Flavonoids reagent

The developed chromatograms are briefly immersed in or evenly sprayed with the appropriate reagent solution. Solution I is employed for flavonoids [1, 3] and solution II for mycotoxins [5, 8, 12], phospholipids, triglycerides and cholesterol [14]. 

To detect glycosides heat the chromatograms to 130—150°C for 15 min. Blue-grey zones are produced (detection limit prunasin 0.3—0.5 pg [18]). Flavonoids are better detected with a modified reagent of the following composition phosphoric acid (85%) — acetic acid — aniline — diphenylamine (20 ml - -100 ml + 5 ml + 5 g). 

Note Flavonoids react with the reagent even at room temperature [1] mycotoxins, steroids, purines, pyrimidines, cardiac glycosides and lipids only react on heating [2, 4-6]. Zirconyl sulfate can be used to replace the zirconyl chloride in the reagent this is reported to result in an increase in the sensitivity to certain groups of substances (e.g. cholesteryl esters, triglycerides) [4]. 

Note Monohydroxybenzene derivatives and vicinal hydroxymethoxybenzene derivatives do not react under these conditions but can be made visible by spraying afterwards with Folin-Qocalteu s reagent [1]. Flavonoids, anthraquinone derivatives and a-nitroso-B-naphthol yield a red color with alkalis alone [1]. Steroids and aromatic amines do not react [1]. 

Compounds with hydroxy groups, such as flavonoids, sugars, or hydroxy acids, often react with 1% diphenyl boric acid (3-aminoethylester in methanol or ethanol. Various colors are formed by many natural products Neu reagent). 

The vanillin method is based on the condensation of the vanillin reagent with proanthocyanidins in acidic solutions. Protonated vanillin, a weak electrophilic radical, reacts with the flavonoid ring at the 6- or 8-position. The vanillin reaction is affected by the acidic nature and concentrations of substrate, the reaction time, the temperature, the vanillin concentration, and water content (Sun and others 1998). 

Hypervalent iodine reagents have shown promising applications in the synthesis of flavonoids. Such an example has been covered in Section II,E (Scheme 60). This section deals with various conversions from flavanone substrates. 

Aqueous or methanolic ferric chloride is a general spray reagent for phenolic compounds and gives a blue-black coloration with flavonoids. Similarly, Fast Blue Salt B forms blue or blue-violet azo dyes. 

For quantitative analyses, scanning of the TLC plate with a densitometer provides good results. The flavonoids, both aglycones and glycosides, in Vaccinium myrtillus and V. vitis-idaea (Ericaceae) were determined after TLC and densitometry at 254 nm. " With suitable spray reagents, detection limits of 20 ng can be achieved by densitometry. ... 

Hiermann, A. and Bucar, E., Diphenyltin chloride as a chromogenic reagent for the detection of flavonoids on thin-layer plates, J. Chromatogr., 675, 276, 1994. 

Swain and Goldstein (6, 7) noted a rather large difference in the molar color yield from different phenols with the Folin-Denis reagent. They attributed this to differences in relative oxidation-reduction potentials of the different phenols, but under their conditions pyrogallol gave about half the color of catechol and more than resorcinol. However, they also reported that the molar absorptivity produced by a flavonoid was approximately equal to the sum of the values for the separate phenolic moieties which it contained. 

Benzene-induced shifts of methoxy protons have proved of value in the structural elucidation of various flavonoids (68JCS(C)2477). Additionally, information on the position of hydroxy groups follows from ASIS shown by the trimethylsilyl ethers of the flavonoids (72P409). Lanthanide shift reagents also contribute usefully to structural assignments, notably with biflavones (75JCS(P1)1563). 

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