New isoelectric buffers for capillary electrophoresis: N-carboxymethylatedpolyethyleneimine as a macromolecular isoelectric buffer

Isoelectric buffers are attractive for electrophoresis because of their low conductivity, and their compatibility with indirect photometric detection in capillary electrophoresis (CE) where they do not interfere with the dete ction by exhibiting competitive displacement of the UV-absorbing probe ion. N-carboxymethylated polyethyleneimine (CMPEI) was prepared by introducing a half molar equivalent of carboxylate groups onto a polyethyleneimine back bone. Its isoelectric point determined by conductometric titration and from the pH of its dilute aqueous solution is approx. 6.8, which allows isoelec tric buffering at a lower pH compared to histidine (pI 7.7). Although the i soelectric point is somewhat diffuse, as expected for a polymeric compound, it exhibits a buffering capacity at a pI point of about twice that of hist idine, Studies of electroosmotic now (EOF) profile at various pH values in fused silica capillaries showed that CMPEI adsorbs onto the fused silica wa ll and reverses the EOF at pH < 6.5. CMPEI was applied as a buffer in an el ectrolyte containing 0.5 mM of the anionic dye tartrazine used as the probe for indirect detection of anions. The separation system exhibited a stable baseline, no system peaks, separation efficiencies of up to 195 000 theore tical plates, and detection limits down to 0.2 <mu>M or 2 amol of injected analyte.