jgg FLAVONOIDS AND RELATED COMPOUNDS 



Methyl ethers are also hydrolyzed to phenols by this procedure so that the position of 

 methoxy groups cannot be determined. However, methoxy groups are retained if 10% 

 barium hydroxide in a hydrogen atmosphere is used for the cleavage. A longer reaction 

 time is required for the latter method, but in some cases it is obviously preferable. Neu 

 and Neuhoff (57) and Dunlap and Wender (58) have applied this technique to microquantities 

 (20-40 fj,g) of flavonoids, using paper chromatography to identify the split products. The 

 position of sugar attachment in glycosides may be determined by methylating all free hy- 

 droxyl groups with methyl suKate, removing the sugar by acid hydrolysis, and locating 

 the position of the now freed hydroxyl group. Paper chromatography can also be applied 

 to identification of the partially methylated aglycone. Chandler and Harper (59) have de- 

 veloped a procedure for identifying the type and location of sugars in flavonoid glycosides. 

 It depends on selective oxidative splitting of the glycoside and identification of the sugar 

 residue that is released. Hydrogen peroxide splits off sugars attached at C-3 whereas 

 permanganate or ozone releases sugars attached to an aromatic system. 



Paper chromatography of flavonoids has been widely used in recent years. The 

 most popular solvent has been butanol-acetic acid-water (4: 5: 1) although water is useful 

 for moving glycosides away from aglycones. In organic solvents the Rf value decreases 

 with hydroxylation of the molecule. Bate-Smith and Westall (60) have discussed the vari- 

 ables affecting chromatographic behavior of phenolic compounds, using the polyphenols of 

 green tea. General reviews on the chromatography of flavonoids have been presented by 

 Harborne (61) and by Roux and Maihs (62). Roux and Evelyn (63) have used chromatogra- 

 phic behavior to estimate molecular weights of condensed tannins. 



Geissman (10) has outlined a routine procedure for examining paper chromatograms 

 of flavonoids: 



1. Note visible spots (anthocyanins, chalcones, aurones). 



2. Examine in long wave ultra-violet light--some substances fluoresce (flavonols, 

 chalcones) others absorb and appear as dark spots against the fluorescence of 

 the paper (flavonol glycosides, anthocyanins, flavones). 



3. Expose to ammonia vapor while examining in ultraviolet light --flavones and 

 flavonol glycosides fluoresce yellow, flavonones appear pale yellow, catechins 

 pale blue. 



4. Reexamine in white light in presence of ammonia vapor --flavones appear yellow, 

 anthocyanins blue-gray, chalcones and aurones orange-red. 



A few other spray reagents are worthy of mention. Diazonium salts react with all phenols 

 to give colored azo dyes. The diazonium salts that have been used most frequently are 

 prepared from benzidine, p-nitroaniline, or sulfanilic acid. Paranitrobenzenediazonium 

 fluoborate is especially convenient since it is a stable compound that can simply be dis- 

 solved in water before use. Compounds other than phenols may react (e. g. histamine), 

 but not many of them are likely to be encountered in flavonoid preparations. 



Flavanones and flavanonols may be detected because they show up as purple spots 

 when sprayed with 4% Rhodamine B in 0. IN HCl. Flavones and flavonols do not react (64). 



The vanillin-hydrochloric acid reaction is valuable for identifying phloroglucinol or 

 resorcinol derivatives which do not have a carbonyl group next to the ring (e. g. catechins 

 and leucoanthocyanidins). This reagent gives a pink color with such compounds (65). 



The phlobaphene reaction of catechins and leucoanthocyanidins is best applied to 

 paper by spraying with p-toluenesulfonic acid and heating (66). The former give brown 

 spots, the latter pink. Other flavonoids may also have their colors intensified by this 

 reagent without heating. 



