Diode array instruments

If the wavelength of maximum absorption of the analyte (Xmax) is known, it can be monitored and the detector may be considered to be selective for that analyte(s). Since UV absorptions are, however, generally broad, this form of detection is rarely sufficiently selective. If a diode-array instrument is available, more than one wavelength may be monitored and the ratio of absorbances measured. Agreement of the ratio measured from the unknown with that measured in a reference sample provides greater confidence that the analyte of interest is being measured, although it still does not provide absolute certainty. 

In order to observe a short-lived species it may be necessary to employ a rapid-scanning spectrometer, such as a diode-array instrument (Sms for a 240nm-800nm spectrum). In addition, the absorbances of electrogenerated species can be very small and signal-averaging or phase-sensitive detection may be necessary to achieve the required signal-to-noise ratio (cf. EMIRS and FTIR). 

Insufficient resolution leads to a decrease in the extinction coefficient across the wavelength axis, and therefore inaccurate quantitation results. The sensitivity of the measurement is also compromised. From a qualitative point of view, the fine features in the spectrum may be lost. The resolution of a UV-Vis spectrophotometer is related to its spectral bandwidth (SBW). The smaller the spectral bandwidth, the finer the resolution. The SBW depends on the slit width and the dispersive power of the monochrometer. Typically, only spectrophotometers designed for high-resolution work have a variable slit width. Spectrophotometers for routine analysis usually have a fixed slit width. For diode array instruments, the resolution also depends on the number of diodes in the array. 

Instrumentation for UV-vis process analysis falls into four categories scanning instruments, diode-array instruments, photometers, and fiber-optic diode-array and CCD instruments. The former two are more typically encountered in at-line or near-line applications, whereas the latter two are better suited to actual on-line analyses. 

UV is the most popular detector but has limited usage and is not universal as not all compounds absorb in that range. Current instruments can be set at specific wavelengths but care must be exercised in the choice of solvent to be used since some solvents are not transparent to UV. The new photo-diode array instruments are very useful because they can acquire a full spectrum and data manipulation can be performed to aid in the detection of unresolved peaks at one specific wavelength. 

Figure 7.15 HPLC-UV diode array instrument and chromatograms, (a) HPLC diode array UV detector system (b) UV spectra recorded at three points on an HPLC peak to enable peak purity to be determined (c) Isometric map obtained by plotting successive spectra from an HPLC separation of polynuclear aromatics. A, naphthalene B, fluorene C, anthracene D, chrysene.

Stray light generated by Fresnel reflection on lens surfaces, air bubbles in glass, and diffraction at aperture edges is less important in diode array instruments compared to conventional spectrometers because of less complex construction and lower optical surfaces. The stray light in diode array... 

Diode array instruments have no main moving optical elements therefore, no mechanical errors or drift arise. Consequently, the widely accepted rule in scanning spectrometry emphasizing that an accurate quantitation requires the use of the absorption maximum as analytical wavelength is no longer critical for diode array instruments. Therefore, such a choice, especially in the case of multicomponent analysis, should be focused only on reasons related to selectivity. 

Diode array instruments have reversed optics (see Figure 11), which means that the sample is placed in front of the narrow-acceptance-angle entrance slit of the polychromator. Consequently, ambient stray light does not essentially affect measurements, even in the case of open sample area. Based on the same assumptions, fluorescence induced in samples affects diode array instruments to a lesser extent compared to the forward optics setup. 

Forward and reversed optics are also discussed in terms of influence on sample stability. Reversed optics increases the probability of decomposition of photolabile compounds, because the whole wavelength range is passed through the sample. These are by no means conclusive results, as it is difficult to compare effects of a single full wavelength fast exposure of the sample (produced with a reversed optics instrument) with those of a cumulative long-term exposure made by a scanning instrument. Both examples of induced sample decomposition as well as successful analysis of well-known photosensitive compovmds by means of diode array instrumentation have been reported in the literature. 

Figure 11 Comparative influence of ambient stray light and sample fluorescence on scanning and diode array instrumentation,...

Recently, diode array systems have been used in fast transient absorption or chemiluminescence measurements due to their capability of providing extensive real-time spectral data. Enzyme kinetics as part of biochemistry relies on fast spectral multiwavelength acquisition. At low costs, diode array instruments are ideal for portable microfluidic bioanalyzers and emerging large-scale integrated microfluidic technologies. 

Online applications are by far the most important utilization of diode array spectrometry. High-performance liquid chromatography, supercritical fluid chromatography, capillary electrophoresis, and flow-injection techniques produce enhanced sensitivity and structure-related information due to coupling with diode-array-based detectors. Emission of the microwave-induced plasma generated in atomic emission detectors for capillary gas chromatography is also analyzed by means of UV-Vis diode array instruments. 

The highest sensitivity and selectivity in vitamin E LC assays are obtained by using fluorescence or electrochemical detection. In the former, excitation at the low wavelength (205 nm) leads to improved detection limits but at the expense of selectivity, compared with the use of 295 nm. Electrochemical detection in the oxidation mode (amperometry or coulometry) is another factor 20 times more sensitive. In routine practice, however, most vitamin E assays employ the less sensitive absorbance detection at 292-295 nm (variable wavelength instrument) or 280 nm (fixed wavelength detectors). If retinol and carotenoids are included, a programmable multichannel detector, preferably a diode array instrument, is needed. As noted previously, combined LC assays for vitamins A, E, and carotenoids are now in common use for clinical chemistry and can measure about a dozen components within a 10 min run. The NIST and UK EQAS external quality assurance schemes permit interlaboratory comparisons of performance for these assays. 

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