Fundamentals of capillary electrochromatography and its coupling with mass spectrometry

Capillary electrochromatography (CEC), an emerging electrokinetic separation technique, combines the versatility offered by HPLC and the high plate efficiency of CZE. It employs electrosmosis for generating eluent flow (EOF) through a packed capillary column where the sample components are separated due to differences in their partitioning between the mobile and the stationary phases as well in their electrophoretic mobilities. In this study the physicochemical phenomena underlying the generation and control of electrosmotic flow as well as the conductance of packed capillary columns were investigated. The conductance of capillaries packed with various stationary phases was evaluated with respect to the open capillary and the data interpreted in the light of Tobias equation. The results suggest that CEC may facilitate the use of relatively long columns packed with small particles that would require high column inlet pressures in micro-HPLC. The capillary columns presently used in CEC are duplex in nature since there is an open segment present after the packed column. An analysis of the column architecture with respect to the electrical and flow properties as well as the selectivity of the chromatographic system is provided. Experiments performed with a home built capillary electrochromatograph capable of operation at voltages up to 60 kV, exhibited an enhancement in the magnitude of electrosmotic flow and enabled measurement of reduced plate heights over a wide range of Peclet numbers. The results illustrate that since the EOF is generated at the charged surface of the stationary phase the van-Deemter curve for CEC is flatter at high reduced velocity than that under identical conditions in micro-HPLC. A CEC unit was constructed to facilitate gradient elution by allowing a controlled change of the mobile phase composition that is pulled into the column electrosmotically. The potential of this approach for the rapid separation of biomolecules is investigated. In most experiments reversed phase CEC with hydrocarbonaceous silica bonded particles was employed for the separation of neutral compounds with hydroorganic eluents at high pH. At low pH, CEC of peptides was carried out on a gigaporous strong cation exchanger. Reduced plate heights of 1.1 and lower were obtained and attributed to intraparticulate EOF in the gigapores at high field strengths. Striving for MS compatibility has become a major design consideration for newly emerging separation techniques. For this reason the potential of coupling capillary electrochromatography with an electrospray ionization time of flight mass spectrometer was investigated for the rapid separation and identification of biomolecules. The results illustrate that internal tapers of CEC columns offer a viable alternative to silica frits in meeting the particularly demanding requirements of CEC/ESI/MS.