High-performance liquid chromatography of proteins

Most of the chromatographic techniques described thus far are usually performed under relatively low pressures, where flow rates through the column are generated by low-pressure pumps (i.e. low-pressure 

One approach that allows increased chromatographic flow rates without loss of resolution entails the use of microparticulate stationary-phase media of very narrow diameter. This effectively reduces the time required for molecules to diffuse in and out of the porous particles. Any reduction in particle diameter dramatically increases the pressure required to maintain a given flow rate. Such high flow rates may be achieved by utilizing high-pressure liquid chromatographic systems. By employing such methods, sample fractionation times may be reduced from hours to minutes. 

The successful application of HPLC was made possible largely by (a) the development of pump systems that can provide constant flow rates at high pressure and (b) the identification of suitable pressure-resistant chromatographic media. Traditional soft gel media utilized in low-pressure applications are totally unsuited to high-pressure systems due to their compressibility. 

Many small proteins, in particular those that function extracellularly (e.g. insulin, GH and various cytokines) are quite stable and may be fractionated on a variety of HPLC columns without significant denaturation or decrease in bioactivity. Preparative HPLC is used in industrial-scale purification of insulin and of IL2. In contrast, many larger proteins (e.g. blood factor VIII) are relatively labile, and loss of activity due to protein denaturation may be observed upon high-pressure fractionation. 

At both preparative and analytical levels, HPLC exhibits several important advantages compared with low-pressure chromatographic techniques  

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SynChropak GPC supports were introduced in 1978 as the first commercial columns for high-performance liquid chromatography of proteins. SynChropak GPC columns were based on research developed by Fred Regnier and coworkers in 1976 (1,2). The first columns were only available in 10-yu,m particles with a 100-A pore diameter, but as silica technology advanced, the range of available pore diameters increased and 5-yu,m particle diameters became available. SynChropak GPC and CATSEC occasionally were prepared on larger particles on a custom basis, but generally these products have been intended for analytical applications. 

Torres, A. R., Edberg, S.C., and Peterson, E. A., Preparative high-performance liquid chromatography of proteins on an anion exchanger using unfractionated carboxymethyl displacers, /. Chromatogr., 389, 177, 1987. 

Lee, A. L., Liao, A. W., and Horvath, Cs., Tandem separation schemes for preparative high-performance liquid chromatography of proteins,. Chromatogr., 443, 31, 1988. 

Coupek, J. and VinS, I., Hydroxyethyl methacrylate-based sorbents for high-performance liquid chromatography of proteins, /. Chromatogr. A, 658, 391, 1994. 

Regnier, E E. and Gooding, K. M., High-performance liquid chromatography of proteins, Anal. Biochem., 103, 1, 1980. 

Bushey, M.M., Jorgenson, J.W. (1990). Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography of proteins. Anal. Chem. 62, 161-167. 

Mal tsev, V.G., Nasledov, D.G., Trushin, T.B., Tennikova, L.V., Vinogradova, I.N., Volokitina, I.N., Zgonnik, V.N. (1990). High-performance liquid chromatography of proteins on short capillary columns. J. High Resolut. Chromatogr. 13, 185-192. 

Harris, D. A. (1992) Size-exclnsion high-performance liquid chromatography of proteins, in Methods in Molecular Biology, vol. 11 Practical Protein Chromatography (Kenney, A. and Powell, S., eds.), Humana, Clifton, NJ, pp. 223-236. 

Goheen, S.C. and Engel horn, S.C. 1984. Hydrophobic interaction high-performance liquid chromatography of proteins. J. Chromatogr. 317 55-65. 

T. D. Schlabach, C. T. Wehr. In T. W. Hearn, F. E. Regnier, C. T. Wehr, eds. HPLC of proteins and peptides. Proceedings of the First International Symposium on High-Performance Liquid Chromatography of proteins and peptides. Academic Press, London, p 221 (1983). 

M de Frutos, A Cifuentes, JC Diez-Masa. Multiple peaks in high-performance liquid chromatography of proteins /3-lactoglobulins eluted in a hydrophobic interaction chromatography system. J Chromatogr A 778 43-52, 1997. 

Protein Fv was isolated and purified by Jean-Pierre Bouvet et al. [1] in 1990 from stool extracts obtained from 43% of patients affected by viral hepatitis C (HCV) and from 35% of patients with hepatitis B (HBV). This factor (originally called protein F ) was found in only 6.7% of healthy subjects. The original name soon became protein Fv (for Fv fragment) because Bouvet and Pillot [2] provided evidence that this factor binds to the variable domain of the heavy chain of immunoglobulins. Molecular sieving by high performance liquid chromatography of protein Fv indicated an apparent molecular... 

For examples of peptide analysis using these techniques, see (a) Hean, M.T.W. Regnies, F.E. Wehr, C.T (Eds.), High Performance Liquid Chromatography of Proteins and Peptides, Academic Press, New York, 1983 (b) Hean, M.T.W. 

Regnier. F.E. (1983b). High Performance Liquid Chromatography of Proteins. In Hirs, C.H.W. and Timasheff, S.N. (eds). Methods in Enzymology, Vol. 91. Enzyme Structure Part 1. Academic Press. New York and London. 

Winkler G, Briza P, Kunz C (1986). Spectral properties of some ion-pairing reagents commonly used in reversed-phase high-performance liquid chromatography of proteins and peptides in acetonitrile gradient systems. J. Chromatogr. A. 361 191-198. 

Yao, K. Hjerten, S. Gradient and isocratic high-performance liquid chromatography of proteins on a new agarose-based anion exchanger. J. Chromatogr. 1987, 385, 87-98. 

Reif OW, Freitag R. Characterization and application of strong ion-exchange membrane adsorbers as stationary phases in high-performance liquid chromatography of proteins. J Chromatogr 1993 654 29-41. 

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