End-column detection

FIGURE 3-24 Electrophoretic separation of catechols with end-column detection. Detection potential, +0.8 V separation capillary, 20 kV The peaks correspond to 4.6 fmol dopamine (1), 4.1 fmol isoproterenol (2), and 2.7 fmol catechol (3). (Reproduced with permission from reference 60.)... 

Electroosmotic flow, 195 End column detection, 89 Energy barrier, 16 Enzyme electrodes, 172, 174 Enzyme immunoassays, 185 Enzyme inhibition, 181 Enzyme reconstitution, 178 Enzyme wiring, 178 Equilibrium potential, 15 Ethanol electrodes, 87, 178 Exchange current, 14... 

The second approach (end-column detection) is best suited for capillaries of 25 pm or less [49]. This mode of detection is illustrated in Figure 27.20. In this case, the electrode is placed at the end of the capillary (but not inside) and no coupler is employed. A 70-cm, 5-pm-i.d. capillary filled with a zwitterionic... 

End-column detection is done by coupling the outlet of the capillary to an external detection cell. The properties of this detection cell (path length, material, etc.) can be tailored to allow for high sensitivity and low analyte limits of detection. The careful selection of coupling and transfer fluidics, such as zero dead-volume unions, ensures that the high resolution afforded by CE is maintained within the end-column detector. 

For all of the various end-column derivatization schemes, it is important that labeling reactions be chosen that exhibit fast kinetics. This ensures that derivatization is complete before the analytes enter the detection zone, which in turn ensures maximum analyte detectability. Common derivates with suitable kinetics for end-column detection include fluorescamine, OPA, and NDA. In addition to kinetics, the selection of an appropriate derivatization reaction can be influenced by other factors, including the potential side reactions for a given set of reagents and analytes, equilibrium and concentration considerations, and the pH at which the derivatization is carried out. 

Online, on-column, or end-column detection makes possible direct monitoring and evaluation of the separation process the whole analytical procedure can be automated. 

One of the main advantages of CE over gel electrophoresis is that the separation is monitored by online, on-column, or end-column detection. In the most frequently employed UV absorption photometric detection, a small part (less than 1mm) of the capillary serves as a detection cell. Micromolar concentrations of proteins are detectable using the low UV detection wavelength of 200-220 nm. A higher sensitivity, up to nanomolar concentrations, is achieved with fluorescence, particularly laser induced fluorescence (LIE) detection. The disadvantage of the LIE detection of proteins is the necessity for their derivatization using a fluorogenic label. The native fluorescence of proteins, mostly due to the presence of aromatic amino acids residues, tryptophan, and tyrosine, can be utilized only when low UV laser... 

Figure 1 CEEC systems with (A) off-column detection and (B) end-column detection.

An alternative (and very widespread) approach to off-column detection is end-column detection (Figure IB). In this case, small-diameter fused sihca capillaries are employed for the separation. It has been shown by Huang et al. that when capillaries with internal diameters of less than 25 xm are employed, most of the voltage is dropped across the capillary [21]. Therefore, if the electrode is placed just outside the end of the capillary, the separation voltage has minimal influence on the apphed detection potential. This obviates the need for a decoupler, making the overall system easier to construct and more rugged because the capillary is made in one piece. 

End-column detection is more popular than off-column detection due to the ease of alignment and elimination of the often fragile fraeture. One potential... 

Ever since Baldwin and Ye [24] first demonstrated that macroelectrodes could be used for end-column detection, this has been a common approach for CEEC. Alignment of macroelectrodes is much simpler because they can just be butted up to the end of the fused silica capillary. However, the sensitivity is not as good as that obtained with off-capiUary or optimized end-column detection. If selectivity rather than sensitivity is the primary goal of the analytical system, this method is the easiest to implement. Limits of detection for end-column detection are usually in the micromolar range. 

An important consideration in the use of end-column detection is the effect of the separation voltage on the detector voltage. Both Matysik and Wallenborg et al. [25,26] have shown that, even with 25 jm i.d. capillaries, the separation field has a considerable effect on the applied voltage at the detector. Therefore, the detection voltage must be adjusted for the separation voltage or the maximum current response will not be obtained. This problan does not occur with off-col-umn detection because in that case the detector is more efficiently isolated from the separation voltage. 

Figure 3 HDVs recorded for dopamine and catechol using end-column detection at cap-illary-to-electrode distances of 20 and 60 pm without (A) and with (B) a fracture decoupler. Symbols dopamine ( ) and catechol ( ) at 20 pm dopamine ( ) and catechol (A) at 60 pm. Open and closed symbols represent different trials. (Reprinted with permission from Ref. 26.)...

The first reports of CEEC described the use of micromanipulators to position the electrode into the end of the fused silica capillary. Since that time, numerous cell holders have been designed [38 1]. In 1998, Kok and coworkers reported the direct coupling of a BAS Unijet cell with a fused silica capillary [39]. More recently, Everett et al. reported the use of a commercial CEEC interface for off-column detection in CEEC [41]. A diagram of this cell configuration is shown in Figure 4. This cell holder is used with electrodes with an i.d. larger than that of the separation capillary. Either off-column or end-column detection can be employed. 

Huang, X. Zare, R.N. Sloss, S. Ewing, A.E. End-Column Detection for Capillary Electrophoresis. Anal. Chem. 1991 63, 189-192. 

The main difficulty in working with amperometry is the need for decoupling of the detection signal to eliminate any interference from the applied electric held, precise electrode positioning, and electrode stability [51]. However, use of decoupler in front of the detector [52,53], or end-column detection [54,55] may resolve these issues. 

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