FAST, HIGH-RESOLUTION (CAPILLARY) ELECTROPHORESIS IN BUFFERS DESIGNEDFOR HIGH-FIELD STRENGTHS

Capillary electrophoresis in conventional buffers and in 50 mu m capil laries permits field strengths as high as 300-500 V/cm with acceptably low thermal zone deformation. However, still higher field strengths ( up to at least 2000 V/cm) can be applied without a decrease in resolut ion if the experiments are performed in the buffers described in this paper. Characteristic of these buffers is their low electrical conduct ivity and yet satisfactory buffering capacity accomplished either (i) by selecting buffer constituents of relatively high molecular weight a nd small net charge or (ii) by fractionation of carrier ampholytes (or iginally introduced for isoelectric focusing experiments) into a serie s of narrow pH range fractions and using these fractions as buffers, o r (iii) by selecting an ampholyte with two acidic groups and one basic group (or one acidic group and two basic groups) and with a pi value close to two of its pK values. In such buffers, aromatic carboxylic ac ids and proteins used as model substances could be analyzed rapidly. F or instance, albumin and transferrin were separated at 30000 V (1.99 m u A) in 15 cm long fused silica capillaries (50 mu m ID) within 40 s a nd the carboxylic acids within 25 s. The resolution was similar to tha t obtained at standard voltage (5000 V; 0.33 mu A), but the analysis t ime was reduced sixfold. Although not verified experimentally we also suggest the use of relatively high-molecular-weight polyoxyethylene de rivatized with one acidic group (for instance, boric acid) and one bas ic group (an amine), both having the same pK value, which should affor d both a very high buffering capacity and very low electrical conducti vity (at low buffer concentrations).