Hydrophobic‐Interaction Chromatography

Robert M. Kennedy1

1 Pharmacia Biotech, Piscataway, New Jersey
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 8.4
DOI:  10.1002/0471140864.ps0804s00
Online Posting Date:  May, 2001
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Abstract

This unit discusses the important parameters in designing and optimizing a separation by hydrophobic interaction chromatography (HIC), including preparing the sample and choosing a matrix, column, and buffer. Protocols are provided for packing and testing a column; determining binding and elution conditions; eluting the sample; and cleaning, regenerating, and storing HIC columns.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Packing a Column with an HIC Matrix of ≥90‐µm Beads
  • Alternate Protocol 1: Packing a Column with an HIC Matrix of ≤34‐µm Beads
  • Basic Protocol 2: Testing the Packed Bed
  • Support Protocol 1: Pilot Experiment to Choose a Matrix and Determine Binding and Elution Conditions for HIC
  • Basic Protocol 3: Elution of Proteins from HIC Columns
  • Support Protocol 2: Regeneration, Cleaning, and Storage of HIC Columns
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Packing a Column with an HIC Matrix of ≥90‐µm Beads

  Materials
  • Appropriate HIC matrix with bead size ≥90 µm (see and protocol 4)
  • Packing solution: distilled H 2O or low‐ionic‐strength buffer (e.g., 20 mM sodium phosphate, pH 7.0)
  • 20% (v/v) ethanol
  • Chromatography column with flow adaptors (see )
  • Sintered‐glass funnel (medium or coarse grade)
  • Carpenter's level
  • Pump (see )

Alternate Protocol 1: Packing a Column with an HIC Matrix of ≤34‐µm Beads

  • Appropriate HIC matrix with bead size ≤34 µm (see and protocol 4)
  • Tween 20
  • Chromatography column rated at 5 bar, with flow adaptors (see )
  • Pressure gauge

Basic Protocol 2: Testing the Packed Bed

  Materials
  • HIC matrices to be tested (e.g., a low‐substitution phenyl, a higher‐substitution phenyl, a butyl, and an octyl matrix; Table 8.4.1)
  • General start buffer: 50 mM Na 2HPO 4/1.0 M (NH 4) 2SO 4, pH 7.0 (prepare ∼1 liter)
  • 5 to 10 mg/ml solution of protein mixture to be purified, in general start buffer
  • 50 mM Na 2HPO 4containing 0.75 M, 0.50 M, 0.25 M and 0 M (NH 4) 2SO 4 (for step gradient only; all solutions at pH 7.0; prepare 1 liter each)
  • 1‐ml chromatography columns
  • Chromatography system with syringes or pump, detector, and test tubes or fraction collector (see )
  • Gradient mixer (unit 8.2) set to deliver descending gradient from 1.0 M to 0 M (NH 4) 2SO 4 in 10 column volumes of Na 2HPO 4, pH 7.0 (for continuous gradient only; if gradient mixer is not built into chromatography system)
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Figures

Videos

Literature Cited

Literature Cited
   Englard, S. and Seifter, S. 1990. Precipitation techniques. Methods Enzymol. 182:285‐300.
   Ericksson, K.‐O. 1989. Hydrophobic interaction chromatography. In Protein Purification: Principles, High Resolution Methods and Applications. (J.‐C. Janson and L. Rydén, eds.) pp. 207‐226. VCH Publishers, New York.
   Franks, F. 1988. Characterization of Proteins. In Characterization of Proteins. (F. Franks, ed.) p. 53. Humana Press, Clifton N.J.
   Hereld, D., Krakow, J.L., Bangs, J.D., Hart, G.W., and Englund, P.T. 1986. A phospholipase C from Trypanosoma brucei which selectively cleaves the glycolipid on the variant surface glycoprotein. J. Biol. Chem. 261:13813‐13819.
   Hjerten, S. 1977. Fractionation of proteins by hydrophobic interaction chromatography, with reference to serum proteins. In Proceedings of the International Workshop on Technology for Protein Separation and Improvement of Blood Plasma Fractionation. pp. 410‐421. Reston, Virginia.
   Hodder, A.N., Aquilar, M.I., and Hearn, M.T.W. 1989. High performance liquid chromatography of amino acids, peptides and proteins LXXXIX. The influence of different displacer salts on the retention properties of proteins separated by gradient anion exchange chromatography. J. Chromatogr. 476:391‐411.
   Klotz, I.M. 1970. Comparison of molecular structures of proteins: Helix content, distribution of apolar residues. Arch. Biochem. Biophys. 138:704‐706.
   Lau, K.H., Freeman, T.K., and Baylink, D.J. 1987. Purification and characterization of an acid phosphatase that displays phosphotyrosyl‐protein phosphatase activity from bovine cortical bone matrix. J. Biol. Chem. 262:1389‐1397.
   Lee, B. and Richards, F.M. 1971. The interpretation of protein structures: Estimation of static accessibility. J. Mol. Biol. 55:397‐400.
   Melander, W. and Horvath, C. 1977. Salt effects on hydrophobic interactions in precipitation and chromatography of proteins: An interpretation of the lyotropic series. Arch. Biochem. Biophys. 183:200‐215.
   Porath, J., Sundberg, L., Fornstedt, N., and Olsson, I. 1973. Salting‐out in amphiphilic gels as a new approach to hydrophobic adsorption. Nature 245:465‐466.
   Porath, J. 1986. Salt promoted adsorption: Recent developments. J. Chromatogr. 376:331‐341.
   Roos, P., Nyberg, F., and Wide, L. 1979. Isolation of human pituitary prolactin. Biochim. Biophys. Acta. 588:368‐379.
   Scopes, R.K. 1994. Protein Purification: Principles and Practice, 3rd ed. Springer‐Verlag, New York.
   Segel, I.H. 1976. Biochemical Calculations. John Wiley & Sons, New York.
   Sparrman, M. 1986. Purification of monoclonal antibodies by hydrophobic interaction chromatography. Presented at the 6th International Symposium on HPLC of Proteins, Peptides, and Polynucleotides. Baden‐Baden, Germany.
   Srinivasan, R. and Ruckenstein, E. 1980. Role of physical forces in hydrophobic interactions chromatography. Sep. Purif. Methods 9:267‐370.
   Tiselius, A. 1948. Adsorption separation by salting out. Arkiv för Kemi, Mineralogi, Geologi 26B:1‐5.
   Visser, J. and Strating, M. 1975. Separation of lipoamide dehydrogenase isoenzymes by affinity chromatography. Biochim. Biophys. Acta. 384:69‐80.
   von der Haar, F. 1976. Fractionation of proteins by fractional interfacial salting out on unsubstituted agarose gels. Biochem. Biophys. Res. Commun. 70:1009‐1013.
Key Reference
   Ericksson, 1989. See above.
  Describes both the theory and practice of hydrophobic‐interaction chromatography and includes a number of references to the theoretical as well as the applications literature.
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