A difference spectrum is an absorbance spectrum obtained by subtracting that of one substance from that of another. For example, a difference spectrum may be plotted of the absorbance spectrum obtain ed from reduced cytochrome c and subtracting the absorbance spectrum from the same concentration of cytochrome c in its oxidised state. The difference spectrum may be used to quantify the amount to which the cytochrome c is reduced. This can be achieved with the aid of a reference spectrum (or spectra) and the least squares method.
MitoPedia methods: Spectrophotometry