.9.1) or prepacked GF column (Table 10.9.3) with appropriate exclusion limit for protein of interest (also see and ).
  • recipeGF desalting buffer (see recipe)
  • Colored marker: 0.2 mg/ml Blue Dextran 2000 or 0.2 mg/ml vitamin B 12
  • recipeVoid marker (see recipe)
  • recipeTotal liquid volume marker (see recipe)
  • Protein sample to be desalted
  • 90°C water bath (optional)
  • Buchner or sintered‐glass funnel
  • GF chromatography column (see and Table 10.9.2) with column extension (optional), adaptors, and buffer reservoir (Fig. )
  • Carpenter's level
  • Peristaltic pump (Fig. )
  • 0.22‐µm filter: any 0.22‐µm filter for buffer; protein‐compatible for sample
  • Detector (optional; Fig. )
  • Chart recorder (optional; Fig. )
  • Sample applicator: sample application loop (e.g., Superloop, Amersham Pharmacia Biotech) or syringe
  • Fraction collector (optional; Fig. ).
NOTE: Small‐scale desalting of multiple samples may be carried out using spin columns and a centrifuge. At present, spin columns are primarily used for preparing microliter‐sized samples of oligonucleotides; however, they are equally useful for small‐volume protein samples. As the procedure is different from that described here, manufacturer's instructions should be consulted (see Table 10.9.3 for suppliers).

Basic Protocol 2: Protein Fractionation

  • GF matrix (see Table 10.9.4) or prepacked GF column (see Table 10.9.6) with appropriate selectivity for protein of interest (also see and ).
  • recipeGF fractionation buffer (see recipe)
  • Colored marker: 0.2 mg/ml Blue Dextran 2000 or 0.2 mg/ml vitamin B 12
  • Low‐molecular‐weight marker (e.g., 5 mg/ml acetone or 2 M sodium chloride)
  • Protein sample to be fractionated
  • GF chromatography column (see and Table 10.9.5)

Basic Protocol 3: Determination of Molecular Size

  • GF matrix or prepacked GF column (see Table 10.9.4, Table 10.9.6, , and )
  • recipeGF fractionation buffer (see recipe; note variations for size determination)
  • GF chromatography column (Table 10.9.5)
  • High‐precision pump
  • Protein sample for size determination

Support Protocol 1: Column Calibration

  • recipeCalibration standards (see recipe and Table 10.9.8)
    Table 0.9.8   Additional Materials (also see protocol 3)   Additional MaterialsMolecular Size Standards for Calibration of Gel Filtration Columns

    Standard Molecular mass (M r) log 10(M r) Molecular size (radius, Å) Supplier
    Thyroglobulin, bovine thyroid 669,000 5.825 85.0 PB
    Ferritin, horse spleen 440,000 5.643 61.0 PB
    Catalase, bovine liver 232,000 5.365 52.2 PB
    Gamma globulin, bovine 158,000 5.199 BR
    Aldolase, rabbit muscle 158,000 5.199 48.1 PB
    Transferrin 81,000 4.908 SI
    Albumin, bovine serum 67,000 4.826 35.5 PB
    Ovalbumin, chicken 44,000 4.643 BR
    Ovalbumin, hen egg 43,000 4.633 30.5 PB
    Chymotrypsinogen A, bovine pancreas 25,000 4.398 20.9 PB
    Myoglobin, equine 17,500 4.243 BR
    Ribonuclease A, bovine pancreas 13,700 4.137 16.4 PB
    Non‐protein standard
    Vitamin B 12 1,350 3.130 BR
    Dextran T‐10 8,100 23 PB
    Dextran T‐40 23,600 40 PB
    Dextran T‐70 33,000 50 PB
    Dextran T‐500 370,000 150 PB
    Dextran 1 1,080 9 PC
    Dextran 5 4,440 18 PC
    Dextran 12 9,980 26 PC
    Dextran 25 21,400 39 PC
    Dextran 50 43,500 55.3 PC
    Dextran 80 66,700 68.4 PC
    Dextran 150 123,600 93.1 PC

     Abbreviations and symbols: —, Not stated by manufacturer; BR, Bio‐Rad; PB, Amersham Pharmacia Biotech; PC, Pharmacosmos; SI, Sigma. Addresses and phone numbers of suppliers are provided in appendix 44.
     The size of proteins may vary according to the source of information and the methodology used for determination—e.g., a value of 67.1 Å has also been assigned to ferritin (de Haen, ).
     Dextran is a polydisperse polymer and values given here—e.g., peak molecular mass—may vary from lot to lot. In cases where peak molecular mass is not stated by the supplier, the column is calibrated as elution volume versus M= (M w⋅M n)1/2, where M w is the weight‐average molecular mass and M n is the average molecular mass. The molecular size of dextran is calculated according to the equation R vis = 0.271 ×M0.498, where R vis is the viscosity radius and M is the molecular mass (Hagel, ).
  • 6 M guanidine hydrochloride (see recipe for recipeGF buffer)
  • In‐line refractive‐index detector
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  •   FigureFigure 10.9.1 Equipment used for gel filtration. (A) Simple setup for desalting using an open‐bed column made with a Pasteur pipet. (B) Column and attachments. (C) Complete automated chromatography system. Courtesy of Amersham Pharmacia Biotech.
  •   FigureFigure 10.9.2 Chromatogram illustrating the separating volume ( Vi) of gel filtration as determined by the void volume ( Vo) and the total liquid volume ( Vt).The total bed volume ( Vc) is equal to the total liquid volume plus the matrix volume. Column efficiency is calculated as N = 5.54( Vr/ Wh)2, where N is the efficiency of the column in terms of number of theoretical plates, Vr is the elution volume of the peak, and Wh is the peak width at half peak height. The symmetry of the peak is calculated at 10% peak height as b/a, where b is the width of the tailing part of the peak and a is the width of the leading part of the peak.
  •   FigureFigure 10.9.3 Chromatogram illustrating desalting of proteins by gel filtration. A 4 × 85–cm column packed with Sephadex G‐25 is used. The sample consists of 400 ml hemoglobin (protein peak; solid line) in NaCl (salt peak; broken line). Note that the sample volume is close to the pore volume of the packed matrix, i.e., 490 ml. No correction for sample volume has been made. Calculation from the figure yields Vo = 560 ml − 200 ml = 360 ml, which is 31% of the geometric column volume ( Vc). Reproduced from Flodin () with permission of the Journal of Chromatography.
  •   FigureFigure 10.9.4 Chromatogram illustrating protein fractionation by gel filtration. A Superdex 75 HR 10/30 column is used. The sample consists of recombinant human growth hormone. This figure is an enlarged section of the original chromatogram displayed to illustrate complete resolution between monomer and dimer. Reproduced from Hagel () with permission of the publisher. Courtesy of B. Pavlu, Kabi‐Pharmacia Peptide Hormones, and H. Lundström, Pharmacia Biotech.
  •   FigureFigure 10.9.5 Calibration of GF column for determination of protein size. A K 26/70 column packed with Sephadex G‐200 SF is used. (A) Chromatogram obtained for six size standards: 1, catalase; 2, aldolase; 3, bovine serum albumin; 4, ovalbumin; 5, chymotrypsinogen A; 6, ribonuclease A. (B) Calibration curve obtained by plotting the logarithms of the molecular radii (log10R) of the standards ( x axis) versus the elution volumes for peaks 1, 2, 4, 5, and 6 from panel A (filled circles). The curve was used for evaluating the size of bovine serum albumin (filled square; elution volume corresponds to peak 3 in panel A), giving an apparent size of 34.8 Å. The excellent agreement with the nominal molecular size value of 35.5 Å may be incidental. Repeating the procedure for peak 4 in panel A showed that a deviation of ∼3 Å from the nominal value may be expected for proteins of globular shape. Panel A reproduced from Pharmacia Biotech () with permission from the publisher.


Literature Cited

Literature Cited
   ASTM (American Society for Testing and Materials). 1978. Annual Book of ASTM Standards. ASTM D 3016‐78. American Society for Testing and Materials, Philadelphia.
   Andrews, P. 1970. Estimation of molecular size and molecular weights of biological compounds by gel filtration. In Methods of Biochemical Analysis, Vol. 18 (Glick, D., ed.) pp. 1‐53. Wiley‐Liss, New York.
   Casassa, E.F. 1967. Equilibrium distribution of flexible polymer chains between a macroscopic solution phase and small voids. J. Polym. Sci. Polymer Letters Part B 5:773‐778.
   de Haen, C. 1987. Molecular weight standards for calibration of gel filtration and sodium dodecyl sulfate–polyacrylamide gel electrophoresis: Ferritin and apoferritin. Anal. Biochem. 166:235‐245.
   Dubin, P.L., Kaplan, J.I., Tian, B.‐S., and Metha, M. 1990. Size‐exclusion chromatography dimensions for rod‐like macromolecules. J. Chromatogr. 515:37‐42.
   Flodin, P. 1961. Methodological aspects of gel filtration, with special reference to desalting operations. J. Chromatogr. 5:103‐115.
   Granath, K.A. and Flodin, P. 1961. Fractionation of dextran by the gel filtration method. Makromol. Chem. 48:160‐171.
   Hagel, L. 1985. Effect of sample volume on peak width in high‐performance gel filtration chromatography. J. Chromatogr. 324:422‐427.
   Hagel, L. 1989. Gel filtration chromatography. In Protein Purification: Principles, High Resolution Methods, and Applications (J.‐C. Janson and L. Rydén, eds.) pp. 63‐106. VCH Publishers, New York.
   Hagel, L. 1992. Peak capacity of columns for size‐exclusion chromatography. J. Chromatogr. 591:47‐54.
   Hagel, L. 1993. Size‐exclusion chromatography in an analytical perspective. J. Chromatogr. 648:19‐25.
   Hagel, L. and Andersson, T. 1991. A simple procedure for calibrating a column in solute radius. Presented at the 11th International Symposium on HPLC of Proteins, Peptides and Polynucleotides, October 20‐ 23, Washington D.C.
   Hagel, L. and Janson, J.‐C. 1992. Size‐exclusion chromatography. In Chromatography, 5th ed., part A: fundamentals and techniques (E. Heftmann, ed.) pp. A267‐A307. Elsevier/North‐Holland, Amsterdam.
   Hagel, L., Lundström, H., Andersson, T., and Lindblom, H. 1989. Properties, in theory and practice, of novel gel filtration media for standard liquid chromatography. J. Chromatogr. 476:329‐344.
   Lathe, G.H. and Ruthven, C.R.J. 1956. The separation of substances and estimation of their relative molecular size by the use of columns of starch in water. Biochem. J. 62:665‐674.
   Lindqvist, B. and Storgårds, T. 1955. Molecular‐sieving properties of starch. Nature 175:511‐512.
   Moore, J.C. 1964. Gel permeation chromatography. I. A new method for molecular weight distribution of high polymers. J. Polym. Sci. Part A 2:835‐843.
   Pharmacia Biotech. 1991. Gel Filtration Principles and Methods (5th ed.), Lund, Sweden.
   Potschka, M. 1987. Universal calibration of gel permeation chromatography and determination of molecular shape in solution. Anal. Biochem. 162:47‐64.
   Porath, J. and Flodin, P. 1959. Gel filtration: A method for desalting and group separation. Nature 183:1657‐1659.
Key References
   Yau, W.W., Kirkland, J.J., and Bly, D.D. 1979. Modern Size‐Exclusion Liquid Chromatography, Practice of Gel Permeation and Gel Filtration Chromatography. John Wiley & Sons, New York.
  Comprehensive overview of theory and use of gel filtration, covering the entire spectrum of applications (of which protein separation is only a small part). Describes in detail analytical determination of molecular size and size distribution of aqueous and nonaqueous polymers.
   Hagel, L. 1998. Gel filtration, In Protein Purification: Principles, High Resolution Methods, and Applications, 2nd ed. (J.‐C. Janson and L. Ryden, eds.) pp. 19‐144. John Wiley & Sons, New York.
  Describes practical implications of gel filtration theory, with special reference to proteins; a good complement to Yau et al. (), and particularly for protein chemists.
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