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Difference spectra have three distinct features as compared to absolute spectra:

• difference spectra may contain negative absorbance values;


• absorption maxima and minima may be displaced and the extinction coefficients are
  different from those in peaks of absolute spectra;


• there are points of zero absorbance, usually accompanied by a change of sign of the
  absorbance values. These points are observed at wavelengths where both species of
  related molecules exhibit identical absorbances (isosbestic points), and which may be
  used for checking for the presence of interfering substances.

Common applications for differenceUV spectroscopy include the determination of the

number of aromatic amino acids exposed to solvent, detection of conformational changes

occurring in proteins, detection of aromatic amino acids in active sites of enzymes, and

monitoring of reactions involving ‘catalytic’chromophores (prosthetic groups, coenzymes).

Derivative spectroscopy

Another way to resolve small changes in absorption spectra that otherwise would

remain invisible is the usage of derivative spectroscopy. Here, the absolute absorption

spectrum of a sample is differentiated and the differentialdxA/dlxplotted against the

wavelength. Since the algebraic relationship betweenAandlis unknown, differenti-

ation is carried out by numerical methods using computer software. The usefulness of

this approach depends on the individual problem. Examples of successful applications

240

0

0

Absorbance

260 280 300 320

240 260 280 300 320

Absorbance

Wavelength (nm)

Fig. 12.7Top: Absolute spectra of ubiquinone (solid curve) and ubiquinol (dotted curve). Bottom: Difference spectrum.

492 Spectroscopic techniques: I Photometric techniques