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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.
Zhang et al. (Thu,) studied this question.