Optimised Extraction and Comprehensive Chromatographic-Spectral Analysis of Anthocyanins from Hibiscus sabdariffa Calyces

Abstract

Anthocyanins are naturally occurring polyphenolic pigments with valuable chromatic, antioxidant, and analytical properties. This study optimized the extraction of anthocyanins from Hibiscus sabdariffa L. calyces and characterized them using low-cost chromatographic and spectroscopic techniques. Methanol proved superior to ethanol as an extraction solvent, yielding 12.4 ± 0.6% crude extract by dry calyx weight, with excellent pigment stability over 72 hours at 4 °C. Total anthocyanin content, determined via the pH differential method, was 185.6 ± 4.2 mg cyanidin-3-glucoside equivalents per 100 g dry calyces. Thin-layer chromatography (TLC) revealed a predominant band with Rf = 0.93 ± 0.02 in the crude extract, with systematic pH-dependent shifts from 0.55 ± 0.06 at pH 1–3 to 0.38 ± 0.04 at pH 12–14, corresponding to flavylium, carbinol, chalcone, and quinoidal forms. Colorimetric evaluation demonstrated reproducible transitions from pink (pH 1–3) to faint pink/light color (pH 4–7) and green hues (pH 8–14), confirming the potential of these pigments as natural pH indicators. UV–visible spectroscopy revealed a characteristic absorption maximum around 520 nm under acidic conditions, with absorbance values ranging from 0.451 ± 0.012 (pH 1) to 0.734 ± 0.015 (pH 2), decreasing at neutral pH and partially recovering under alkaline conditions. These results demonstrate that simple solvent extraction combined with TLC and UV–visible spectroscopy provides an effective framework for the quantitative and qualitative analysis of roselle anthocyanins, supporting their application in food, sensor, and educational settings.