For nicotinamide nucleotides, flavin nucleotides, and cytochromes, the absorption spectrum for the reduced carrier differs from that of its oxidized counterpart (Figure 15.5).

From the absorption spectrum of a mixture of these carriers, the proportions of each in the oxidized and reduced states can be ascertained. The sensitivity of the technique is increased if a difference spectrum is obtained (Figure 15.8). To obtain a difference spectrum, the sample cuvette contains the mixture of electron carriers under study, and the reference cuvette is filled with not a blank, but an equimolar concentration of carriers in a known state (i.e., entirely oxidized). Thus, any small absorbance changes, either positive or negative, result from reduction of a portion of the carriers in the test sample.