New approaches in the design of capillary electrophoresis experiments.

This paper gives a survey of recent methodological studies of capillary electrophoresis at the Department of Biochemistry, Uppsala University. Several methods for on-and off-tube concentration of solutes have been developed. These methods also permit desalting of the sample, which is necessary for the creation of narrow starting zones in zone electrophoresis and to reduce the risk of precipitation and narrowing of the separation window in isoelectric focusing (IEF). A unique method designed particularly for desalting of samples for IEF is also discussed. To obtain high resolution the adsorption of solutes to the capillary wall must be eliminated. A pH stable, hydrophilic polymer coating has, therefore, been developed. Although polymer solutions give lower resolution than do gels they are often used as molecular-sieving media because they are replaceable and thus permit repetitive automated analyses in the same capillary. We have shown that low-melting methoxylated agarose gels are also replaceable and give the same high resolution as do polyacrylamide and dextran gels. Following an electrophoresis the methoxylated agarose gels can be displaced in the stationary capillary past the detection window. This scanning technique has several advantages. A capillary with strong electroendosmosis permits, in principle, separation of both acidic and basic proteins in one run. However, depending on whether the capillary wall is negatively or positively charged either the basic or the acidic proteins most often migrate with strong tailing caused by the electrostatic interaction with the capillary wall. The distortion of the zones is much less if the electroendosmotic flow is replaced by a hydrodynamic flow and the electrophoresis is performed in a coated electroendosmosis-free capillary to suppress adsorption of both basic and acidic proteins. By a unique technique we can prepare chromatographic beds with diameters as small as 5-25μm. These beds, which are more homogeneous than conventional beds built up of preformed beads, can be used with advantage for electrochromatography and for different modes of capillary chromatography of fractions obtained from micropreparative HPCE experiments. There is a general trend and desire to decrease the analysis time without sacrificing resolution. Therefore, we have developed low-conductivity buffers with satisfactory buffering capacity which permit field strengths as high as 2, 000V/cm with attendant very short run times. Many enantioselective agents gave strong UV-absorption. Therefore, the experimental conditions must be chosen so that they do not pass the detection window. Otherwise, the noisy background is very disturbing. When a zone migrates from a straightinto a curved section of a capillary the molecules at the outer lane will lag behind those at the inner lane. The attendant loss in resolution, which all commercial apparatus exhibits, can be decreased considerably by coiling the capillary into the figure-of-eight or a serpentine path. A straight capillary is, however, preferable. Methods for the determination of pH and electrical conductivity in small volumes are discussed. A technique for micropreparative HPCE and HPLC is presented. An HPCE apparatus designed according to new principles is described (straight capillary; one electrode vessel is closed to avoid hydrodynamic flow in the capillary; electroendosmotic pump for washing of the capillary; thermostated slit for the UV beam to obtain a straight base line; only a small part of the capillary is not cooled actively; thermal application of the sample; the sample rests as a small droplet on a carousel; the droplet is covered by a cap which can be lifted by a magnet; effective insulation for field strengths up to at least 2, 000V/cm).