Separation of anthocyaninic and non-anthocyaninic flavonoids by liquid-liquid extraction based on their acid-base properties: a green chemistry approach

Abstract

Anthocyanins are natural pigments belonging to the flavonoid family that originate the red, blue and purple colors of fruits, vegetables and flowers. Their structure and color are dependent of various factors, like the pH. Anthocyanins appear red in acidic, purple in neutral, and blue in basic solutions, making them suitable to be used as natural pH indicators. Thus, petals of flowers could be an attractive pedagogic tool for laboratory activities due to the highly content of anthocyanins, but the presence of copigments such as flavonols and flavones modify the anthocyanins characteristic colors in pH studies. Therefore, a simple and ecological liquid-liquid extraction was developed for the separation of anthocyaninic and non-anthocyaninic flavonoids, based on the different acid-base properties of these two classes of pigments. Treatment with NaOH allowed the detection of non-anthocyanin flavonoids in the organic phase through the development of a deep yellow color. Colorimetric analysis of the aqueous anthocyaninic fractions enabled visualization of the typical colors from different forms of anthocyanins, demonstrating the efficacy of the separation method. It was also possible to distinguish if the red pigments of the flower were anthocyanins or betalains. Thus, the beautiful colors of flowers make them an accessible and attractive source of pigments that can be used to demonstrate acid-base base properties of organic molecules, solid-liquid and liquid-liquid extraction techniques, intermolecular interactions and solubility properties. This protocol could be implemented in practical classes of chemistry and biochemistry degrees, being particularly useful to teach fundamentals of organic chemistry and color chemistry.