Large zone size exclusion

The purpose of this chapter is to review the principles of large zone size exclusion chromatography (large zone SEC) as applied to the study of self-association of macromolecules, specifically proteins. This selfassociation is not only of fundamental scientific interest, but also has practical consequences with respect to protein purification and to maintenance of biological function. The recent development of recombinant DNA technology has made available substantial amounts of sosie proteins, that were formerly available in very limited amounts if at all. These recombinant proteins must be obtained in a state that is fully biologically active. In some cases proteins are active as monomers, but in others the active protein exists as a dimer or an oligomer. Clearly it is of practical interest to understand the self-association of proteins under a variety of conditions. 

Size exclusion column chromatography (SEC), a physical method employed less widely than some of the other techniques, can be used in either an equilibrium or non-equilibrium (transport) mode. In the transport mode, the sample may be applied either in a small volume or in a large volume (large zone) relative to the volume of the column. Large zone size exclusion chromatography is the method that will be discussed in this chapter. 

A wealth of information can be gleaned from a large zone size exclusion experiment. By studying the behavior of the leading boundary over a range of concentrations, one can immediately ascertain whether a rapid equilibrium exists under the conditions being studied. If there is an equilibrium, the stoichiosietry of the self-association can be deduced, and finally the equilibrium constaut can be determined. 

An extensive discussion of the theoretical basis of large zone size exclusion chromatography has been published in a series of papers and review articles by Ackers and his co-workers (ref. 1-3). The goal here is not to present an exhaustive review of the theory behind this approach and the reader is referred to the references cited above. However, a brief overview of the basic principles will be given. The following discussion is a greatly simplified version of the theoretical discussion of Valdes and Ackers (ref. 2). 

Fig. 1. Chromatograms illustrating difference between a small zone (a) and a large zone (b) size exclusion experiment. In the saull zone experiment, the elution volume, V, is taken as the apex of the peak In a large zone experiment, the elutxon volume is the centroid volume, V, of the leading boundary. The shaded areas represent the volume loaded onto the column in each case.

With optical techniques, vibrational dynamics are probed on spatial scales much greater than molecular sizes, or unit cell dimensions in crystals, commonly encountered. The scale is directly related to the wavelength of the incident radiation (in the range fl om 1 to 10(X) p,m in the infrared or about 0.5 p,m for Raman). Oscillators at very short distances, compared to the wavelength, are excited exclusively in phase. For molecules, only overall variations of the dipole moment or polarizability tensor can be probed. In crystals, only a very thin slice of reciprocal space about the centte of the BriUouin zone (k 0) can be probed. This corresponds to in-phase vibrations of a virtuaUy infinite number of unit cells. With optical techniques, band intensities are largely determined by symmetry-related selection rules, although these rales hold only in the harmonic approximation. 

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