Optical Methods

Optical detection methods are more widely employed than any other detection means. Commercial CE instruments with optical absorption detectors were introduced in 1989 and are available from a variety of instrument manufacturers. The detectors employed have often been adapted from devices used in HPLC and this may be part of the reason for the prevalence of the ultraviolet (UV) absorption detection method.

Table 1 Main detection methods for capillary electrophoresis

Method Features Detection limitsa (mol L 1)

Table 1 Main detection methods for capillary electrophoresis

Method Features Detection limitsa (mol L 1)

UV/Vis absorption

Readily available commercially

10"

-7

Indirect UV/Vis absorption

Compromise with poorer detection limits for nonabsorbing species

10"

-5

such as most inorganic ions

Fluorescence

Good detection limits but most species require derivatization;

10"

-9

available commercially

Laser fluorescence

Elaborate; excellent detection limits; available commercially

10"

-11

Conductometry

Good for small ions; available commercially

10"

-6

Amperometry

Simple, but only possible for electroactive ions; not available

10

-8

commercially

Mass spectrometry

Provides information on peak identity; expensive; interfaces

10

-8

available commercially aThe values given should be considered as rough guides only, as these are often very much dependent on species and instrumental set-up. UV/Vis, ultraviolet-visible.

available commercially aThe values given should be considered as rough guides only, as these are often very much dependent on species and instrumental set-up. UV/Vis, ultraviolet-visible.

Fluorescence-based detectors are not as widely used but are also on the market.

To carry out on-column detection the usual poly-imide protection coating has to be removed from the column by burning, by dissolution with hot sulfuric acid, or by mechanical scraping, to form a window into the capillary. The material is fairly brittle, so that care has to be taken to avoid breakage once the protective cladding has been removed. Fused silica capillaries are transparent even below 200 nm, so that the near-UV range is readily accessible.

The basic cell arrangement for absorbance measurement through a capillary is illustrated in Figure 1. Generally, besides the light source, there is a monochromator or optical filter to define the wavelength employed, a lens and aperture, and a photodetector. Variations of this arrangement are possible. Most commonly wavelengths in the UV range from about 250 nm down to 185 nm are employed, using different types of sources such as deuterium lamps, but instruments that include the visible range are also available. Variable wavelength as well as fixed wavelength arrangements are in use. It is important to get a high light intensity transmitted onto the detector for best S/N ratio. The usual UV light sources, such as deuterium lamps, are larger than the optical cell and it is only possible to focus

Figure 1 Schematic representation of absorbance detector. 1, Light source; 2, monochromator or optical filter; 3, lens; 4, aperture; 5, capillary; 6, photodiode or photomultiplier tube.

a small fraction of the radiation emitted through the cell even with the best available lenses. Ball lenses, mounted directly adjacent to the capillary, are often employed. Apertures are required to minimize the amount of stray light reaching the detector. Optical fibres can be used for transmission of the radiation as this allows efficient electrical shielding of the photodetector and at the same time the distal ends form the optical apertures. Absorbance detectors based on light-emitting diodes (LEDs) and laser diodes have also been demonstrated. These devices give high baseline stability because of the absence of flicker noise present in discharge lamps and allow the construction of battery operated instruments because of their low power consumption. However, these devices are not available for the UV wavelength range.

The circular cross-section of the capillary is far from ideal for absorbance measurements because it is not possible to pass collimated light through the interior of the tube without refraction. This means that changes in the refractive index of the solution are a potential source of interference. In practice, however, the only serious limitation appears to be the short optical pathlength, which leads to low sensitivity according to the Lambert-Beer law. For this reason the largest capillary diameters that allow efficient cooling are usually employed in absorbance detection, typically with an internal diameter of 50-75 |im. Different methods of increasing the sensitivity in absorbance detection have been described. These include the use of rectangular capillaries, capillaries bent in a Z-shape to obtain a longitudinal light path, multipass cells by multiple reflections in silver-coated capillaries, and so-called bubble cells formed on the capillary itself. Only the last approach is reasonably easily implemented, and it appears to be the only one that is commercially available (albeit at a cost much higher than that of ordinary capillaries).

The internal diameter of the capillary is widened in the detector region by a factor of about three, thereby increasing the sensitivity by the same magnitude.

A different approach to increase the sensitivity of absorption measurements is the use of thermooptic methods. Here the heat evolved following the absorption of light is sensed indirectly. In the thermal lens method the refraction of a laser light beam is measured, using a relatively simple arrangement. Two light beams perpendicular to each other are employed. One of the beams is of a wavelength that is absorbed by the analytes. The heat evolved through absorption of light leads to a refractive index gradient in the capillary which is monitored by the second beam. A variation on this technique has been reported that uses intensity-modulated light. This leads to a vibration of the capillary that may again be detected with a second probe beam. Ordinary refractive index detection has also been described using the deflection of a laser beam but neither of these two techniques has gained much acceptance.

Most organic analytes possess chromophoric groups that show intrinsic absorbance in the near-UV range, so most methods are based on this wavelength region. It has been demonstrated that for these species it is preferable to use wavelengths that are as short as possible (below 200 nm) for the best sensitivity. Photodiode-array detection is also possible. This technique yields additional qualitative information on the identity of the detected species and allows peak inhomogeneity to be detected. However, the S/N ratio and therefore the detection limit, which is always critical in CE, are degraded because of the reduced integration time available, and the method requires considerable computing power, because of the large amount of data acquired. Ions that do not show absorbance in the UV/Vis range, such as inorganic species or completely saturated organics, may be determined by indirect methods. These methods rely on the displacement of dye molecules of equal charge as the analyte species (to maintain electroneu-trality) so that a decrease in absorbance is detected. This is more demanding on the stability of the system than the direct absorbance method and the detection limits are generally higher. However it is the only method employing optical absorbance detectors to be available for most inorganic anions. Chromate is often used as the background ion but other species, some for the visible wavelength range, have also been reported. Inorganic cations can also be detected by indirect means, but many of them are best determined via the formation of coloured complexes using non-discriminating ligands.

Fluorescence detection is also possible and is commercially available. However, few species display in trinsic fluorescence, so derivatization reactions have to be employed. Derivatization may be classified as pre-column, on-column or post-column according to the scheme employed. Fluorescence has the great advantage of much higher sensitivity than absorbance measurements. Detection limits approaching single molecule detection have been achieved. Lasers appear to be ideal light sources for fluorescence measurements, as the light is produced in a tightly focused beam well matched to capillaries, but inexpensive sources are not available for the UV range and available lasers are often plagued by insufficiently stable output intensities. This limits their use, especially for the more universal indirect detection scheme. Nevertheless, impressive results have been obtained for microbiological applications (e.g. in neuroscience) that include the analysis of single cells. Chemiluminescence detection is usually based on the influence of analytes on the efficiency of one of several available chemiluminescence reactions. The achievable sensitivities are very high, a feature this method has in common with fluorescence. Its implementation is similar to post-column fluorescence detection in that a pumped reagent stream has to be merged with the column effluent in a suitable small-scale mixing device prior to detection in a light-tight enclosure with a photomultiplier tube.

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