General considerations and theoretical background
Molecular absorption in the UV and visible region arises from energy transitions that involve the outer orbital or valency electrons. Spectra in liquid media are usually broad, relatively featureless bands (Fig. 1C), a result of the large number of closely spaced vibrational and rotational transitions. The fundamental band shape approximates Gaussian or log normal Gaussian curves. Given the broad, over lapping profiles commonly encountered, the shape and precise location of individual bands are of limited usefulness in qualitative analysis. However, any fine structure detected in the spectra, coupled with changes due to solvent and pH effects (see Fig. 1), can be of diagnostic value. More informative spectra can be obtained for some volatile molecules of toxicological interest, such as benzene and polynuclear aromatic hydrocarbons. With examination in the vapour phase, vibrational and rotational fine structure can readily be seen superimposed on the broad spectral profiles. This is illustrated in Fig. 1 for 1,2,4,5-tetrazine. However, most drugs, metabolites and related compounds are relatively nonvolatile and hence spectra of these substances are measured in solution, or possibly in the solid phase by reflectance, or by compression to form a KBr disk, as used in infrared (IR) spectrophotometry.
One of the primary uses of UV and visible spectrophotometry is in quantitative analysis. For substances which do not absorb strongly in the UV-visible region of the radiation spectrum, the scope of absorption spectroscopy can be extended significantly by the use of colour reactions. Such reactions are used to modify the spectrum of an absorbing molecule so that it can be detected in the visible region, well separated from other interfering components in the UV spectrum, often with a concomitant increase in sensitivity and/or selectivity. Moreover, chemical modification can be used to transform an otherwise nonabsorbing molecule into a stable derivative that possesses significant absorption.
Figure 15.1 Absorption spectra of 1,2,4,5-tetrazine.
Spectral selectivity can be enhanced further by a number of chemical or instrumental techniques, which include difference, higher derivative and dual-wavelength spectrophotometry. Such methods, and certain graphic techniques such as the Morton–Stubbs method, can contribute in different ways to reducing the general problem of spectral interference in quantitative spectroscopy. Morton and Stubbs (1946) developed a method to take account of absorption by substances other than vitamin A in the quantitative analysis of this vitamin in cod-liver oil, where the absorption by the interfering substances was unknown and variable. Spectral interference can arise from so-called ‘irrelevant’ nonspecific absorption, and also from absorption by other materials and impurities that may be present. When interference arises specifically from the spectral overlap of two or more well defined components, a number of methods can be applied to measure the individualconcen trations. These methods include the Vierordt multiwavelength technique, least-squares deconvolution and second- or higher-derivative spectrophotometry.
Spectral selectivity, and in some cases detec tion sensitivity, can be enhanced significantly by the various chemical and instrumental tech niques outlined above. Such methods should, of course, be validated by applying the conventional analytical criteria of accuracy (against a reference method), linearity, precision and independence from interfering substances. The scope of UV and visible (UV-visible) spectrophotometry can be further extended when combined with a chromatographic separation step, such as high-performance liquid chromatography (HPLC). The LC eluent is directed post-column to a UV-visible spectrophotometer to enable the detection of substances absorbing in the UV-visible region. Traditionally, absorption was measured at a single wavelength, but the development of rapid-scanning detectors based on linear photodiode arrays permits spectra to be acquired during the elution of peaks. Computer-aided manipulation of these spectra has led to new strategies for the examin ation of chromatographic peak purity, based on classic techniques in spectroscopy. Figure 2A shows the relatively limited information available from measurement at a single wavelength compared with the additional information available from photodiode array detection (DAD) (Fig. 2B) (Lindholm et al. 2003). Spectral libraries of UV-visible spectra are available commercially and these, combined with computers enable the development of archive retrieval methods for spectral characterisation.
Figure 2 HPLC analysis of a sample taken from the genetically modified bacterial fermentation of the steroid progesterone to produce 9a-hydroxyprogesterone. (A) HPLC chromatogram from absorbance measured at 245 nm. (B) HPLC chromatogram from absorbance measured with diode-array detector (DAD) between 220 and 400 nm. Monitoring absorbance only at 245 nm did not permit the peak due to the product to be identified. The 3D plot from HPLC-DAD allowed the peak for 9a-hydroxyprogesterone to be clearly identified. (Reprinted from J. Lindholm et al., Use of liquid chromatography–diode-array detection and mass spectrometry for rapid product identification in biotechnological synthesis of a hydroxyprogesterone, J. Chromatogr. A, 2003, 992, 85–100 with permission from Elsevier.)
