Investigating Flavonoids by HPTLC Analysis Using Aluminium Chloride as Derivatization Reagent

This is the first study to report on high performance thin layer chromatography (HPTLC) generated spectrophotometric data to systematically capture flavonoid compounds using optimized derivatization with either AlCl₃ or NaNO₂-AlCl₃-NaOH as visualisation reagents. While the traditional AlCl₃ colorimetric method using UV–Vis analysis provides valuable insights into the presence of flavonoids and allows derivation of the total flavonoid content (TFC) of a sample, HPTLC fingerprints obtained after spraying with AlCl₃ or NaNO₂-AlCl₃-NaOH enable the visualization of the various flavonoids present in a sample based on their respective absorption shifts, thus complementing the traditional TFC assay. In this study, 40 different flavonoids representing different classes (flavonols, flavanolols, flavan-3-ol, flavones, flavanones, and isoflavonoids) were analysed. Upon derivatization with AlCl₃ most of the investigated flavonoids recorded bathochromic shifts, yielding characteristic λ_max values between 370 and 420 nm, while spraying with NaNO₂-AlCl₃-NaOH triggered hyperchromic shifts, and thus an increase in absorbance intensity in flavonoids with particular substitution patterns. A few non-flavonoid components with structural similarities to flavonoids (e.g., rosmarinic acid, gallic acid, aspirin, salicylic acid) served as the negative control in this study to determine whether the derivatization reagents allowed exclusive detection of flavonoids. The method was then applied to the analysis of flavonoid containing supplements as well as red clover honey to demonstrate the method’s application in the analysis of natural products.