Sheath flow cuvette

Extremely low level detection work is being performed ia analytical chemistry laboratories. Detection of rhodamine 6G at 50 yoctomole (50 x lO " mol) has been reported usiag a sheath flow cuvette for fluorescence detection foUowiag capiUary electrophoresis (9). This represeats 30 molecules of rhodamine, a highly fluoresceat molecule (see Electhoseparations, electrophoresis Spectroscopy, optical). 

Two high-voltage power supplies are used to drive the separation. The first power supply applies high voltage through a platinum electrode to the injection buffer reservoir for the first dimension separation. The second power supply applies potential to the interface through its buffer reservoir. The sheath flow cuvette is held at ground potential. 

After this prerun, the voltage is programmed to periodically pulse a plug of analyte into the interface. This fraction is then drawn into the second capillary for further separation. In our current configuration, the separation window in the CSE dimension is roughly 200 s in duration, and roughly 200 pulses of 1 s duration are required for the contents of the CSE capillary to be transferred to the second dimension. A constant potential is applied across the second dimension capillary, typically 10,000-20,000 V. Under this constant voltage, any analyte present within the interface is driven into the second dimension capillary for separation. Detection is by laser-induced fluorescence in a postcolumn sheath-flow cuvette. 

Amino acids Postcolumn sheath flow cuvette 500 amol (for valine) 83... 

Swerdlow, H., Wu, S. L., Harke, H., and Dovichi, N. J., Capillary gel electrophoresis for DNA sequencing. Laser-induced fluorescence detection with the sheath flow cuvette, J. Chmmatogr., 516,... 

A sheath-flow cuvette increases S/N ratios and decreases detection limits. 

Lastly, the most popular detection technique for subcellular analysis is LIF due to the extremely low LODs that can be attained. Mass LODs on the order of attomoles can be achieved with commercial CE-LIF instruments and custom-built instruments, in conjunction with sheath-flow cuvettes, can reach down to the yoctomole (10 " mole) scale. LIF detection thus allows the quantification of minute amounts of analyte, and is the only detection method that has been used to detect individual organelles. We will therefore describe off-column LIF detection in further detail. 

Sheath-flow cuvettes were originally developed for flow cytometry, but were adapted for use with CE by Dovichi. " Figure 20.16 illustrates LIF detection in conjunction with a sheath-flow cuvette. As displayed, the capillary is surrounded by sheath-flow buffer inside the cuvette. As organelles migrate out of the capillary, they are hydrodynamically focused by the sheath flow into a narrow stream. The excitation source is focused beneath the capillary outlet to excite the organelles once they have been focused. Since the diameter of the sample stream is dependent on the difference between the volumetric flow rates of the sample stream and the sheath flow, the sample stream can be narrowed to the desired width by increasing the sheath volumetric flow. 

FIGURE 20.16 Diagram of the sheath-flow cuvette used for LIF detection. The capillary is inserted into the sheath-flow cuvette and the laser beam is focused several micrometers away from the outlet. Sheath fluid focuses the sample as it migrates out of the capillary into a narrow stream, in which it is excited by a focused laser beam. The fluorescence is collected at a 90 angle from the excitation source by a high N.A. objective. (Reprinted from Johnson, R. et al.. Anal. Bioanal. Chem., In Press. Copyright 2006. With kind permission from Springer Science and Business Media.)... 

Cheng, Y. F., Wu, S., Chen, D. Y, and Dovichi, N. J., Interaction of capillary zone electrophoresis with a sheath flow cuvette detector. Anal. Chem., 62, 496-503, 1990. 

Aspiration of a single cell typically requires a pulse of 11 kPa vacuum to the distal end of the capillary for 1 or 2 s. Control of such modest pressure is not trivial. Krylov developed an elegant method for generating an extremely reproducible vacuum. The distal end of the capillary is connected to a sheath-flow cuvette, and the waste stream of the cuvette is connected to a three-way valve. In normal operation, the valve directs the waste to a receiving reservoir. To inject a sample, the valve directs flow to a solenoid valve, which is connected to a water-filled piece of tubing that terminates in a beaker placed 1 m below the level of the injection block. A timer circuit is used to open the solenoid valve for a precisely timed period, during which time the injection end of the capillary is connected to a water column of 1 m high, which applies vacuum necessary to aspirate the cell within the capillary. 

FIGURE 21.18 Instrumentation for two-dimensional CE. Two capillaries arejoined through an interface, that allows fractions to be transferred by manipulation of the voltage applied to the capillaries. In our system, the sheath-flow cuvette detector is held at ground potential. 

FIGURE 21.20 Typical timing diagram used in two-dimensional CE for chemical cytometry. The distal end of capillary 2 is held in the sheath-flow cuvette, which is at ground potential. 

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