Separation of Anthocyaninic and Nonanthocyaninic 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 generate the red, blue, and purple colors of fruits, vegetables, and flowers. Their structure and color are dependent on various factors, like the pH. Anthocyanins appear red in acidic, purple in neutral, and blue in basic solutions, making them suitable for use as natural pH indicators. Thus, petals of flowers could be an attractive pedagogic tool for laboratory activities due to the high content of anthocyanins, but the presence of copigments such as flavonols and flavones modifies the characteristic anthocyanin colors in pH studies. Therefore, a simple and ecological liquid–liquid extraction was developed for the separation of anthocyaninic and nonanthocyaninic flavonoids, based on the different acid–base properties of these two classes of pigments. Treatment with NaOH allowed the detection of nonanthocyanin 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 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 for teaching fundamentals of organic chemistry and color chemistry.

Composition and Antioxidant Activity of Anthocyanins and Non-Anthocyanin Flavonoids in Blackberry from Different Growth Stages

“…Anthocyanin and non-anthocyanin flavonoid fractions of blackberry were separated according to a previous study [ 14 ]. Samples (2 g) were ground in a grinder for 2 min, extracted with 20 mL of acetone solution for 2 h, and centrifuged (8000× g , 15 min) to collect a supernatant.…”

Chalcones: Potential Chemotherapeutic Compounds and Educational Tools for Closing the Loop in STEM

“…Our study is not the first time an undergraduate teaching lab has infused research into its curriculum. Dias has studied anthocyaninic and nonanthocyaninic flavonoids and a specific way to isolate them via liquid–liquid extraction, and their biological properties are noted . An integrated, multiuniversity undergraduate lab-based study of the cytotoxic properties of chalcones with respect to B16 murine melanoma cells was conducted by Lee and co-workers.…”

Resin Assisted Purification of Anthocyanins and Their Encapsulation

“…The unique property of this group of plant pigments, i.e., the pH-dependent color profile, has been successfully demonstrated as a simple and impressive experiment for high school and undergraduate students to teach the fundamentals of chemistry of pigments. 1,2 It is of more interest to the graduate and postgraduate students to learn the purification techniques of anthocyanins from the crude plant extracts. The interest in purified anthocyanins is not only their use as a natural food colorant, but also their excellent antioxidant properties.…”