Chromatography, of proteins

S. Yamamoto, K. Nakanishi and R. Matsuno, Ion-exchange Chromatography of Proteins, Chromatographic Science Series Vol. 43, Marcel Dekker, New York, 1988. ISBN 0824779037. 

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. 

Protein-Pak packings are designed for the size exclusion chromatography of proteins and related compounds. They are based on silica, which is deactivated with glycidylpropylsilane. The diol function prevents the interaction of the target analytes with the silica surface. However, because coverage of the silica surface is always incomplete, residual acidic silanols can interact with the analytes. For this reason, most applications are carried out with a salt concentration above 0.2 mol/liter, which eliminates the interaction of analytes with surface silanols. Protein-Pak packings are stable from pH 2 to pH 8. 

This section discusses in detail the column types that are available for the size exclusion chromatography of both polar and nonpolar analytes. It first discusses the various columns available for standard nonaqueous size exclusion chromatography. It then reviews the columns available for general size exclusion chromatography using aqueous mobile phases. Finally, it examines the columns designed for size exclusion chromatography of proteins and peptides. 

Special Considerations for the Size Exclusion Chromatography of Proteins... 

For the size exclusion chromatography of proteins on silica-hased diol packings, it is generally recommended to use fully aqueous mobile phases with a salt concentration between 0.1 and 0.3 M. In general, a phosphate buffer around pH 7 is used as the mobile phase. Under these circumstances, the tertiary structure of most proteins is preserved without difficulty and the interaction of proteins with each other is minimized. However, other inorganic buffers or combinations of buffers with organic solvents can be used without difficulties for special applications. 

Fig. 3. Cation-exchange chromatography of protein standards. Column poly(aspartic acid) Vydac (10 pm), 20 x 0.46 cm. Sample 25 pi containing 12.5 pg of ovalbumin and 25 pg each of the other proteins in the weak buffer. Flow rate 1 ml/min. Weak buffer 0.05 mol/1 potassium phosphate, pH 6.0. Strong buffer same +0.6 mol/1 sodium chloride Elution 80-min linear gradient, 0-100% strong buffer. Peaks a = ovalbumin, b = bacitracin, c = myoglobin, d = chymotrypsinogen A, e = cytochrom C (reduced), / = ribonuclease A, g = cytochrome C (oxidised), h = lysozyme. The cytochrome C peaks were identified by oxidation with potassium ferricyanide and reduction with sodium dithionite [47]...

Ion-Exchange Chromatography of Proteins, Shuichi Yamamoto, Kazuhiro Naka-nishi, and Ryuichi Matsuno... 

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Figure 18 Very-high-speed gradient anion exchange chromatography of proteins. Column 0.46 x 3.5 cm ZipSep AX, 3 p. Eluent Tris-HCl, pH 8.0, operated on a gradient from 0-0.5 M NaCl. Flow rate 2ml/min. Detection UV absorbance at 280 nm. (1) Ribonuclease A, (2) carbonic anhydrase, (3) conalbumin, (4) bovine serum albumin. (Reproduced from Hatch, R. G., J. Chromatogr. Sci., 31, 469,1993. By permission of Preston Publications, A Division of Preston Industries, Inc.)...

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Hatch, R. G., Very-high-speed anion-exchange chromatography of proteins using monodisperse nonporous polymer particles, /. Chromatogr. Sci., 31, 469 1993. 

Kundu, A., Shikla, A. A., Barnthouse, K. A., Moore, J., and Cramer, S. M., Displacement chromatography of proteins using sucrose octasulfate, BioP-harm., 64, May 1997. 

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Xu, W. and Regnier, F. E., Electrokinetically driven cation-exchange chromatography of proteins and its comparison with pressure-driven high-performance liquid chromatography, /. Chromatogr. A, 853, 243, 1999. 

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

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