, 2000; online edn, Oxford Academic , 12 Nov. 2020 ), https://doi.org/10.1093/oso/9780199638130.003.0011, accessed 8 Sept. 2024.
Navbar Search Filter Mobile Enter search term Search Navbar Search Filter Enter search term SearchChapter 1 describes the principles and practice of spectrophotometry, and in this chapter we will consider how this technique can be used to measure the amount of analyte in solution, when the product of a reaction between the analyte and a ‘very useful reagent’ produces a measurable change in absorbance. Some good examples of this type of reaction are given together with various protocols for routine applications such as the measurement of protein concentration. If there are no contaminating species absorbing at the wavelength of interest, it may be possible to measure the analyte directly simply by taking absorbance measurements. Spectrophotometry has also proved to be fundamental for rate measurements using a wide variety of enzyme-catalysed and non-catalysed reactions. We will also see that it is often the case that we will use an enzyme reaction that has gone to completion to measure the amount of an analyte that is limiting in the reaction. Furthermore, in some cases we can get the reaction to cycle so that the amplified rates can provide a highly sensitive assay, see, for example, the Tietze method (1) for measuring glutathione (Section 3.3). We will also briefly cover the use of so-called ‘plate readers’ as these are being used increasingly as ‘multi-cuvette spectrophotometers’. Finally, ‘plate readers’ will be compared with ‘centrifugal analysers’, much beloved in clinical chemistry labs. The reader should note that some methods are dealt with in detail here to allow selected protocols to be used directly. Also, a number of useful reference works for enzyme assays are given, including (2-4), which should be consulted. With a little thought novel assays can often be developed based on the various ‘coupling’ methodologies described below. The design of standard spectrophotometers has been covered in Chapter 1 and we should simply mention that most modern ‘plate readers’ work in essentially the same way. The major difference is in their beam geometry. In spectrophotometers, samples are read through cuvettes with a horizontal light path which is normally 1 cm in length allowing the Beer-Lambert Law (see Section 1.2) to be applied in its simplest form (constant light path).
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