To achieve highly sensitive analysis without labor-intensive experimental procedures in capillary electrophoresis (CE), large-volume sample stacking with an electroosmotic flow pump (LVSEP)-field-amplified sample injection (FASI) was combined with a dynamic coating technique. In this study, poly(vinyl pyrrolidone) (PVP) was employed for the dynamic coating additive. Since a standard fluorescent dye, fluorescein, was well concentrated in a conventional LVSEP, the PVP dynamically-coated capillaries can be also applied to the LVSEP-FASI analysis. In our home-made CE apparatus, however, current breakdown was often caused, especially at a longer electrokinetic injection time due to bubble formation. To avoid the interference of bubble formation, the distance between the tips of the electrode and the capillary in the vertical direction was changed from 0 to 2.5 cm under the magnetic stirring condition. This allowed for a long electrokinetic injection time of up to 20 min, resulting in a sensitive enhancement factor (SEF) of 34900 for fluorescein. The developed method was applied to the chiral analysis of amino acids in CE. As a result, leucine (Leu) was successfully separated in LVSEP-FASI with SEFs of 6420 and 4500 for the D-and L-Leu peaks, respectively.
To achieve an on-line coupling of the sample preconcentration by a large-volume sample stacking with an electroosmotic flow pump (LVSEP) with microchip gel electrophoresis (MCGE), a sample solution, a background solution for LVSEP and a sieving solution for MCGE were loaded in a T-form channel and three reservoirs on PDMS microchips. By utilizing the difference in the flow resistance of the two channels, a low-viscosity sample and a viscous polymer solution were easily introduced into the LVSEP and MCGE channels, respectively. Fluorescence imaging of the sequential LVSEP-MCGE processes clearly demonstrated that a faster stacking of anionic fluorescein and successive introduction into the MCGE channel can be carried out on the T-channel chip. To evaluate the preconcentration performance, a conventional MCZE analysis of fluorescein on the cross-channel chip was compared with LVSEP-MCGE on the short T-channel chip, and as a result that the value of sensitive enhancement factor (SEF) was estimated to be 370. The repeatability of the peak height was good with the RSD value of 3.2%, indicating the robustness of the enrichment performance. In the successive LVSEP-MCGE analysis of φX174/HaeIII digest, the DNA fragments were well enriched to a sharp peak in the LVSEP channel, and they were separated in the MCGE channel, whose electropherogram was well-resembled with that in the conventional MCGE. The values of SEF for the DNA fragments were calculated to be ranging from 74 to 108. Thus, the successive LVSEP-MCGE analysis was effective for both preconcentrating and separating DNA fragments.
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