Table 810

Chemical Tests for Anthocyanin Characterization

Reaction Conditions (anthocyanin in)

Ethanol dissolved with HCl and

Mg (Wilstatter reaction) Ethanolic solution with AgNO3

(12% in water) Paper chromatography and treatment with ammoniac vapors Sodium methoxide (2.5% in methanol)

Sodium acetate (solid) AlCl3 (5% in methanol)

AlCl3 (5% in methanol) with HCl solution

Detection

Structural Characteristic Observed Characteristic y-Benzopyrone structure

(all flavonoids) Flavonoids with o-hydroxyl groups Flavonoids

Flavonoids with hydroxyl groups at 3- and/or 4'-positions Hydroxyl at 7-position Flavonoids with o-hydroxyl and/or 5-OH and/or 3-OH Flavonoids with o-hydroxyl

Colors from red to green

Formation of a silver mirror

Dark or fluorescent colors

Bathochromic change of spectra, and if intensity of visible bands does not decrease, then a 4'-OH is present Bathochromic change of the UV band Bathochromic change of spectra

The bathochromic change reverts after HCl addition

Source: With permission of CRC Press.

techniques for structural elucidation of anthocyanins will continue to be important, in particular when complex molecules must be analyzed.1,4,44,45

Chemical tests. For the characterization of anthocyanins, pigments are isolated and separated. They are hydrolyzed with hydrochloric acid to disrupt the glycosidic bonds; resultant anthocyanidins are further hydrolyzed to eliminate the methylated groups and their structure is verified by chemical tests (Table 8.10).1 The presence of aliphatic acids in the anthocyanin structure can be detected by electrophoresis because they form zwitterion structures.3 In anthocyanin structure it is frequent to have phenolic acids as esters; for their determination, concentrated extracts are hydrolyzed with 6 N HCl (100°C/60 min). The solution is extracted with ethyl acetate, evaporated under vacuum, and the residue prepared for analysis by HPLC.45 Reflectance colorimetry has been employed to produce a numerical value representing color. This approach has shown a good correlation with the chemical composition and pigment concentration.3,46

7. Anthocyanins as Food Colors

In the United States, extracts of grape are the only anthocyanin source approved by the FDA as a food colorant. The commercial preparations are enocyanin and lees (sediment of grape juice tanks). The grape color extract is approved to be used in nonbeverage foods, whereas grape skin extract (enocyanin) is permitted in beverages. Both of these grape extracts are used in agreement with GMP.47 In the Codex

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