Abstract
In the past, reverse-phase high-performance liquid chromatography (RP-HPLC), which separates solutes based on hydrophobic interactions, has been established as the most commonly used method for separating peptides and proteins. Its advantages include excellent efficiency and resolving power along with reasonable elution times, and the availability of volatile mobile phases that eliminate the need for a desalting step. The application of this method, however, is limited when a low selectivity or the presence of complex protein mixtures does not permit satisfactory separation of individual components. In such cases, methods are successfully applied that are based on separation mechanisms other than hydrophobic interactions, like electrostatic (see Chapter 3) or hydrophilic interactions or different molecular size (Chapter 5).
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References
Alpert, A. J. (1990) Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. J. Chromatogr. 499, 177–196.
Lindner, H., Sarg, B., and Helliger, W. (1997) Application of hydrophilic-interaction liquid chromatography to the separation of phosphorylated H1 histones. J. Chromatogr. A. 782, 55–62.
Zhu, B.-Y., Mant, C. T, and Hodges, R. S. (1991) Hydrophilic-interaction chromatography of peptides on hydrophilic and strong cation-exchange columns. J. Chromatogr. 548, 13–24.
Zhu, B.-Y., Mant, C. T, and Hodges, R. S. (1992) Mixed mode hydrophilic and ionic interaction chromatography rivals reversed phase liquid chromatography for the separation of peptides. J. Chromatogr. 594, 75–86.
Oyler, A. R., Armstrong, B. L., Cha, J. Y, et al. (1996) Hydrophilic interaction chromatography on amino-silica phases complements reversed-phase high-performance chromatography and capillary electrophoresis for peptide analysis. J. Chromatogr. A. 724, 378–383.
Mant, C. T, Kandejewski, L. H., and Hodges, R. S. (1998) Hydrophilic interaction/cation-exchange chromatography for separation of cyclic peptides. J. Chromatogr. A. 816, 79–88.
Mant, C. T, Litowski, J. R., and Hodges, R. S. (1998) Hydrophilic interaction/cation-exchange chromatography for separation of amphipathic alpha-helical peptides. J. Chromatogr. A. 816, 65–78.
Litowski, J. R., Semchuk, P. D., Mant, C. T, and Hodges, R. S. (1999) Hydrophilic interaction/cation-exchange chromatography for the purification of synthetic peptides from closely related impurities: serine side-chain acetylated peptides. J. Peptide Res. 54, 1–11.
Lindner, H., Sarg, B., Meraner, C, and Helliger, W. (1996) Separation of acetylated core histones by hydrophilic-interaction liquid chromatography. J. Chromatogr. A. 743, 137–144.
Lindner, H., Sarg, B., Hoertnagl, B., and Helliger, W. (1998) The microheterogeneity of the mammalian H1(0) histone-Evidence for an age-dependent deamidation. J. Biol. Chem. 273, 13,324–13,330.
Mizzen, C. A., Alpert, A. J., Levesque, L., Kruck, T. P. A., and McLachlan, D. R. (2000) Resolution of allelic and non-allelic variants of histone H1 by cation-exchange-hydrophilic-interaction chromatography. J. Chromatogr. B 744(1), 33–46.
Feste, A.S. and Khan, I. (1993) Separation of glucooligosaccharides and polysaccharide hydrolysates by gradient elution hydrophilic interaction chromatography with pulsed amperometric detection. J. Chromatogr. 630, 129–139.
Yu, J. and El Rassi, Z. (1994) Preparation of amino-zirconia bonded phases and their evaluation in hydrophilic interaction chromatography of carbohydrates with pulsed amperometric detection. J. High Resolut. Chromatogr. 17, 773–778.
Alpert, A. J., Shukla, M., Shukla, A. K., et al. (1994) Hydrophilic interaction chromatography of complex carbohydrates. J. Chromatogr. A. 676, 191–202.
Churms, S. C. (1996) Recent progress in carbohydrate separation by high-performance liquid chromatography based on hydrophilic interaction. J. Chromatogr. A. 720, 75–91.
Zhang, J. and Wang, D. I. C. (1998) Quantitative analysis and process monitoring of site-specific glycosylation microheterogeneity in recombinant human interferon-gamma from Chinese hamster ovary cell culture by hydrophilic interaction chromatography. J. Chromatogr. B. 712, 73–82.
Alpert, A. A. (1983) Cation-exchange high-performance liquid chromatography of proteins on poly(aspartic acid)-silica. J. Chromatogr. 266, 23–37.
Alpert, A. A. (1988) Cation-exchange chromatography of peptides on poly(2-sulfoethyl aspartamide)-silica. J. Chromatogr. 443, 85–96.
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© 2004 Humana Press Inc.
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Lindner, H., Helliger, W. (2004). Hydrophilic Interaction Chromatography. In: Aguilar, MI. (eds) HPLC of Peptides and Proteins. Methods in Molecular Biology™, vol 251. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-742-4:75
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DOI: https://doi.org/10.1385/1-59259-742-4:75
Publisher Name: Springer, Totowa, NJ
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