Anthocyanins are a large group of biochemical pigments responsible for many red to dark purple colours in fruits and vegetables. They are potent antioxidants when consumed in the diet, largely found to scavenge prooxidants via hydrogen or electron transfers. Furthermore, foods with bright colours are suggested to stimulate appetite and digestion and are considered markers of fresh produce quality to consumers. Berries, red cabbage, and purple/black carrots are some of the produce rich in anthocyanins, from which pure standards of the pigments are often sourced. Anthocyanins are flavonoids that structurally consist of aglycones (anthocyanidins), sugars, and often acyl group(s). They are highly reactive molecules and face reversible chemical structure changes depending on pH. This alters the visual coloration emitted by the pigments – and thus their respective absorbance spectra – depending on the medium in which they are suspended. For this reason, anthocyanins may be esteemed suitable alternatives to synthetic indicators. The pH differential method determines the total monomeric anthocyanin content of a sample from the chemical structure differences observed between pH:1 (bright red) and pH:4.5 (purple-blue). In acid environments, the absorption spectra of anthocyanins within the visible range are between 465 and 550 nm, the wavelength of maximum absorption being 520 nm. This pH differential method protocol was adapted from Hamilton-Amachree & Etasi (2019), for use with a spectrophotometer.

This protocol is a part of research modelling the pigment-colour relationships between image analysis data and wet-lab pigment quantification of 16 carrots varieties of different colours. Associated to this research is a literature review (with its protocol).