TERPENOIDS AND STEROIDS 161 



CHA RA CTERIZA TION 



There is no single test which will distinguish terpenoids and steroids as a group 

 from all other plant constituents. The closest approach to this goal is to describe them 

 all as unsaponifiable lipids although such an operational grouping will include a few other 

 types of compounds. There is no simple test to distinguish between the volatile terpenoids 

 and such unsaponifiable aromatic compounds as eugenol or cinnamaldehyde. Classically 

 hydrogenation to known cyclohexane derivatives or pyrolysis of terpenoids to form iso- 

 prene have been used to make such a differentiation. Infra-red spectra also indicate 

 whether a compound is aromatic or aliphatic. Characterization of the lower terpenoids 

 usually depends on their functional groups rather than on the carbon skeleton. A stand- 

 ardized procedure for identification of sesquiterpenes using distillation and chromatography 

 on alumina has been developed by Pliva et al. (63). Paper chrortiatographic identification 

 is based on recognition of functional groups. Because of their volatility and non-polar 

 nature ordinary paper chromatography of the lower terpenoids is difficult. Carbonyl com- 

 pounds may be chromatographed as their bisulfite complexes or reversed phase chroma- 

 tography used with silicone impregnated paper. A method using glass strips coated with 

 adsorbent rather than paper has been developed by Kirchner e/ al. (64). The best ad- 

 sorbent layer was found to be silicic acid and the best general purpose solvent 15% ethyl 

 acetate in hexane. Most compounds could be detected by spray reagents specific for func- 

 tional groups. Unreactive materials were detected by charring with a sulfuric-nitric 

 acid mixture and heat. This technique has been applied to terpenes oi Mentha piperita 

 (65). A general spot test for essential oils has been proposed by Hayashi and Hashimoto 

 (66). They touch a spot of oil on filter paper with 20% sodium bisulfite and then with 10% 

 antimony pentachloride in chloroform. Different constituents give rise to characteristic 

 colors. Vapor phase chromatography will probably find increasing application for sepa- 

 rating small quantities of volatile terpenoids. However, identification must depend on 

 comparison with the elution time of knowns or on isolation and characterization by other 

 techniques such as infrared spectrophotometry (67,68). 



The various structural types of the higher terpenoids and the steroids have classi- 

 cally been recognized by the aromatic hydrocarbons formed on dehydrogenation. Dehy- 

 drogenation is carried out either catalytically with palladium or by heating with sulfur or 

 selenium at about 300° to form hydrogen sulfide or selenide as the other product. Yields 

 in such reactions are usually very low; but if an identifiable product is obtained, it may 

 be enough to determine the ring structure of the starting material. This procedure has 

 already been mentioned in discussing the sesquiterpenoids where naphthalene and azulene 

 types are recognized according to which of these two ring systems is produced on de- 

 hydrogenation. The azulenes are readily recognized by their blue color or specific ab- 

 sorption spectra. The different types of azulenes must be identified by comparison with 

 knowns. Similarly, the tetracyclic triterpenoids yield 1, 2, 8-trimethylphenanthrene, 

 pentacyclic terpenoids yeild picene or naphthalene derivatives, and steroids yield Diet's 

 hydrocarbon (methylcyclopentanophenanthrene) along with several other products. It will 

 be noticed that tertiary methyl groups are lost as a result of aromatization. 



Eudesmol Eudalene 



