Light Spectroscopy by Dr David Harris;

Author:Dr David Harris;
Language: eng
Format: epub
ISBN: 9781000140934
Publisher: CRC Press (Unlimited)
Published: 2020-01-15T00:00:00+00:00

6.8 How to improve resolution

The resolution of a spectrophotometer is its ability to distinguish between two closely spaced absorption bands. As noted above, this is maximized by (i) choosing a sufficiently narrow slitwidth (spectral bandwidth) so that the observed peak width is no broader than its natural bandwidth (Section 6.4.2) and (ii) choosing an appropriate scan speed and response time (Section 6.4.3). However, in systems whose spectra are complex because (i) multiple absorbing components are present and/or (ii) significant light scattering occurs, additional measures may be needed to resolve individual components.

6.8.1 Difference spectra

The most widely used technique for enhancing resolution is to measure a difference spectrum. In principle, the method is identical to that described above for subtracting a baseline; the spectrum of a reference is subtracted from that of the sample under study either in a single measurement (using a dual beam instrument) or sequentially, by storing the reference data in the instrument prior to measuring the sample (in a single beam instrument). The difference in method lies in the composition of the two solutions used: to measure a difference spectrum both sample and reference contain the chromophore under study, but in the sample, the chromophore has been chemically modified. Thus, for example, a difference spectrum of reduced minus oxidized cytochrome c is shown in Figure 6.7a, in comparison with the spectrum of each individually (see Chapter 1, Figure 1.8). The difference spectrum clearly shows that, on reduction, a peak appears at 550 nm, while absorbance falls at about 535 nm. Difference spectra (light minus dark) of chloroplasts reveal both cytochrome oxidation and the photochemical reaction centers, which are bleached (at 682 and 700 nm) on illumination (Figure 6.7b). Similar observations can be made, for example, during the reaction of a protein with a chromogenic reagent (Figure 6.8).

The increase in resolution resulting from this approach is clearly seen in Figure 6.9, where the spectrum of a highly turbid yeast suspension is shown. The ‘absolute’ spectra of the suspension, in both reduced or oxidized state, are dominated by light scattering from the solution and, as a result, appear featureless and very similar. However, in the reduced minus oxidized difference spectrum, a number of peaks are resolved, corresponding to various cytochromes within the cell (the peak at 550 nm representing cytochrome c, for example). The levels and redox potentials of these individual components in vivo can therefore be studied.

Referring back to Figures 6.6–6.8, features of difference spectra can be seen to include positive peaks (higher absorbance under sample conditions: appearance of a product), and negative peaks (higher absorbance under reference conditions: disappearance of a reactant). There are also points where absorbance is identical in both sample and reference conditions, namely, ΔA = 0 over the change studied and the difference spectrum crosses the baseline. These are isosbestic points (iso, equal; sbestos, extinguished) (see Section 1.4). Isosbestic points occur when there are only two species involved in the change, that is, a single reactant produces a single product. (Note that the presence

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