64 AROMATIC COMPOUNDS 



if oxygen, ultraviolet light and traces of metal ions are present (42). All simple phenols 

 show strong absorption of ultraviolet radiation in the range 270-280 m/i. 



Most of the common coumarins are strongly fluorescent when exposed to ultraviolet 

 light. Sen and Bagchi (43) determined the absorption spectra of various coumarins and 

 chromones, concluding that these two similar classes may be distinguished from each 

 other on the basis of their spectra. The paper chromatography of coumarins and furocou- 

 marins has been studied by Grujic-Vasic'(44). When coumarins are separated by paper 

 chromatography they may be detected by fluorescence or by spraying with Emerson's 

 reagent (0. 5% NazCOg, 0.9% of 4-aminoantipyrine, 5. 4% K3Fe(CN)6) (45). The fluorescent 

 spots may also be cut from the paper, eluted, and absorption spectra of the eluates deter- 

 mined for identification. Since coumarins (and other phenols) often occur as glycosides 

 it may be advisable to submit plant materials to acidic hydrolysis before attempting to 

 detect free coumarins. 



Furanocoumarins may be identified by the fact that oxidation with hydrogen peroxide 

 in sodium hydroxide produces furan-2, 3-dicarboxylic acid. Since all natural pyranocou- 

 marins have a 2, 2-dimethylpyran structure, they may be identified by the fact that ace- 

 tone is formed by prolonged alkaline hydrolysis. Alkaline hydrolysis does not affect the 

 furan ring of furanocoumarins. 



There is no simple test to distinguish lignans from other natural phenolic compounds 

 Lignans have been separated by paper chromatography using as solvents mixtures of for- 

 mamide with several other organic liquids (31, 46). Detection of the spots can be done 

 using diazotized sulfanilic acid or antimony pentachloride. 



The presence of lignin in plant tissues is easily recognized by such simple reactions 

 as the appearance of a bright red color when moistened with a saturated solution of phlo- 

 roglucinol in concentrated hydrochloric acid. Other cinnamaldehyde derivatives give the 

 same reaction but they can usually be removed by a preliminary extraction with acetone, 

 which does not dissolve lignin. Lignin containing sinapyl groups may be recognized by 

 the Maule reaction -- formation of a red color when treated successively with chlorine 

 water and ammonia (47). Gymnosperm lignin, which contains only coniferyl units, gives 

 a brown color in this test. For more complete identification of the units present in lignin 

 oxidation with alkaline nitrobenzene is used to degrade the lignin to benzaldehyde deriva- 

 tives. Stone and Blundell (48) developed a method using 50 mg. samples of wood placed 

 in stainless steel bombs with nitrobenzene and sodium hydroxide at 160° for 2. 5 hours. 

 When reaction was complete, 0. 2 ml. of reaction mixture could be spotted directly onto a 

 paper chromatogram and the products detected by spraying with 2, 4-dinitrophenylhydra- 

 zine. All lignins form vanillin by this treatment. Dicot lignins show syringylaldehyde as 

 well, and grass lignins usually form p-hydroxybenzaldehyde. 



One of the best -known tests for tannins is their precipitation of gelatin. A 0. 5% 

 solution of tannin is added to an equal volume of 0. 5% gelatin. All tannins show some de- 

 gree of precipitation, but other phenolic compounds may also give a positive test. The 

 sensitivity of the reaction may be increased by adjusting the pH to about 4 and adding some 

 sodium chloride. Other precipitation reactions with amines or metal ions have often been 

 used to characterize tannins. Like other phenolic compounds the tannins give blue-violet 

 colors with ferric chloride. 



Several tests are available to distinguish between the hydrolyzable tannins (gallotan- 

 nins) and condensed tannins (catechin tannins). Addition of 2 volumes of 10% acetic acid 

 and 1 volume of 10% lead acetate solution to a filtered 0. 4% tannin solution forms a precip- 

 itate with gallotannins within 5 minutes, but condensed tannins remain in solution. Other 

 special tests can be used to distinguish between different types of hydrolyzable tannins. 

 Several groups of investigators have applied paper chromatography to tannin mixtures. 



