Protein separation, temperature effect

Cohen, K. A., Schellenberg, K., Benedek, K., Karger, B. L., Grego, B., and Hearn, M. T. W., Mobile-phase and temperature effects in the reversed phase chromatographic separation of proteins, Anal. Biochem., 140, 223, 1984. 

As is evident from the preceding discussion, the retention behavior of a polypeptide or protein P- expressed in terms of the capacity factor k is governed by thermodynamic considerations. Peak dispersion, on the other hand, arises from time-dependent kinetic phenomena, which are most conveniently expressed in terms of the reduced plate height he, . When no secondary effects, i.e., when no temperature effects, conformational changes, slow chemical equilibrium, pH effects, etc. occur as part of the chromatographic distribution process, then the resolution Rs, that can be achieved between adjacent components separated under these equilibrium or nearequilibrium conditions can be expressed as... 

There are several reasons to measure the effect of pressure on a wide variety of thermodynamic systems. Perhaps the most important argument is that one can separate the effects of volume and thermal energy changes, which appear simultaneously in temperature experiments . Moreover, high pressure can induce unfolding of protein in a different way from thermal denaturation. The pressure studies have considerably increased in the last decades" . 

However, an analogy with condensed matter physics is useful. If one simply measures the resistivity of some material at room temperature, it is not possible to understand the mechanism responsible for impeding the flow of electrons because there are many contributions to the resistivity, from phonons, defects, and other electrons. However, studies as a function of a temperature can separate the effects of phonons, defects, and interaction with other electrons because the temperature dependence of each of these contributions is characteristically different. We believe that the approach of condensed matter physicists has much to teach us in biophysics, particularly in the study of proteins in which the reaction... 

Many factors, such as solvent viscosity, diffusion rates, and column type, contribute to temperature effects in protein RP-HPLC. Temperature may also directly affect protein conformation or aggregation. For example, whereas fresh extracts of maize zein resolved well upon RP-HPLC [42], even at low temperature, purified zeins, especially after lyophilization and storage, separated poorly [53] (Fig. 5). Resolution was restored, however, at 70 C. These results suggest that zeins may aggregate and polymerize via hydrogen bonds between glutamine... 

An increase in temperature usually decreases retention in RPC. However, since temperature changes may also affect protein structure the effect on RPC behaviour of a protein is less predictable—it may, for instance, lead to an increase in retention. Such changes may be manipulated to improve separation (see Section 8.1) or may be a source of irreproducibility which may be eliminated by thermostatting column and injector. 

A. Gerstner, Z. Csapo, M. Sasvari-Szekely, and A. Guttman, Ultrathin sodium dodecyl sulfate gel electrophoresis of proteins Effect of gel composition and temperature on the separation of sodium dodecyl sulfate-protein complexes, Electrophoresis, 21, 834 (2000). 

Chloupek, R.C., Hancock, W.S., Marchylo, B.A., Kirkland, J.J., Boyes, B.E., Snyder, L.R. (1994). Temperature as a variable in reversed-phase high-performance liquid chromatographic separations of peptide and protein samples, n. Selectivity effects observed in the separation of several peptide and protein mixtures. J. Chromatogr. A 686, 45-59. 

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