A high voltage power supply for CE should deliver voltages up to 30 kV and currents up to 200-250 ¡iA. Because of the direction of the EOF, which in silica capillaries is usually toward the cathode, the common polarity is with the anode at the injection end of the capillary and the cathode close to the detection window. CE separations are generally carried out at constant potential, but constant current may be preferable, especially when the control of temperature of the capillary is not efficient. In fact, increases in temperature due to uncontrolled Joule heating can determine changes in the buffer viscosity of analytes, and, consequently, cause changes in their in migration velocity. Under constant current conditions, the system tends to compensate for temperature changes, because to maintain a constant current in the system, increases of temperature, with consequent reduction of the electric resistance in the capillary, are automatically balanced by a corresponding decrease in the voltage. Gradients or steps in the voltage, though not very popular, may be useful in some cases to allow the analysis in a reasonable time of mixtures containing components that differ greatly in electrophoretic mobility.
Attention is also to be paid to the process of water decomposition taking place at both the electrodes: protons and oxygen are liberated at the anode, while at the cathode hydrogen and hydroxide ions are produced. It follows that care should be given to the buffering capacity of the background buffer, to avoid pH changes during an analysis or from analysis to analysis. Besides, venting of the electrode jars may be necessary, to avoid pressurization of the buffer jars by the released gases.
In general, detectors in CE have to cope with problems of three types: small mass (picogram levels) of analytes injected (due to the limitations in the volumes), which can be loaded into the capillary, limited peak volumes, and inadequately separated peaks.
22.214.171.124 Absorbance Detection UV(-visible) radiation absorption is universally present in CE. To avoid any possible postcolumn band broadening (which would minimize resolution and sensitivity), detection is accomplished through a window in the capillary. One disadvantage of this method is that the path length will be minimal, and; thus, described by Beer's law relating sensitivity to path length, a path length of 25 to 100 mm, such as would be found in CE, will provide less sensitivity than cases in which the optical path length can be about 100 times greater. Also, the cylindrical geomatry of the capillary provides poor optical characteristics to the cell design. Two consequences of these limitations are that in this type of column UV detection, only moderate concentration sensitivity (e.g., 10~4-10~6 M) and a narrow linear range (s about 0.5 AU) will be obtained. In contrast because of nanoli-ter volumes injected, the mass sensitivity of CE can be high and, as a result, picogram amounts can be directly measured with UV detectors. This difference between sensitivity in terms of mass and sensitivity in terms of concentration is often a source of misunderstanding and possible disappointment for beginners in this field.
A way of increasing the optical path length, and sensitivity, is to use axial illumination (instead of the orthogonal illumination) of the capillary, with the light beam passing through a Z-shaped capillary. With a proper choice of the length of the middle branch of the capillary, which is axially illuminated (about 3 mm), the sensitivity can be increased substantially (i.e., 10 times or even more), with only a minimal loss of resolution (see Li, 1994). An alternative to increasing the optical path length without bending the capillary is the "bubble cell" design, which is now available commercially (Heiger, 1992). This design provides only a moderate increase in sensitivity (about 3-4 times), but, reportedly, there is no sacrifice in resolution.
Analogously to HPLC, photodiode array or multiwavelength fast scanning detectors can be used to increase the quantity and quality of information. These detectors allow the analyst to evaluate the on-line UV(-visible) spectra of the separated zones, and, by comparison with recorded reference spectra, to investigate peak purity and peak indentity.
126.96.36.199 Fluorlmetrlc, Electrochemical Detection, and Other Detection Modes Other detection techniques have successfully been implemented in CE, with advantages in sensitivity and/or selectivity. Laser excitation allows the focusing of high radiation energy into the capillary, and consequently laser-induced fluorescence achieves much better sensitivity: up to 10~12 M. Since, however, not all analytes are naturally fluorescent, fluorescence detection often requires derivatization (e.g., for amino acid analysis). The precolumn derivatization procedures used for HPLC are also used in CE. In contrast, the use of postcolumn derivatization in CE is limited because it is difficult to add and mix derivatizing reagents directly in the capillary after the separation without causing unacceptable band spreading.
Electrochemical detection has also been applied in CE. However, a problem not yet satisfactorily resolved is the need to keep the high voltage (with resulting high currents) used for separation from apart from voltages used in the detection compartment, where much smaller currents are generated and measured. This separation is generally accomplished by inserting a porous conductive joint before the detection end of the capillary. The joint is connected with the cathode, closing the high voltage circuit before the end of the capillary, whereas a carbon fiber, representing the working electrode of an amperometric detector, is inserted into the capillary end, a short distance downstream from this joint. Thus, the high electric current (up to 100150 raA) applied to accomplish the electrophoretic separation does not interfere with the faradaic current generated at the electrode by the oxidation-reduction process. Unfortunately, the conceivable complexity of this scheme hampers the utility of electrochemical detectors, which otherwise would be sensitive (about 10"8 M).
Also, conductimetric detection has been used, by applying two electrodes into the capillary. In this detection mode, the separation of the high voltage part of the capillary (under dc) from the detection zone (under ac) can be accomplished also by electronic filtering. A capillary electropherograph, with a conductimetric detector, is commercially available.
A variety of other detection techniques have been applied in CE, including laser-based thermooptical detection, refractive index detection, radioisotope detection, and, notably, mass spectrometric (MS) detection.
188.8.131.52 Mass Spectrometric Detection Electrospray has proven to be the method of choice for CE/MS (Smith et al., 1991). With electrospray, solutions containing the analytes are nebulized and subjected to high electric field. Following solvent evaporation and successive Coulombic explosions, pseudomolecular ions of the dissolved analytes are formed. These ions are focused into the mass spectrometer, which separates them according their mass-to-charge ratios. In this method, the end of the CE capillary is connected to the electrospray probe carrying 3 to 4 kV. Some limitations, regarding the choice of Ce buffers remain, however. For example, borate and phosphate salts are not recommended above 20 mAf. Among the other MS interfacing methods, ion spray-atmospheric pressure ionization and continuous flow-fast atom bombardment have been coupled to CE, also in a tandem MS (MS/MS) arrangement (Johansson et al., 1991).
184.108.40.206 Indirect Detection Indirect detection is suitable for the determination, without derivatization, of ionic compounds (e.g., inorganic ions, organic acids) that do not absorb UV radiation and are not fluorescent or electrochemi-cally active. In this detection mode, the running buffer is added with an ionic compound, which is easily detected at low concentrations by the detector (including UV, fluorimetric, or electrochemical), thus determining a high background signal. To maintain the electroneutrality, during the CE separation, this additive is displaced from the zones occupied by the ionic analytes, resulting in "negative" peaks in the background signal. Thus, analytes are detected as "holes" of UV absorbance (or fluorescence or electrochemical activity) of the background electrolyte, without any need of derivatization. It is obvious that the choice of detection mode is not dependent on the characteristics of the analyte, but only the buffer additive used.
To optimize sensitivity in indirect detection, the concentration of the mobile phase additive should be kept as low as possible and the ratio of background signal to background noise (dynamic reserve) should be as large as possible. Another important factor is the transfer ratio, the number of molecules of the background buffer additive that are displaced by a molecule of analyte. For the best sensitivity, this ratio should be large.
Indirect detection is "universal," although limited to ionic compounds; however, its nonspecificity can also become a limitation, because of ionic components of a mixture can potentially interfere with the analytes of interest. Thus, lacking detection selectivity, the whole selectivity of the method relies on separation. Other drawbacks of this detection mode are inferior sensitivity (almost constantly lower than with the corresponding "direct" mode) and a rather narrow range of linearity.
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