Colorimetric or spectrophotometric methods have been used over the past
few decades for rapid and convenient estimation of certain classes of flavonoids in
fruits, vegetables, grains, raw herbal material, herbal formulations, and nutraceuticals.
This has resulted in a surge in the numbers of research articles discussing the use of
these methods for comparison between numbers of samples of the same kind, such as
analysis to find differences between various tea samples, food articles, raw drug
powders, etc. However, these methods are not selective since several factors influence
color development. Also, the reagents used to form the colored complex are not
specific to a certain class of compounds. There are studies performed where all
compounds belonging to a particular class do not react uniformly to the reagents used
in the method. Chelation using AlCl3
was used to develop deep yellow-colored
complexes of the flavonoids and absorbance was subsequently measured at 420 nm,
using quercetin as the standard. In a modification, potassium acetate was added after
the addition of AlCl3
, and the absorbance was measured at 415 nm, again against
standard quercetin solutions, wherein only flavones and flavonols were estimated. A
study conducted by our team proves that all flavonoids do not form complexes that
absorb at 420 nm, and each flavonoid shows variation in absorption maxima. Only
flavonoids with o-dihydroxy systems show good results, while others absorb at either
higher or lower wavelengths. This research work has been one of the top 20 most
downloaded articles in flavonoid chemistry since its date of publication. Catechins,
flavanones, and anthocyanins cannot be estimated using this method, due to either
inability to bind with AlCl3
in an appropriate manner or due to differences in
absorption maxima of the complex formed. Flavanones like naringenin, naringin, and
hesperidin have been estimated using the 2,4-dinitrophenyl hydrazine method. The
method does not work for flavonols and flavones. Estimation of catechins in tea
samples has been described where caffeine is removed from solution using extraction
by chloroform, and the absorbance of the aqueous phase is taken at 274 nm. The
technique however is flawed since the aqueous extract will also contain phenolic acids
like gallic, protocatechuic, and syringic acids, and a good amount of flavonols such as
quercetin and kaempferol, which also absorb around 274 nm. These phenolic acids and flavonols need to be removed before the estimation of catechins. The reaction of
flavanols like catechin and epicatechin with vanillin in presence of H2SO4
yields redcolored complexes that show absorptions around 500 nm, but certain matrices
interferences of proanthocyanins. Many flavonoid compounds occur in the form of
glycosides, where the presence of sugar molecules like glucose, rhamnose, galactose,
etc. can hamper complex formation responsible for color development. The effect of
hydrolysis can yield better results to remove the sugar moieties, and the aglycones can
be estimated. Another widely used method is the Folin-Ciocalteu method for estimation
of phenolics, developed by Folin and Denis in 1915, and modified by Singleton and
Rossi in 1965, where a blue-colored complex due to reduction of molybdenum by
phenolate ions formed in a basic medium. One major drawback of this method is that
the absorption maxima of the complex formed varies between 620 and 765 nm. Studies
also confirm that this assay is not specific to only phenolics, but can also react to
interferences of ascorbic acid, reducing sugars, certain metals, amino acids, and
reducing agents like NaHSO3
. Most results published in thousands of research papers
worldwide are erroneous due to a lack of knowledge of the actual chemical reactions
taking place in the estimation methods, and how the flavonoids react with the reagents.
Keywords: AlCl3 chelation, Anthocyanins, Flavones, Flavonols, Flavanols, Flavanones, Folin-Ciocalteau, UV-Vis Spectrophotometry.
