The Detection Challenge

The internal diameters of the capillaries employed in CE range from 100 |im down to about 5 |im and a single analyte zone is approximately 1 mm long. Because the detection volume has to be smaller than the peak volume available, detection volumes range from about 1 pL to 1 nL. In high performance liquid chromatography (HPLC), in contrast, detection volumes of at least 1 | L are available. One would therefore expect sensitivities for CE to be several orders of magnitude lower than those in HPLC and, as a consequence, the detection limits to be much inferior. However, in CE the sample does not experience significant dilution before it reaches the detector, as is the case in HPLC, because of the flat flow profile in CE. Therefore, the sensitivities are not in fact as significantly degraded in comparison with HPLC as might be expected. Nevertheless the issue of detection limits is still critical in CE and detector sensitivity is not always adequate. Preconcentration by electros-tacking is sometimes advocated, but this method is only possible for samples with low ionic strength and generally leads to poor precision unless an internal standard is employed.

Because of the small detection volumes, on-column detection schemes are required to avoid band broadening, rather than detector cells attached in an off-column arrangement as is the case in chromatogra-phy. A unique property of detection in electrophor-esis, which is not shared with chromatography, is the fact that there is a dependence of the peak area (expressed on a time basis) on migration velocity. However in practice this is usually of no concern. Detection methods may be grouped according to whether a bulk property of the solution (such as conductivity, refractive index) or a specific attribute of the analytes (such as optical absorption or fluorescence, redox activity or membrane permeability) is monitored. Detectors used in the first case tend to be more universal, but generally suffer from the presence of a large background signal against which small changes have to be distinguished. This often leads to poor signal-to-noise (S/N) ratios and hence relatively high detection limits. The exploitation of specific interactions is generally better in this regard, but each method is usually only applicable to a certain class of analytes. Some of the specific detection methods also allow additional information on the analyte to be gathered, which may be desirable as migration times can never be taken as absolute proof of identity. These detectors may be termed 'information rich', and include for example mass spectrometers, photodiode arrays and voltammetric detectors.

Important general characteristics of detectors are their sensitivity, dynamic range, and linearity. The term sensitivity generally denotes the gradient of the calibration curve but the precision of the measurement (S/N ratio) has to be considered as well for a complete evaluation. Often, the term sensitivity is used to indicate the lowest concentration that may be detected (limit of detection, LOD) and these parameters are of course interrelated. In CE detection limits are sometimes quoted in terms of the detectable mass or number of moles, as impressive figures in the pico- or atto-gram or -mole range can be given because of the small sample volumes used. However, the standard concentration limits are much more useful and meaningful. The dynamic range is encompassed by the detection limit and by a maximum where a loss of sensitivity occurs. Wide dynamic ranges are desirable as they simplify sample preparation, but they often go hand-in-hand with relatively poor precision. The upper concentration limit in capillary electrophoresis is generally determined by the ionic strength of the background buffer (typically 1-10 mmol).

The choice of detector is guided by the requirement of the application in terms of detection limit, selectivity and information requirements but to a large degree also by commercial availability, cost, robustness and ease of use. Some features of the major detection methods are summarized in Table 1.

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Solar Panel Basics

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