Micro determination of eugenol, thymol and vanillin in volatile oils and plants

1. E. Y. Backheet Article first published online: 21 DEC 1998 DOI: 10.1002/(SICI)1099-1565(199805/06)9:3<134::AID-PCA398>3.0.CO;2-9

Copyright 1998 John Wiley & Sons, Ltd.

Issue

Phytochemical Analysis Volume 9, Issue 3, pages 134140, May/June 1998

Abstract
Two simple, rapid and sensitive spectrophotometric methods are described for the determination of some phenolic compounds in volatile oils and plants. The first method is based on their reaction with aqueous sodium nitrite (5%) and hydrochloric acid (10%) solutions, followed by the addition of sodium hydroxide (10%) solution to give redyellow coloured products measured at 450, 396 and 385 nm for eugenol, thymol and vanillin, respectively. The method is highly selective for eugenol: Beer's law is obeyed typically in the concentration range 1100 g/mL. The second method depends on the addition of a known excess of standard potassium permanganate in neutral solution and measuring (at 520 nm) the excess unreacted after 5 min at 25 5C. The decrease in absorbance was found to be proportional to the amount of eugenol, thymol and vanillin reacted: Beer's law is obeyed typically in the concentration range 0.510 g/mL. The methods could be applied to the determination of eugenol in clove oil and dried flower buds, thymol in thyme dried herb, and vanillin in vanilla pods. The results obtained were comparable with those from the official methods. Menthol, anisole and methyl salicylate were found not to interfere with the proposed method. 1998 John Wiley & Sons, Ltd.

A method for the assay of eugenol in caraway oil has been suggested by Thorns.1) The method is based on the separation of eugenol as benzoyl eugenol. It enjoys the advantage that a melting point determination reveals identity as well as purity of the separated phenol. Somewhat later Thorns2) modified this method by removing the sesquiterpenes, which interfere at times, before adding the benzoyl chloride. He enlarged upon it by taking into consideration the eugenol acetate occurring in the oil. Assay of total eugenol. 5 g. of oil of cloves, contained in a 150 cc. beaker, are saponified with 20 g. of 15 p. c. caustic soda solution by heating on a water bath for half an hour. When still warm the contents of the beaker are transferred to a separating funnel with a short tube. After complete separation of the two layers, the solution of eugenol sodium is transferred back to the beaker. The sesquiterpenes remaining in the separating funnel are washed twice, each time with 5 cc. of 15 p. c. caustic soda solution and the lye in each case added to the eugenol sodium solution. 6 g. of benzoyl chloride are now added and the mixture shaken thoroughly until a uniform mixture has been obtained. In a few minutes the ester formation has taken place with appreciable rise of temperature. Any excess of benzoyl chloride is destroyed by brief heating on the water bath. After cooling 50 cc. of water are added, the mixture heated until the crystalline ester has been liquified, and again allowed to cool. The supernatant, clear liquid is removed by filtration, allowing the crystalline cake to remain in the beaker. Again 50 cc. of water are added, heated on a water bath until the ester has melted, cooled and filtered. Finally, the ester is washed a third time in like manner with 50 cc. of water. The excess of caustic soda and sodium salts are then regarded as having been removed. 1) Berichte d. deutsch. pharm Ges. 1 (1891), 278. 2) Arch, der Pharm. 241 (1903), 592. Any crystalline particles that have been cought in the filter are retransferred to the beaker. The benzoyl eugenol, while still moist, is dissolved in 25 cc. of alcohol (90 p. c. by weight) with the aid of the heat of the water bath and gentle agitation. Even after the beaker has been removed from the water bath, the shaking is continued until the benzoyl eugenol has crystallized out. This takes place within several minutes. The temperature of the mixture is lowered to 17, the precipitate collected on a filter 9 cm. in diameter, and the filtrate collected in a graduated cylinder. About 20 cc. of filtrate are thus obtained. The alcoholic solution that has been retained by the crystalline magma in the filter is displaced by 90 p. c. (by weight) alcohol until the filtrate amounts to 25 ccm. The moist filter and precipitate are then transferred to a weighing dish (the latter together with the filter dried at 101 having been weighed previously), and dried at 101 until of constant weight. In as much as 25 cc. of 90 p. c. alcohol dissolve 0,55 g. of pure benzoyl eugenol at 17, this amount should be added to the amount weighed. If a stands for the amount of benzoic ester found, b for the amount of clove oil employed (about 5 g.), and if 25 cc. of alcoholic solution of ester are removed by filtration in accordance with the above directions, then the percentage of eugenol in clove oil is indicated by the formula

This formula results from the two equations: (Benzoyl eugenol) (Eugenol) 268 : 164 = (a + 0,55): the amount of eugenol found.

Assay of free eugenol. 5 g. of clove oil dissolved in 20 g. ether are quickly shaken out in a separating funnel with 20 g. of 15 p. c. caustic soda solution. The eugenol sodium solution is transferred to a beaker and the ethereal solution of sesquiterpenes is washed twice with 5 g. each of caustic soda solution of like strength. The united alkaline solutions are heated on a water bath for the purpose of driving off the ether and the benzoylation carried out in the manner described above. Thus the free eugenol as well as that present as ester can be determined quantitatively. Thorn's method can be applied equally well to other eugenol-containing oils provided they contain no free alcohols.