The Diode Array Detector

The diode array detector also utilizes a deuterium or xenon lamp that emits light over the UV spectrum range. Light from the lamp is focused by means of an achromatic lens through the sample cell and on to a holographic grating. The dispersed light from the grating is arranged to fall on a linear diode array.

The resolution of the detector (AX) will depend on the number of diodes (n) in the array, and also on the range of wavelengths covered (X2 — Xi). Thus:

Figure 3 The multiple-wavelength dispersive UV detector. Courtesy of the Perkin Elmer Corporation.

tion of the intensity of the transmitted light. The detector is usually fitted with a scanning facility that, by arresting the flow of mobile phase, allows the spectrum of the solute contained in the cell to be obtained. Due to the limited information given by most UV spectra, and the similarity between UV spectra of widely different types of compounds, the UV spectrum is not usually very reliable for structure elucidation.

UV spectra are, however, useful for determining the homogeneity of a peak by obtaining spectra from a sample on both sides of the peak. The technique is to normalize both spectra, then either subtract one from the other and show that the difference is close to zero, or take the intensity ratio across the peak and show that it is constant throughout the peak.

A common use of the multiple-wavelength detector is to select a wavelength that is characteristically absorbed by a particular component or components of a mixture to enhance the sensitivity of the detector to those particular solutes. Alternatively, by choosing a characteristic absorption wavelength of the solute, the detector response can be made specific to the solute(s) and thus not respond significantly to other substances in the mixture that are of little interest.

The early multiple-wavelength, dispersive UV detector proved extremely useful, providing adequate sensitivity, versatility and a linear response. It was found, however, to be bulky (due to the need for a relatively large internal optical bench), required mechanically operated wavelength selection and a stop/flow procedure to obtain spectra 'on-the-fly'. The alternative diode array detector has all the ad

It is seen that the ultimate resolving power of the diode array detector will depend on the semiconductor manufacturer and on how narrow the individual photocells can be commercially fabricated.

A diode array detector is shown in Figure 4. Light from the broad emission source is collimated by an achromatic lens, so that the total light passes through the detector cell on to a holographic grating. In this way the sample is subjected to light of all wavelengths generated by the lamp. The dispersed light from the grating is then allowed to fall on to a diode array. The array may contain many hundreds of diodes, and the output from each diode is regularly sampled by a computer and stored on a hard disk. At the end of

Figure 4 The diode array detector.

Figure 5 Dual-channel plot from a diode array detector confirming peak purity. Chlorthalidone was isolated from a sample of tablets and separated by a reversed-phase (C18) column 4.6 mm i.d., 3.3 cm long, using a solvent mixture consisting of 35% methanol, 65% aqueous acetic acid solution (water containing 1% acetic acid). Flow rate: 2 mL min-1. Courtesy of the Perkin Elmer Corporation.

Figure 6 Separation of some aromatic hydrocarbons. Separation was carried out on a 3 cm long, 4.6 mm in diameter column packed with a 3 jim C18 sorbent. Courtesy of the Perkin Elmer Corporation.

Figure 5 Dual-channel plot from a diode array detector confirming peak purity. Chlorthalidone was isolated from a sample of tablets and separated by a reversed-phase (C18) column 4.6 mm i.d., 3.3 cm long, using a solvent mixture consisting of 35% methanol, 65% aqueous acetic acid solution (water containing 1% acetic acid). Flow rate: 2 mL min-1. Courtesy of the Perkin Elmer Corporation.

the run, the output from any diode can be selected and a chromatogram produced using the UV wavelength that was falling on that particular diode.

Most instruments will allow at least one diode to be monitored in real time, so that a chromatogram can be generated as the separation develops. This system is ideal, as by noting the time of a particular peak, a spectrum of the solute can be obtained by recalling from memory the output of all the diodes at that particular time. This directly produces the spectrum of the solute. The diode array detector can be used in a number of unique ways: one example is to use it to verify the purity of a given solute, as shown in Figure 5. The chromatogram, monitored at 274 nm, is shown in the lower part of Figure 5. As a diode array detector was employed, it was possible to ratio the output from the detector at different wavelengths and plot the ratio simultaneously with the chromatogram monitored at 274 nm. Now, if the peak were pure and homogeneous, the ratio of the absorption at the two wavelengths (those selected being 225 and 245 nm) would remain constant throughout the elution of the entire peak.

The top part of Figure 5 shows this ratio plotted on the same time scale as the elution curve, and it is seen

Figure 6 Separation of some aromatic hydrocarbons. Separation was carried out on a 3 cm long, 4.6 mm in diameter column packed with a 3 jim C18 sorbent. Courtesy of the Perkin Elmer Corporation.

that a clean rectangular peak is produced, confirming a constant absorption ratio at the two wavelengths. The wavelengths chosen to provide the confirming ratio will depend on the UV absorption characteristics of the substance concerned. Another interesting example of the use of the diode array detector to confirm the integrity of an eluted peak is afforded by the separation of the series of aromatic hydrocarbons shown in Figure 6.

It is seen that the separation appears to be satisfactory and, without further evidence, it would be reasonable to assume that all the peaks were pure. However, by plotting the absorption ratio, 250 nm/255 nm, for the anthracene peak it becomes evident that the tail of the peak contains an impurity as the clean rectangular shape of the ratio peak is not realized. The absorption ratio peaks are shown in Figure 7.

The presence of an impurity is confirmed unambiguously by the difference in the spectra obtained for

Figure 7 Curves relating the absorption ratio and elution time.

195 245 295

Figure 8 Superimposed spectra taken at the leading and trailing edges of the anthracene peak.

195 245 295

Figure 8 Superimposed spectra taken at the leading and trailing edges of the anthracene peak.

the leading and trailing edges of the peak. Spectra taken at the leading and trailing edge of the anthracene peak are shown superimposed in Figure 8. Further work identified the impurity to be 5% t-butylbenzene.

The diode array detector is now generally considered the most versatile and useful detector for everyday use in liquid chromatographic analyses. The performances of both types of multiple-wavelength detectors are very similar and typically have a sensitivity of about 1 x 10~7 g mL"1 and a linear dynamic range of about three orders of magnitude: 1 x 10~7 to 5 x 10~4 g mL-1. The device automatically records a spectrum at each sampling point and thus is extremely rapid. It is very suitable for use with fast separations completed in a few seconds.

The use of UV detection in capillary electro-chromatography and for detection in capillary elec-trophoresis has enjoined a novel cell design. As the peaks are only a few nanolitres in volume, the sensor volume must be commensurately small. A practical detector has been constructed by removing the polyamide coating from the surface of a short length of quartz capillary tubing, as shown in Figure 9.

UV light from a suitable lamp traverses the tube window and falls on to a photocell. As the solutes migrate across the window they are detected by light absorption in the usual manner. Considering the expression given in eqn [1] for the output of the detector, it appears that, due to the very short pathlength of the cell (c. 300 |im), the detector would be very insensitive. However, the loss of sensitivity caused by the reduced pathlength is partly compensated by the relatively high solute concentration in the peaks resulting from the very high column efficiencies achieved in capillary electrochromatography (c. 105 theoretical plates). The electronic circuitry used with

UV lamp

UV lamp

Figure 9 UV detector cell for capillary electrochromatography.

the microcell is basically the same as that used in the conventional larger cell detectors.

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