Sub-Millisecond Electrophoresis

Instrumentation for sub-millisecond electrophoretic separations is described, and the data represent an 100-fold decrease in analysis time over previous methods. High-speed electrophoretic separations have been performed in capillaries using optical gating for sample introduction,1 and on microchips using a simple cross geometry.2 Both approaches had analysis times on the order of 100 ms. Several issues must be addressed to enhance the separation performance in capillary electrophoresis. Most importantly, the separation field strength must be optimized to reduce the analysis time and minimize dispersion due to diffusion and thermal convection. In addition, the spatial extent of the injection plug and detector observation region must be minimized. The injection plug length can be minimized by fabricating narrow channel dimensions for the injection valve and confining the sample volume within the injection valve using electric fields. Similarly, the detector observation length can be minimized for fluorescence detection by having a small excitation volume or tight spatial filtering. The channel manifold can be designed to reduce the potential drop in areas not contributing to the separation so that high separation field strengths can be achieved with modest applied potentials. To achieve this design goal in a single etch step, narrow channels were fabricated for the injection valve and separation channel, and wide channels for all other sections of the channel manifold. This enabled separation field strengths of 6.1 V cm-1 per volt of applied potential.

Figure 1 shows a schematic of the microchip designed for high-speed microchip electrophoresis. The fabrication included a single etch step with constant channel depths of 7.0 m. Consequently, the different resistivities per unit length result from different channel widths. The wide channels are 440 m wide, and the narrow channels are 26 m wide. The 26 m linewidth was the narrowest, high quality line that could be produced with our in-house direct-write system. The relative resistivity between the narrow and wide channels is 16.9 per unit length, and consequently, the potential drop along the wide channel is 16.9 times less than along the narrow channels for a given length. This design gives a separation field strength of 6.1 V cm-1 per volt of applied potential. If a microchip of similar dimension were fabricated with constant width channels, the applied potential would have to be over 8 times greater to achieve equivalent separation field strengths. For example, the highest separation field strength used in this work was 53 kV cmwhich would require 71 kV of applied potential for a microchip with a uniform width channel design. Such high potentials are clearly not practical.

Figure 2 shows an electrophoretic separation from a single injection of the binary mixture of rhodamine B and dichlorofluorescein resolved in 0.8 ms. The separation was monitored 200 m downstream from the injection valve, and a separation field strength of 53 kV cm-1 was used (8.6 kV applied to the microchip). The heat dissipated in the separation channel for the 53 kV cm-1 separation field strength was estimated to be 172 W m-1 assuming a potential drop of 2.8 kV, a current of 32 A, and a length of 520 m. This value is considered to be exceedingly high for conventional capillary electrokinetic separations, but the separation efficiency was not adversely affected.

The initial demonstration of sub-millisecond separations is extremely promising. Straightforward improvements in the separation efficiency and analysis time can be made by reducing the contributions of the injection plug width and Joule heating to the plate height. High-speed microchip electrophoresis could be a useful tool for ultra-high throughput drug discovery, monitoring millisecond time-scale kinetics for chemical and biochemical reactions, or as the final dimension to multi-dimensional separation systems.

References

1 Moore Jr., A.W.; Jorgenson, J.W. Anal. Chem. 1993, 65, 3550.

2 Jacobson, S.C.; Hergenrder, R.; Koutny, L.B.; Ramsey, J.M.Anal. Chem. 1994, 66, 1114.

Figure 1. Schematic of microchip used for high-speed electrophoretic separations. Inset. Enlargement of the injection valve and separation channel. The wide channels are 440 m wide, and the narrow channels are 26 m wide.

Figure 2. High-speed electropherogram of rhodamine B (RB) and dichlorofluorescein (DCF) resolved in 0.8 ms using a separation field strength of 53 kV cm-1 and a separation length of 200 m. The start time is marked with an arrow at 0 ms.

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