Scanning Detector

For fluorescent detection in multiple channels on microchips, scanning detectors and CCD cameras have been used. For instance, scanning LIF detection was first performed using a galvanoscanner which sequentially probed 16 channels [265] and 48 channels [557], 

FIGURE 7.3 The rotary confocal fluorescence scanner used to detect pCAE chip separations. Laser excitation at 488 nm (100 mW) is directed up through the hollow shaft of a computer-controlled stepper motor, deflected 1.0 cm off-axis by a rhomb prism, and focused on the electrophoresis lanes by a microscope objective. The stepper motor rotates the rhomb/objective assembly just under the lower surface of the microchip at five revo-lutions/s. Fluorescence is collected along the same path and spectrally, and is spatially filtered before impinging on the four-color confocal detector [980]. Reprinted with permission from the American Chemical Society. 

CCD cameras have also been used for multichannel fluorescent detection. However, the use of the CCD detector suffers from the need to read out the entire image content information in order to determine the pixel intensity at the flow channel location. Accordingly, a CMOS imager was used. This method offers direct control over individual pixels, and can provide much faster response times and longer integration times for those desired pixels [252]. 

On the other hand, the scanning confocal fluorescent detector has better sensitivity than the CCD detector because the confocal detector provides better spatial filtering of the fluorescent background of the substrate (e.g., glass) [108]. 

---

Potential of zero charge, 20, 23, 25, 66 Potential scanning detector, 92 Potential step, 7, 42, 60 Potential window, 107, 108 Potentiometry, 2, 140 Potentiometric stripping analysis, 79 Potentiostat, 104, 105 Preconcentrating surfaces, 121 Preconcentration step, 121 Pretreatment, 110, 116 Pulsed amperometric detection, 92 Pulse voltammetry, 67... 

Quantitative HPLC analysis was carried out on a Spectraphysics 8720 chromatography system, a rapid scan detector by Barspec on a Zorbax ODS column with acetonitrile water 75/25 as the eluent. 

A more popular approach for detemining peak purity has been the use of diode-array detectors which were first introduced in 1982 [50]. Peak homogeneity of similiar benzodiazepines [51] and theophylline have been determined by this technique [52]. Rapid-scan detectors are also useful in confirming the presence of known components such as colorants, which are commonly used in drug products [53,54] and identification of related substances [55,56]. 

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. 

The scope of ultraviolet and visible spectrophotometry can be further extended when combined with a chromatographic separation step such as HPLC. The development of rapid-scanning detectors based on the linear photodiode array permits spectra to be acquired during the elution of peaks. Computer-aided manipulation of these spectra has led to new strategies for the examination of chromatographic peak homogeneity, based on classical techniques in spectroscopy. The use of microcomputers enables the development of archive retrieval methods for spectral characterisation (A. F. Fell etal, J. Chromat., 1984, 316, 423-440). 

A spectrum in a specified ranalogue signals from eadi photodiode are digitised and transferred to a computer, where they e corrected for dark current response and transformed to absorbance. A number of digital techniques are available to increase sensitivity and to extend the use of rapid-scanning detectors to multicomponent analysis, reaction kinetics, tablet dissolution tests, process control, and detection in HPLC (A. F. Fell et al, Chrom-atographia, 1982, 16, 69-78). 

With diode array detectors or scanning detectors, the spectral peak homogeneity can be investigated. However, such an approach requires a difference in both the spectra and in the retention time of the coeluting substances. If this is fulfilled, detection of inhomogeneities with commercially available software is easy, if the concentration difference is not too large (Fig. 3B). However, impurities below 1% are difficult to recognize (Fig. 3C). 

Using DAD or scanning detectors it is possible to determine the purity of a peak by spectral peak purity analysis. Peak homogeneity can be demonstrated by a variety of methods. However, the most commonly used technique normalises and compares UV spectra from various peak sections. 

The detector cell is filled with the anthracene solution. The measuring wavelength is varied between 248 and 254 nm in 1 nm steps. The resulting absorbance is documented and its maximum is determined. As an alternative approach if a scanning detector is used, the UV spectrum can be registered and attached to the documentation. 

The limited availability of affordable commercial RSSF instruments has been an important factor that has prevented the widespread application of RSSF spectroscopy to the study of biological systems. However, in the past year, a significant change in the availability of commercial instrumentation hats come about. There currently are at least five manufacturers of computerized rapid-scanning detector systems. The choices in commercial instrumentation range from a mechanically scanned system with a single photomultiplier detector to photodiode array detector systems. This review includes descriptions of the currently available commercial systems. Because the authors experience in the field of RSSF spectroscopy is limited to the use of diode array detector systems and because most of the commercial instruments have appeared on the market just within the past 12 months, it has not been possible to make detailed performance evaluations and comparisons of the new commercial systems. 

UV diode array and fast scanning detectors not only enable the generation and use of libranes of known natural product standards, but can also enhance chromatographic resolution. This enhancement is due to peak punty assessment, which allows the researcher automatically to compare UV spectra at different time points across a peak of interest and thus detect the presence or absence of multiple poorly resolved components. This feature is generally included in the standard software of most commercially available diode array detectors. 

Closed-open systems can also be used for individual and differential kinetic determinations [56,57], a relatively unexplored area In which rapid scan detectors have great potential. 

---