FLAVONOIDS AND RELATED COMPOUNDS 199 



Horhammer and Miiller (67) have recommended a zirconium oxychloride reagent to 

 distinguish flavonols from flavones. The 3-glycosides of flavonols do not react with this 

 reagent and may therefore be distinguished by chromatogramming before and after hydrol- 

 ysis and applying this reagent. Spada and Cameroni (68) identified a 3-glucoside of myri- 

 cetin in this way. 



By combining spectrophotometric methods with paper chromatography, it is fre- 

 quently possible to make a positive identification of a flavonoid aglycone or glycoside in 

 all its structural detail. The amount of material in a chromatogram spot can be enough 

 for spectral measurements and frequently it is not even necessary to elute the spot. All 

 of the flavonoids have a more or less intense absorption band at about 220-270 m^ and 

 another strong band at a longer wavelength. Additional weaker bands may also be present. 

 Approximate locations of the long wavelength band for different flavonoids are as follows: 



anthocyanins 500-530 m/j. 



flavones and flavonols 330-375 m^. 



chalcones and aurones 370-410 m^ 



flavanones 250-300 mp. 



leucoanthocyanidins and 



catechins ca. 280 niju 



iso-flavones 250-290 m/i (very weak) 



Detailed presentation if spectral curves are given in the review of Geissman (10) which 

 also lists extensive references to other papers. The infra-red spectra of many different 

 flavonoids have recently been presented by Inglett (69). 



Addition of alkali causes characteristic spectral shifts with most flavonoids. Sodium 

 acetate and sodium ethylate have been used for this purpose (70, 71). Flavonols with free 

 hydroxyl groups in positions 3 and 4' are decomposed by alkali, and this can be followed 

 by the decrease in absorption at the long wavelength band. Sodium acetate causes a shift 

 of the short wavelength band to shorter wavelengths if a free 7-hydroxyl group is present. 

 Other examples are given in the review by Geissman (10). Harborne (72, 73) has given 

 a full description of the procedures for identifying anthocyanins by a combination of spec- 

 trophotometric methods with paper chromatography. 



Various methods have been developed for locating hydroxyl groups on the flavonoids 

 by utilizing reagents which produce spectral shifts with different hydroxylation patterns. 

 Jurd (74) has presented a method for detecting orthu dihydroxyl compounds by adding 

 borate which, by complexing with such groups, produces characteristic shifts in the long 

 wavelength band. H'orhammer and HUnsel (75) have used boron complexes in a similar 

 way for analysis of flavones, flavonols and chalcones. Aluminum chloride has also been 

 applied as a useful complexing agent. Roux (76) has measured absorption spectrum of 

 spots on chromatograms before and after treatment with 0. 2% aluminum chloride and used 

 the spectral shifts for identification of structures. Shifts of 20 or more m/i are charac- 

 teristic of urtliu dihydroxyl compounds. Flavanones can be distinguished from isoflavones 

 by the differences in spectral shifts which they show with aluminum chloride (77). 



ME TABOLIC PA THWA YS 



The primary precursors of the flavonoids proper are known beyond any doubt as a 

 result of many tracer experiments. Still unknown are the precursors of the less common 



