Sheath flow detection

FIGURE 16.17 Postelectrophoresis sheath-flow detection of DNA fragments. (Reprinted with permission... 

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). 

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. 

Electrospray ionization (ESI) is ideally suited as a detection technique for the online interfacing of liquid-phase separations (HPLC and CE) to MS, because it facilitates the transfer of analytes from the liquid phase of the HPLC or CE column to the gas phase of the MS. Also, it allows the detection of high molecular weight species, such as peptides. Three interface designs have been developed in the past 18 years for coupling CE with MS. The first CE-MS interface, coaxial sheath flow, was introduced by Smith and his group in 1987 (Olivares et al., 1987) and was improved upon in later work (Smith et al., 1988). Coaxial sheath flow is formed using two concentric metal capillaries, whereby the CE terminus and the makeup flow line are inserted into the... 

Many authors claim that the dilution by the sheath flow would not significantly affect the detection sensitivity, because it is completely evaporated in the spray process. Moreover, it has been discussed that in this layered-flow approach, preferably the inner layer of the spray enters the collector opening. If this were true, the composition of sheath liquid would be less important. Anyhow, it has to be stated that there is a dilution problem in the sheath-flow approach. In addition, it has been proven many times that ESI is a concentration-sensitive, not mass-sensitive, process. Knowing this, it makes sense to reduce the sheath liquid flow rate to the minimum required for stable spray conditions. 

The sheathless interface (Fig. 2c) is known since the first CE/MS attempt by Olivares et al. (10). In this system the CE capillary was sleeved in a metal tube, whereas in modem sheathless interfaces the capillary exit is carefully sharpened or pulled to a fine point (14). The outer surface of the capillary tip is coated with metal, usually gold, which is readily accessible for electrical contact. This setup enables the maintenance of both electrical circuits from CE and ESI as well. The advantage of the sheathless approach over the coaxial sheath flow interface is that the eluting CE zone is not diluted by makeup flow and therefore the obtainable sensitivity can be quite high, especially when small-ID capillaries (e.g., 10 yarn) are used. Detection limits in the low fmol range have been demonstrated (13). A... 

To overcome limitations of end-channel detection systems (see Section 34.1.3), a design with sheath-flow channels (1cm long) that flank the separation channel and join it just before the channel exit has been reported [42]. At the end of these auxiliary channels, buffer reservoirs are created by inserting pipette tips into the drilled access holes for generating a gravity constant flow into the detection reservoir. 

P. Ertl, C.A. Emrich, P. Singhal and R.A. Mathies, Capillary electrophoresis chips with a sheath-flow supported electrochemical detection system, Anal. Chem., 76 (2004) 3749-3755. 

Capillary isoelectric focusing can be applied as a micropreparative tool for protein analysis by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) [69,70]. The exact timing of the collector steps in the interface is based on determining the velocity of each individual zone measured between two detection points close to the end of the capillary. During the collection a sheath flow fraction collector is used to maintain the permanent electric current. 

Most forms of detection in High-Performance Capillary Electrophoresis (HPCE) employ on-capillary detection. Exceptions are techniques that use a sheath flow such as laser-induced fluorescence [1] and electrospray ionization mass spectrometry [2],... 

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