Chromatography protein phases

Walhagen, A., Edholm, L.E. (1989). Coupled-column chromatography on immobilized protein phases for direct separation and determination of drug enantiomers in plasma. J. Chromatogr. 473, 371-379. 

These systems rely on various combinations of size-exclusion chromatography, reversed-phase chromatography, and zone electrophoresis to characterize amines, peptides, and proteins (Yamamoto etal., 1989 Bushey and Jorgenson 1990 Larmann et al., 1993, Moore and Jorgenson, 1995 Optick and Jorgenson, 1997). Haleem Issaq reviews these separations in Chapter 16 of this book. 

Ahamed, T., Ottens, M., van Dedem, G.W.K., van der Widen, L.A.M. (2005). Design of self-interaction chromatography as an analytical tool for predicting protein phase behaviour. Journal of Chromatography A, 1089, 111-124... 

In immunoaffinity chromatography adsorption phase is performed in physiological conditions followed by a wash with high ionic strength buffers to eliminate nonspecifically adsorbed proteins. Elution of human IgG is obtained by a deforming buffer such as a 0.2 M glycine-HCl, pH 2.7. 

Analyses of phase boundaries reveal evidence for polymer saturation in the presence of excess protein. Phase boundaries also facilitate comparisions of the behavior of various proteins. The failure of net surface charge density as a universal parameter for protein-polyelectrolyte interaction is believed to be related to the existence of "charge patches" on the protein surface. The determination of a more realistic protein charge parameter possesses great importance, since the ionic interactions of proteins are exploited in a variety of applications, including protein purification via ion exchange liquid chromatography. 

Names such as gel filtration chromatography (mobile phase is water), used by biochemists, and gel permeation chromatography (mobile phase is an organic solvent), used by polymer chemists, describe this technique. Size exclusion chromatography, however, is the recommended term. Molecular weight distribution of polymers can be obtained by this technique, and proteins, enzymes, peptides, nucleic acids, hormones, polysaccharides, and so on can be separated. 

Liquid Chromatography Stationary phase Amino Acid Derivatives Low-Mass Synthetic Selectors Poly(saccharide) Derivatives Cyclodextrin Derivatives Glycopeptides Metal Chelates Proteins Helical Polymers... 

Proteins High Performance Liquid Chromatography Reverse Phase Liquid Chromatography 44 ... 

Sample preparation and extraction procedures are an important prepurification step. Different methods are used in the purification of a target protein, such us precipitation, preparative centrifugation, two-phase systems, electrophoresis, or chromatography. In chromatography, proteins are separated on the basis of their different affinity to a stationary or a mobile phase. 

The disadvantage of precolumn derivatization is increased manipulation of the sample before it can be chromatographed. Most autosamplers today can automate the derivatization reaction, thus freeing lab personnel for other activities. However, the derivatization reaction will usually not occur on the still very acidic hydrolyte. This necessitates the prior removal of the acid and water from the sample. This is commonly done by a vacuum. To completely remove the acid from the sample, the sample often has to be dried, ledissolved (usually in water), and then dried a second time. Once dried, the sample is ledissolved using a solvent compatible with the derivatization reaction. The additional time required by these steps can offset the savings achieved by using reversed-phase chromatography. Gas-phase hydrolysis of pure proteins eliminates the need to remove the acid from the sample. 

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