Using Spectrophotometry to Measure Anthocyanin Concentration: The pH Differential Method
We can take advantage of the pH-dependency of the anthocyanin absorption spectrum to accurately and rapidly measure the total anthocyanin concentration in complex mixtures like wines and fruit juices. Wines and fruit juices often contain both degraded pigments and additives that artificially enhance color. At a pH of 1.0, some of these substances absorb 520 nm light just as anthocyanins do. However, unlike anthocyanins, at a pH of 4.5, they do not undergo the structural changes that anthocynains undergo and do not become colorless; they continue to absorb 520 nm light. Thus, by taking into account the unique pH-dependency of anthocyanins, we can selectively measure anthocyanin concentration and eliminate the other interfering substances from the measurement.
The method used to do this is called the pH differential method. It is a simple method of measuring the absorption of 520 nm light (the Amax wavelength) by anthocyanin standards and wine and fruit juice samples at two pHs, 1.0 and 4.5. The absorbance values obtained at a pH of 1.0 are due to ALL substances in the sample that absorb 520 nm light, including anthocyanins. However, the absorbance values obtained at pH 4.5 are only due to the other substances, and not anthocyanins. To determine the absorbance by anthocyanins, one simply subtracts the absorbance values at 4.5 from absorbance values at 1.0.