Analyst, March, 1974, Vol. 99, fq5. 153-155 153 Detection of Curcuma zedoaria and Curcuma aromatica in Curcuma longa (Turmeric) by Thin-layer Chromatography BY A. R. SEN, P. SEN GUPTA AND N. GHOSE DASTIDAR (Central Food Laboratory, 3 Kyd Street, Calcutta-16, India) It is difficult to determine the genuineness or otherwise of powdered turmeric (Curcuvna longa) when it is admixed with C. zedoaria or C. aromatica. A simple and rapid thin-layer chromatographic technique is described that involves a three-step colour sequence for the detection of camphor and camphene, the active principles of these adulterants, which are absent in turmeric. TURMERIC is the dried rhizome or bulbous root of Curcuma Zonga Linn., a perennial herb of the ginger family (Zingiberaceae), which is extensively cultivated in India, as well as in China and the East Indies.It is not a true spice, but rather a condiment that is used to a very large extent in the preparation of curries, pickles and many spicy Indian foods. It is also one of the chief ingredients of curry powder. The adulteration of ordinary spices and condiments is exceedingly prevalent in India and probably the most subject to admixture is turmeric. The fact that this condiment is fre- quently offered for sale in a ground condition furnishes an opportunity for the incorporation of various cheaper vegetable substances. The use of a microscope is important for the detection of these additions, but when the adulterants belong to the same genus (Curcuma), even experts in microscopy find it difficult to decide whether or not a sample of turmeric is genuine.In general, turmeric can readily be identified by the brilliant greenish yellow colour of the “paste balls,” oleoresin cells that contain the valuable yellow colouring principle, curcumin. Curcumin is, however, also found in several other species of Curcuma,l e.g., C. aromatica Salisb. and C. domestica Valeton. “The Wealth of India”2 and the “Glossary of Indian Medicinal plant^",^ however, appear to be confused and class C. domestica with C. Zonga, while Watt* and Bailey5 did not mention it. Guenther6 categorically differentiated C. domestica as a different species. It may be mentioned here that similar confusion had also arisen when Guibourt’ classed C. caesia Roxb. as C. Zoaga. Watt4 suspected that so-called forms of C.Zonga may prove to be the tubers of different species. “The Wealth of India”8 has mentioned its substitution with C. aromatica, as well as its close resemblance to C. zedoaria Roscoe, and from long experience we have found that both of these species, particularly the latter (local name Kachura, Soti), are being used extensively as adulterants. Although the latter species do not contain curcumin, this fact is of little significance when they are mixed with C. Zonga in the powdered form. Parry9 suggested that because the starch grains are usually more or less gelatinised as a result of the method of preparation, the presence of some well defined starch grains may indicate the presence of an adulterant. However, although most of the starch is swollen, grains may occasionally be found that still exhibit the characteristic scitaminaceous shape; moreover, while scalding is a very commonly used process, it is not obligatory and hence the presence of long, lens-shaped unaltered starch grains cannot be taken as indicative of adult eration.The statutory chemical standards prescribedlO are of minor importance, as all of these species belong to the same genus. Consequently, the detection of adulteration of turmeric will remain very difficult until their characteristic differences are established. The flavouring substances present in the essential oils of C. aromatica and C. zedoaria contain sufficient amounts of camphor and camphene (both of which are absent in C. Zonga) to permit their identification, thus providing a useful indication of the presence of C.aromatica @ SAC and the authors.154 SEN et al. : DETECTION OF Curcuma xedoaria [Analyst, Vol. 99 or C. xedoaria. The relatively simple and rapid thin-layer chromatographic technique des- cribed below is suitable for differentiating these compounds and is of value for quality control purposes. A number of solvent systems, as well as different spraying reagents, have been tried. Although the solvent with which optimum separation was achieved is not new,ll the detection by means of a three-step colour sequence has not previously been attempted. EXPERIMENTAL AND RESULTS SOLVENT SYSTEM- Ethyl acetate - n-hexane (3 + 17). (1) Concentrated sulphuric acid (50 ml) - nitvic acid (as oxidising agent) (0.5 ml). (2) Anisaldehyde - sulphuric acid reagent-A freshly prepared mixture containing 0.5 ml of anisaldehyde, 9 ml of ethanol (95 per cent.), 0.5 ml of concentrated sulphuric acid and 0.1 ml of glacial acetic acid.THIN-LAYER CHROMATOGRAPHY- Any convenient type of applicator can be used. Clean 10 x 20-cm thin-layer chromato- graphic plates with detergent and rinse them thoroughly. Coat them to a thickness of 500 pm with a 1 + 2 slurry of silica gel G in water. (An adsorbent layer of this thickness results in higher sensitivity and less background, and does not tend to peel when sprayed.) Air-dry the coated plates in a vertical position for half an hour and then dry them for 1 hour at 110 "C so as to activate the layers. Cool, wipe the backs and edges of the plates free from excess of silica gel G and store them in a desiccating storage cabinet until needed.The adsorbent layer must be uniform from plate to plate in order to obtain reproducible results. Steam distillation (as recommended by the British Phannacopoeia12) for 4 hours is the most convenient method of separating the essential oils from other plant constituents. The separated oils (from 20 to 25 g of sample) are mixed with a volatile solvent (benzene) so as to make an approximately 1 per cent. solution and a 10-pl spot is applied to the plate 2 cm from the bottom edge. Spots are identified by com- parison with volatile oils from authentic materials run alongside. The developing tank should be prepared at least 3 hours before use, so as to allow the tank to become saturated and to shorten the developing time.The solvent level should not be higher than the spotting line, a depth of 1 cm being generally sufficient. As the solvent travels rapidly (10 to 12 minutes), the plate must be removed from the tank as soon as the solvent front reaches the previously marked line, 10 cm from the origin. IDENTIFICATION- The identification of the substances was achieved by means of a three-step sequence: colour produced with sulphuric acid - nitric acid (chromogenic reagent No. l), fluorescence and the subsequent colour produced with anisaldehyde - sulphuric acid (chromogenic reagent No. 2), as described below. After allowing the solvent to evaporate, the chromatogram is sprayed with chromogenic reagent No. 1. The entire chromatographed portion must be uniformly sprayed in order to achieve optimum results (10 ml of reagent are sufficient).The plate is allowed to stand for 1 minute, so as to enable the layer to become saturated with the reagent. Although the patterns obtained are somewhat similar near the origin, the spots obtained with C. longa with R, values of 0.55 and above are different from those obtained with the other two varieties (see Table I). Inspection under ultraviolet light (365 nm) showed that with C. zedoaria and C. aromatica the spots fluoresce at R, 0.55 while with C. longa there is no such fluorescence in that zone, hence both species can be clearly differentiated from turmeric in this way. If the chromatogram is then further sprayed with chromogenic reagent No. 2, a contrasting pattern of colour differentiation is revealed.Orange (R, 0.72) and deep pink (R, 0.55) spots of camphene and camphor are characteristic of C. xedoaria and C. aromatica. Neither of these spots is given by oils of C. longa, which show only one bluish violet spot (R, 0.75) and another dirty brown spot (R, 0.60) closer to the region of the camphene and camphor spots (see Table I). CHROMOGENIC REAGENTS- Different samples can be spotted 2 cm apart.March, 19741 AND Curcuma aromatica IN Curcuma Zonga 155 The spots can also be made visible by spraying the plate with chromogenic reagent No. 2 alone, a similar pattern being observed after heating the plate for 2 minutes at 110 “C. The colour reactions described generally depend on the substance, amount of reagent used, temperature and duration of heating.The R, values obtained should not be regarded as absolute as they may be influenced by the presence of other extractives from the sample. For this reason, the identity of an isolate should be confirmed by re-chromatographing the suspected material against known standards. For identification purposes, a 5 per cent. ad- dition of C. zedoaria or C. aromatica to C. Zonga can be clearly distinguished from the C. Zonga by the R, values, the colour of the spots and their fluorescence. TABLE I COLOUR REACTIONS IN THE DIFFERENT DETECTION METHODS Figures in parentheses are R, values Chromogenic reagent No. 1 Daylight Ultraviolet C. aromatica C. longa C. avomatica C. longa A A I \ t \ C. zedoaria and Violet (0.77) Bluish violet Pink (0.72) Light pink (0.64) Orange (0.60) Brown (0-60) C.zedoaria and (0.75) Dirty green (0.55) Greenish violet (0.46) Violettish pink (0.40) Bluish violet (0.33) Pink (0.23) Bluish fluorescence (0.55) Light blue Violet (0.40) Bluish green Light pink (0.23) (0:46) (033) Chromogenic reagents No. 1 and No. 2 (daylight) r A 1 C. zedoaria and C. avomatica C. longa Violet (0.77) Bluish violet Orange (0.72) Light Pink (0-64) Blue (0.60) Dirty brown Deep pink (0.55) Violet Greenish violet (q.46) (0.46) Light pink Pinkish brown (0.40) (0.40) Bluish violet Bluish green (q.33) (0.33) Violet (0.23) Brown (0.23) (0.75) (0.60) It may be further added that the above test can be applied to check for the presence of C. caesia and C. domestica,6 as both of these species contain camphor, and the former also contains camphene.The authors are grateful to Shri T. V. Mathew, Director, Central Food Laboratory, Calcutta, for his keen interest in this study and kind permission to publish this work. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. REFERENCES Rao, B. S., and Shintre, V. P., J . SOC. Chem. Ind., Lond., 1928, 47, 54T. “The Wealth of India,” Volume 11, “Raw Material,” Council of Scientific and Industrial Research, Chopra, R. N., Nayar, S. L., and Chopra, I. C., “Glossary of Indian Medicinal Plants,” Council of Watt, G., “A Dictionary of the Economical Products of India,” Volume 11, Department of Revenue Bailey, L. H., “Manual of Cultivated Plants,” The Macmillan Co., New York, 1949. Guenther, E., “The Essential Oils,” Volume V, Van Nostrand Co. Inc., New York, 1952, p. 123. Watt, G., op. cit., p. 658. “The Wealth of India,” Volume 11, “Raw Material,” Council of Scientific and Industrial Research, Parry, E. J., “Food and Drugs,’’ Volume 1, Scott, Greenwood and Sons, London, 1911, p. 245. “The Prevention of Food Adulteration Rules, 1955,” Government of India, Ministry of Health Miller, J. M., and Kirchner, J. G., Analyt. Chem., 1953, 25, 1107. “The British Pharmacopoeia 1968,” The Pharmaceutical Press, London, 1968, p. 1275. New Delhi, 1950, p. 402, Scientific and Industrial Research, New Delhi, 1956, p. 85. and Agriculture, Government of India, 1889, p. 665. New Delhi, 1950, pp. 401 and 406. Publication, Appendix B, Item 05.20.01 (Substituted by GSR 1633, dated July Sth, 1968). Received June 21st, 1973 Accepted September 25th, 1973