Paper Chromatography of Monohydroxyphenols in Vanilla Extract M. H. ANWAR Research Department, Pepsi-Cola Co., long Island City, N.
b Chromatographic separation and purification of monohydroxyphenols in vanilla extract were studied. Various methods of separation, concentration, and purification were employed. Millon's reagent, used as a spray, reacted with monohydroxyphenols on the paper chromatogram, producing colored compounds which facilitated their identification and estimation. Of the seven phenols separated, three had been reported previously and three others were identified in this study. A hypothesis was proposed for the formation of these simple phenols by the hydrolysis and oxidation of glucosides in vanilla beans.
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Y.
_ _ - LlOUlD FRGNT-
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0
CONIFERYL A L C O H O L - T E N T A T I V E
0
UNKNOWN
0
S A L I C Y L I C ACID
0
I
4
P.HYDRoxYBENZALDEHYDE
VANILLIN
0 0
P-HYDROXYBENZOIC ACID VANILLIC ACID
liquid constantly circulating through t h e bean. The first two extractions, which usually contained 70 t o 75% of the total strength, were not treated further. The final three evtracts n ere concentrated under vacuum a t about 50" C. The concentration was carried to such an extent that when the two portions were mixed, a fivefold extract was obtained, each fold corresponding to 10 grams of bean in 100 ml. of final volume. This study was confined to tn-o type. of bean, Mexican and Bourbon. The Tahiti bean \vas found to differ markedly from the above two and is therefore not discussed in this paper. Duplicate samples of the following grades of beans were extracted and investigated. Mexican. Buena, medium, ordinary, and
P
chromatography has been successfully employed in the past for identification of phenolic compounds in various systems. Armstrong ( 2 ) identified a large number of phenolic acids in human urine. Various solvents and detection techniques have been used in the separation and identification of phenols, aromatic acids, and related compounds in naturally occurring substances (3, 4. 6-8). Vanilla extract is a rich source of phenols, flavones. anthocyanins, etc. From the point of view of flavor, the monohydroxyphenols play a unique role in vanilla extract. These phenols contain other functional groups in their molecules, such as aldehyde, acid, and alcohol. Only a fen- of them, however, have been identified and evaluated. Aside from vanillin, vanillic acid has been shown to be present, especially in aged vanilla extracts. The presence of p-hydroxybenzaldehyde and its quantitative estimation have been demonstrated by Chovin et al. (6). During the study of authentic vanilla eltracts by paper chromatography, the presence of four, as yet unknown, monohydroxyphenols was observed. I n this study an attempt was made to separate and identifj- these compounds. APER
EXPERIMENTAL
Equipment. Chromatography was performed in a square t s n k equipped with accessories for descending techniques. Whatman S o . 1 paper was used exclusively. Perkin-Elmer automatic instruments were employed for 1974
ANALYTICAL CHEMISTRY
S T A R T I N G POINT
i
Figure 1 . Chromatogram of monohydroxyphenols present in vanilla extract
cuts Bourbon. Second, third, fourth, and cuts Some of the sample> nere freshly prepared and others had been aged a t room temperature for 4 to 5 pears.
PROCEDURES
Separation a n d Concentration of Monophenol Fractions. Since the presence of some of the water-soluble compounds (such as sugars, polyhydroxyphenols, gluco-ide.. etc.) distort3 t h e chromatographic pattern of monohydroxyphenols under study, by reducing the Millon reagent 7%it11 subsequent formation of black and brown area
