Separation of Protein Mixtures by Two Dimensional Techniques

Another broad field is the separation of complex protein mixtures. While separations using monodimensional techniques have had only limited success, applications of two-dimensional techniques are emerging (Giddings, 1984; Davis and Giddings, 1985; Giddings, 1987).

The first attempt at a two-dimensional approach involved the coupling of HPCE with HPLC. Because coupling of CZE with HPLC results in incompatibility of the time constants of the two separation steps, HPLC (gel permeation chromatography) should always be the first separation step, followed by the electrokinetic separation. Lemmo and Jorgenson (1993) developed such a system for two-dimensional separations, combining HPLC gel chromatography and CZE, and it exhibits practical applicability. The size exclusion was carried out in a 1 m X 250 mm microcolumn. The effluent from this size-exclusion chromatography microcolumn filled a sample loop on a computer-controlled six-port valve. The contents of the loop were transferred past the grounded end of the electrophoresis equipment capillary for electromigration injection. Detection was by UV absorbance at 214 nm. The success of this arrangement was based on the extremely fast separations in the HPCE section. The overall electrophoresis separation should take 4 minutes or less, to allow for sufficiently fast sampling of the gel column effluent.

Another problem for two-dimensional separations occurs when the first dimension involves silica-based size-exclusion chromatography of proteins, performed at high salt concentration, often of the order of 0.5 M. Such salt concentrations are not compatible with electrophoresis systems, which in most cases are run in an environment that includes 50 mAi buffer. To solve the problem, Zorbax GF 450, which has relatively low excess surface charge, was proposed as a suitable packing material for the size-exclusion step (Lemmo and Jorgenson, 1993). This addition permitted the size-exclusion separation step to be carried out at a low salt concentration, which was compatible with subsequent electrophoresis. However, this change limited the method to the separation of proteins with pi values less than 8.23. When the pi was higher, there was adsorption of proteins both to the size-exclusion column packing and to the capillary wall of the electrophoresis equipment, distorting the separations.

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