Effective electrokinetic field-amplified sample injection occurring at the capillary inlet from a sample volume equivalent exceeding that of the capillary up to 10-fold is described and demonstrated to provide over 1000-fold sensitivity enhancement. Successful application of this head-column field-amplified sample stacking approach to the analysis of positively chargeable, hydrophobic compounds in binary system capillary electrophoresis is shown to require an initially introduced low-conductivity zone (water plug) of >1 mm length, a sample injection voltage <20 kV, and an injection time interval <60 s. Following these conditions for more than 1500 runs with capillaries of 50 μm i.d. and about 20 cm effective length, damaging heat production during electroinjection within the low-conductivity zone at the column inlet (boiling of solvent and possible deposition of solutes or fusing of capillary walls) could be prevented. The solute amount injected by head-column field-amplified sample stacking is further shown to be dependent on the organic fraction and the buffer in the sample solution. High content of organic solvent, low conductivity, and the presence of a small amount of H(+) (50-100 μM) provides the highest sensitivity for analysis of positively chargeable model substances, including amiodarone and desethylamiodarone. Solutes present at the nanomolar level can thereby be accumulated from a sample volume equivalent of about 4 μL (with injection of about 20 nL of sample solvent into the capillary) and measured by UV absorption detection. To prevent disturbances caused by electrolysis, sample vials should be employed only once. The data obtained further show that quantitation can be reliably performed using internal calibration based on peak height (RSDs for inter- and intraday determinations are on the 2% level). However, due to variation of the roughness of the capillary walls and cuts, the time interval between operational steps, and trace adsorption onto the capillary walls, the length of the water zone drawn by capillary action on the inlet side is not constant, and external calibration therefore cannot be employed for quantitation.

A new dynamic computer model permitting the combined simulation of the temporal behavior of electroosmosis and electrophoresis under constant voltage or current conditions and in a capillary which exhibits a pH-dependent surface charge has been constructed and applied to the description of capillary zone electrophoresis, isotachophoresis, and isoelectric focusing with electroosmotic zone displacement. Electroosmosis is calculated via use of a normalized wall titration curve (mobility vs pH). Two approaches employed for normalization of the experimentally determined wall titration data are discussed, one that considers the electroosmotic mobility to be inversely proportional to the square root of the ionic strength (method based on the Gouy-Chapman theory with the counterion layer thickness being equal to the Debye-Hückel length) and one that assumes the double-layer thickness to be the sum of a compact layer of fixed charges and the Debye-Hückel thickness and the existence of a wall adsorption equilibrium of the buffer cation other than the proton (method described by Salomon, K.; et al. J. Chromatogr. 1991, 559, 69). The first approach is shown to overestimate the magnitude of electroosmosis, whereas, with the more complex dependence between the electroosmotic mobility and ionic strength, qualitative agreement between experimental and simulation data is obtained. Using one set of electroosmosis input data, the new model is shown to provide detailed insight into the dynamics of electroosmosis in typical discontinuous buffer systems employed in capillary zone electrophoresis (in which the sample matrix provides the discontinuity), in capillary isotachophoresis, and in capillary isoelectric focusing.

In capillary electrophoresis, head-column field-amplified sample stacking (FASS) provides the largest sensitivity enhancement of all electrokinetic concentration techniques (Zhang, C.-X.; Thormann, W. Anal. Chem. 1996, 68, 2523). Application of head-column FASS to the analysis of closely related opioids by capillary zone electrophoresis in binary systems with ethylene glycol is described. It is shown that sample condensation is further increased about 2-fold by introduction of a preinjection plug, i.e., introduction of a short solution plug of high conductivity, high pH, and high viscosity at the capillary tip prior to injection. The preinjection plug acts as a temporary trap for solutes. Its effective length is shown to be limited to a few millimeters. The highest sample stacking efficiency in head-column FASS is obtained via optimization of the electric field strength and the effective electrophoretic mobility of the solutes within the sample and the adjacent zones and is thus strongly dependent on the compositions of both the sample matrix and the preinjection plug. Binary sample solutions of low conductivity and low viscosity containing small amounts of a weak acid are demonstrated to be most effective for the stacking of positively charged opioids. The procedure developed for capillary zone electrophoresis of opioids in binary systems with ethylene glycol and UV absorbance detection is documented to provide a 3 orders of magnitude sensitivity enhancement, exhaustive sample injection from an external reservoir of up to 20 microL (i.e., from a volume that is more than 20-fold the volume of the capillary employed), and a lowest detectable concentration of 0.1 ng/mL (S/N = 3). Using internal calibration, typical intraday and interday imprecisions of solute concentrations between 3 and 10 ng/mL and between 0.5 and 1.5 ng/mL are < or = 5% and < or = 15%, respectively. The stacking approach has been successfully applied to the analysis of dihydrocodeine in extracts of 20 microL of human plasma and is shown to permit the precise (imprecision < or = 10%) determination of dihydrocodeine plasma levels of pharmacological interest (3-300 ng/mL or 10-1000 nM). Furthermore, using a modified protocol for micellar electrokinetic chromatography, head-column FASS is shown to provide a 400-fold sensitivity enhancement for a number of opioids. The stacking procedure is based on insertion of a surfactant-free preinjection plug and temporary application of reversed voltage after electroinjection.

A stepwise mobilization strategy has been developed for the elution of complex protein mixtures, separated by capillary isoelectric focusing (CIEF) for detection using on-line electrospray ionization mass spectrometry (ESI-MS). Carrier polyampholytes are used to establish a pH gradient as well as to control the electroosmotic flow arising from the use of uncoated fused-silica capillaries. Elution of focused protein zones is achieved by controlling the mobilization pressure and voltage, leaving the remaining protein zones focused inside the capillary. Protein zones are stepwise eluted from the capillary by changing the mobilization conditions. Stepwise mobilization improves separation resolution and simplifies coupling with multistage MS (i.e., MSn) analysis since it allows more effective temporal control of protein elution from the CIEF capillary. We also describe a modified configuration for coupling CIEF with ESI-MS using a coaxial sheath flow interface that facilitate the automation of on-line CIEF-ESI-MS analyses. The stepwise mobilization strategy is demonstrated for the analysis of standard protein mixtures and soluble E. coli lysate proteins using CIEF-ESI-MS. These results indicate that inlet pressure or voltage programming to control the elution of the protein zones from the capillary (i.e., gradient mobilization) may allow for the optimization of the mobilization conditions and provide higher resolution for CIEF separation of complex mixtures with on-line MS.