Head-column field-amplified sample stacking
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The fundamentals of electrokinetic injection of the weak base methadone across a short water plug into a phosphate buffer at low pH were studied experimentally and with computer simulation. The current during electrokinetic injection, the formation of the analyte zone, changes occurring within and around the water plug and mass transport of all compounds in the electric field were investigated. The impact of water plug length, plug injection velocity, and composition of sample, plug and background electrolyte are discussed. Experimental data revealed that properties of sample, water plug and stacking boundary are significantly and rapidly altered during electrokinetic injection. Simulation provided insight into these changes, including the nature of the migrating boundaries and the stacking of methadone at the interface to a newly formed phosphoric acid zone. The data confirm the role of the water plug to prevent contamination of the sample by components of the background electrolyte and suggest that mixing caused by electrohydrodynamic instabilities increases the water plug conductivity. The sample conductivity must be controlled by addition of an acid to prevent generation of reversed flow which removes the water plug and to create a buffering environment. Results revealed that a large increase in background electrolyte concentration is not accompanied with a significant increase in stacking.
Part I on head-column field-amplified sample stacking comprised a detailed study of the electrokinetic injection of a weak base across a short water plug into a phosphate buffer at low pH. The water plug is converted into a low conductive acidic zone and cationic analytes become stacked at the interface between this and a newly formed phosphoric acid zone. The fundamentals of electrokinetic processes occurring thereafter were studied experimentally and with computer simulation and are presented as part II. The configuration analyzed represents a discontinuous buffer system. Computer simulation revealed that the phosphoric acid zone at the plug-buffer interface becomes converted into a migrating phosphate buffer plug which corresponds to the cationically migrating system zone of the phosphate buffer system. Its mobility is higher than that of the analytes such that they migrate behind the system zone in a phosphate buffer comparable to the applied background electrolyte. The temporal behaviour of the current and the conductivity across the water plug were monitored and found to reflect the changes in the low conductivity plug. Determination of the buffer flow in the capillary revealed increased pumping caused by the mismatch of electroosmosis within the low conductivity plug and the buffer. This effect becomes elevated with increasing water plug length. For plug lengths up to 1% of the total column length the flow quickly drops to the electroosmotic flow of the buffer and simulations with experimentally determined current and flow values predict negligible band dispersion and no loss of resolution for both low and large molecular mass components.
Head-column field-amplified sample stacking of cations from a low conductivity sample followed by enantiomeric separation using negatively charged chiral selectors was studied experimentally and with computer simulation. Aspects investigated include the direct electrokinetic injection of the analytes into the background electrolyte, the use of a selector free buffer plug, the contribution of complexation within the buffer plug and the application of an additional water plug between sample and buffer plug. Attention was paid for changes of ionic strength which is known to have a significant impact on complexation and thus effective mobility. Racemic methadone was selected as a model compound, randomly substituted sulfated β-cyclodextrin as chiral selector and phosphate buffers (pH 6.3) for the background electrolyte and the buffer plug. Results confirm that the buffer plug is providing a spacer between cationic analytes and the negatively charged selector during electrokinetic injection. Simulation predicts the required length and composition of the plug for a given injection time to avoid an interference with the selector. A short water plug added between the low conductivity sample and a high conductivity buffer plug is demonstrated to provide best conditions to achieve high sensitivity in enantioselective drug assays with sulfated cyclodextrins as selectors.
