Simultaneous separation of acidic and basic proteins using gemini pyrrolidinium surfactants and hexafluoroisopropanol as dynamic coating additives in capillary electrophoresis

The separation of acidic and basic proteins using CE has been limited in part due to the adsorption of proteins onto the capillary wall. In this work, the efficient control of EOF and the simultaneous separation of acidic and basic proteins are achieved by use of C 18 -4-C 18 PB as a dynamic coating additive, which is a representative surfactant for 1,1′-(butane-1,s-alkyl)bis(1-alkylpyrrolidinium) bromide (C n -4-C n PB, n   =   10, 12, 14, 16 and 18). C 18 -4-C 18 PB exhibits a powerful capability in the reversal of EOF, and a low concentration even less than 0.001   mM is sufficient to reverse EOF at the tested pH values (3.0–9.0). Baseline separation of eight proteins with sharp peaks and high efficiencies (54,000–297,000 plates/m) is obtained with 30   mM NaH 2 PO 4 buffer (pH 5.0) containing 4   mM C 18 -4-C 18 PB. At the same buffer condition, the C n -4-C n PB with shorter alkyl chain ( n   =   10, 12, 14, 16) cannot achieve the same effective protein separation as C 18 -4-C 18 PB. However, the combined use of small amounts (≤0.5%, v/v) of hexafluoroisopropanol (HFIP) and C n -4-C n PB ( n   =   10, 12, 14, 16) as additives can completely separate all eight proteins with high efficiencies of 81,000–318,000 plates/m. The RSDs of migration time are less than 0.80% and 5.84% for run-to-run and day-to-day assays ( n   =   5), respectively, and the protein recoveries are larger than 90.15%. To the best of our knowledge, this is the first report on the simultaneous separation of acidic and basic proteins using C n -4-C n PB surfactants or C n -4-C n PB surfactants combined with HFIP as dynamic coating additives.