Detailed discussion of the use of HPCE for separation of proteins are available in specialized reviews (Karger, 1989; Rohlicek and Deyl, 1989; Steuer et al., 1990; Mazzeo and Krull, 1991; Deyl et al., 1994a, 1994b; Li, 1994).
In general, three methods may be used for protein separation by HPCE. It is possible by exploiting the charge differences to separate proteins according to their effective charge in acid or alkaline media. Acid buffers are usually preferred for complex protein mixtures, even though the runs may easily take an hour because of the decreased EOF. In alkaline pH the peaks as a rule get sharper and the runs shorter (about 30 min), but fused peaks are frequently observed. The other possibility is to exploit the differences in molecular dimensions and perform protein separations in gel-filled capillaries. Here the separations can be related to molecular mass. The advantage of these separations is an easy interpretation; the disadvantage is that most gel-filled capillaries can withstand only a limited number of runs. This disadvantage has been overcome by using replaceable gels, where the sieving medium is simply added to the run buffer (Cohen et al., 1993; Werner et al., 1993). The third possibility is to add suitable ampholytes to the background electrolyte and to run the capillary under capillary isoelectric focusing (CIEF) conditions. In this operating mode, proteins, after they have been focused, stop, and their zones sharpen. To reach this stage, EOF must be abolished by suitable coating of the capillary (Yao and Regnier, 1993).
Protein in untreated capillaries tend to stick to the inner capillary walls. Several approaches have been used to abolish or at least minimize this problem (McCormick, 1988; Bushey and Jorgenson, 1989; Green and Jorgenson, 1989; Emmer et al., 1991; Kajiwara, 1991). The easiest approach is to perform the separations at very high or very low pH values. In the former case, dissociation of the free amino groups is suppressed and, consequently, interactions between the dissociated silanol groups of the capillary surface and the free amino groups of the protein are minimized. At extremely acid pH, the opposite
effect can be introduced; namely, hindrance of dissociation of the silanol groups of the capillary, accompanied by nearly complete dissociation of the free amino groups of the proteins. As a result, the ionic interactions between the protein and the capillary wall are minimized.
Other techniques for suppressing or decreasing the dissociation of both the separated solutes and silanol groups of the capillary include increasing the ionic strength of the run buffer—for example, by adding metal salts (Green and Jorgenson, 1989). However, the increase of conductivity resulting from higher salt concentrations in the buffer requires the use of lower voltages and capillaries of smaller diameter, to provide adequate heat dissipation. Another way of avoiding protein adsorption to the capillary wall is to use zwitterionic buffers, which permit the use of high salt concentrations without excessive heating during the separation (Bushey and Jorgenson, 1989).
Hydrophobic domains in protein molecules may constitute another cause of adsorption to capillary walls. Such interactions can be abolished by using fluorosurfactant buffer additives; however, charge reversal can occur at the surface of the capillary (Epimer el al., 1991). Proteins at a pH below their pi are repelled from the wall. High efficiency separations can be obtained in this arrangement at low ionic strengths. Hydrophobic interaction electrophoresis can be performed in the presence of amphophilic polymers (e.g., stearoyl dextran), together with ethylene glycol. These additives change the degree of the hydrophobic interaction between the protein and the capillary wall, improving resolution (Kajiwara, 1991).
Alternatively, protein sticking may be prevented by modification of the capillary surface. Successful separations can be obtained with capillary columns coated with glycidoxy-propyltrimethoxysilane and polyethylene glycol (Bruin et al., 1989). This approach is limited to separations at pH 3 to 5 because at higher pH values the coatings becomes unstable. Other coatings are represented by polyethyleneimine (Towns and Regnier, 1990) and treatment with Tween and Brij (Towns and Regnier, 1991). These coatings can be used successfully over a wide pH range, typically 3 to 15. The deactivation of the capillary surface through aryl pentafluoro modifiers represents another possibility (Swedberg, 1990; Maa et al., 1991). Also C8 and Ci8 modifications have been successfully used (Dougherty et al., 1991). Modification of the inner surface of the capillary with vinyl-bonded polyacrylamide (Cobb et al., 1990) not only eliminates sorption of the separated proteins but eliminates the EOF as well. Similar effects were observed with polymethylglutamate-coated capillaries (Bentrop et al., 1991). Hydrophilic coatings with hydroxylated poly-ether functionalities represent another way of successful shielding of the capillary wall (Nashabeh and El Rassi, 1991).
In using this approach to prevent sticking, the problem of coating stability must be considered. Therefore the procedure referred to as dynamic coating was introduced (Wu and Regnier, 1991). A number of commercially available coated capillaries are currently marketed.
Protein sorption to the capillary wall is not an issue with gel-filled capillaries because the wall is shielded by the gel filling. Gel-filled capillaries can be either prepared in the lab or purchased. An easy and inexpensive approach is the dynamic filling of the capillary with a diluted gel solution (typically 4% linear polyacrylamide) (Wu and Regnier, 1992).
Whereas in free capillaries the addition of SDS to protein samples before separation results in a loss of resolution, with gel-filled capillaries the runs can be performed in the presence of SDS, and separations result from the sieving effect of the gel.
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