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duced. The length of H from the central axis of C to the point of attachment to A in the apparatus is about 14 cm. The degree of vacuum may be regulated by adjusting the height of the mercury column-i. e., raising or lowering the mercury dish and/or decreasing the weight, D. With the apparatus described, evacuated to 12 mm. of mercury with a glass water pump, it was possible to produce a fluctuation of vacuum ranging from 5 mm. to several centimeters. The same fluctuations could be produced as well a t lower vacuums. The frequency of the operation could be adjusted from 8 to 20 phases per minute. Inasmuch as a lowering of vacuum raises the boiling point of a substance, fluctuation of vacuum in this apparatus will produce a corresponding fluctuation in the boiling point of the solvent. The same precaution should be taken with this apparatus as with a manually controlled apparatus-i. e., heat should be applied to the distillation flask in such a way that, with the decreases in vacuum, the temperature does not rise
Vol. 13, No. 2
above the decomposition point of the material being treated. With this apparatus a water bath was used, the temperature of which was maintained a t a point below the decomposition temperature of the most heat-sensitive substance in the solution being distilled. If it is desired to carry on the distillation in the presence of an inert atmosphere (carbon dioxide was used in this case), appropriate connections may be made from the gas cylinder or generator to E. If desired, capillary tubes may be used in F. With badly foaming solutions, little advantage was realized, the tubes tending to “blow up” bubbles formed. If an inert gas is used, gas connections must also be made to the capillary tubes. The apparatus has been successfully used in the distillation of water and 50 per cent water-alcohol extracts of animal tissues. The speed of the distillation is approximately the same as for a carefully controlled manual operation.
New Color Reactions for Cannabis sativa Resin WILLIAM JOHN BLACKIE, Department of Agriculture, Suva, Fiji
T
HE recognition of the resinous constituent of Cannabis sativa (Cannabis indica) has been rendered difficult by the absence of reliable and specific chemical tests. A clue to the nature of suspected material can often be obtained by microscopic examination; the odor developed by heating the residue of a petroleum ether extract a t the temperature of the water bath is also of diagnostic value, provided the residue is free from lipoid and other materials extracted with the resin during toxicological investigations. Beam (1) developed a delicate test, using alcoholic potassium hydroxide, and for many years this test has been of distinct service t o the analyst in the East. Doubt has been expressed in regard to its specificity, and moreover it has failed on occasions with genuine plant material and pharmacopoeia preparations. Recently, several new color reactions have been developed, notably those of Munch (4) and Ghamrawy (3) using p dimethylaminobenzaldehyde and ( 2 ) Duquhois and Moustapha using vanillin and acetaldehyde in the presence of ethyl alcohol. I n the Duquenois and Moustapha method the colors are developed in the presence of hydrochloric acid, and in the technique of Ghamrawy and of Munch sulfuric acid is used. In this laboratory, a considerable number of samples of ganja, charas, and bhang come under observation and it was considered that an examination of these new methods would be of distinct service in connection with forensic chemical work. The reactions of Ghamrawy, Duquenois and Moustapha, and Munch were given by fifteen samples of Cannabis sativa resin examined in this laboratory and appear to be delicate tests for the resin; definite color reactions are also obtained with benzaldehyde, o-nitrobenzaldehyde, and salicylaldehyde; the color is best developed with hydrochloric acid; the reaction is not obtained with aliphatic aldehydes and therefore appears to be specific for aromatic aldehydes of the type studied; the reaction can be carried out in the presence of solvents other than ethyl alcohol; the presence of an aromatic aldehyde group is essential for the reaction as described in this paper; and finally of 104 substances tested by the technique, 15 samples of Cannabis sativa resin alone gave the reaction.
In this paper detection methods only are considered although it is believed that the test as carried out by the author using vanillin, salicylaldehyde, and p-dimethylaminobenzaldehyde could be developed into a quantitative colonmetric estimation. The samples of Cannabis sativa resin examined in this investigation were obtained by the extraction with petroleum ether of ten separate samples of plant material of Indian origin and five samples of the crude drug seized by local custom and police officials. The color changes recorded in Table I were given by the crude resin, the resin after the passage through a Tswett chromatographic absorption column of activated alumina, and the resin af&r treatment with activated carbon.
Experimental I n Table I are recorded color changes using benzaldehyde, vanillin, o-nitrobenzaldehyde, p-dimethylaminobenzaldehyde, and salicylaldehyde. The materials and reagents were tested in the following manner : A volume of petroleum ether extract containing 2 mg. of the resin was either spotted onto a spotting porcelain plate with depressions, or added t o a microtest tube and the solvent allowed to evaporate. A quantity of the aldehyde corresponding to 3 mg. contained in 0.5 cc. of the solvent was added to the resin and the material was dissolved by manipulation with a microspatula. Concentrated hydrochloric acid was then added drop by drop from a micropipet until maximum color intensity was developed. The test was carried out also by smearing a minute portion of the resin on filter paper, adding a small drop each of benzyl alcohol and benzaldehyde, and developing the color with concentrated hydrochloric acid. The reaction under these conditions is very striking and is of value in the examination of efains and minute quantities of the resin.
Color Changes I n general under the conditions of testing described above benzaldehyde gave an intense violet color which faded rapidly. Vanillin gave a persistent green-blue-green coloration and in some cases violet tinges were noted. The bluegreen color faded gradually, but in all cases the color was clearly perceptible after 20 minutes. o-Nitrobenzaldehyde,
TABLEI. COLOR CHANGES OF Cannabis sativa RESIS Alcohol Solvent Methyl alcohol
Reagent A B C D
E
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Color Violet Green-blue green Little change Violet red Deep green
Persistency 1 minute Slow change
.......
Slow change Slow fade
Remarks Fades rapidly Perceptible green after 20 minutes Yellow-green precipitate Cloudy violet after 20 minutes Yellow green after 20 minutes
material with petroleum ether and other plant products which might appear in extracts were tested by the methods described above. The following substances failed to give the reactions recorded in Table I.
A L K A L o I D s . Atropine, aconitine, berberine, brucine, caffeine, cinchonine, codeine, colchicum, cocaine, ecgonine, homatropine, hydrastine, morphine, narceine, nicotine, narcotine, papavIsobutyl alcohol A Violet 5 minutes Fades rapidly to slate erine, strychnine, theobromine. B Green-blue green Slow fading Blue green after 20 minutes 5 minutes Slow fading t o yellow C Brown yellow ESSENTIAL OILS.Rosemary, ti-tree, D Violet red Slow fading Pink after 10 minutes croton, verbena, vetiver, nutmeg, amygE Deep green Slow fading Green after 20 minutes dalus, bay, citronella, lemongrass, 1 minute Complete fading in 5 minutes A Cloudy violet Acetone eucalyptus, cajuput, lavender, clove, B Cloudy green Slow fading Light green after 20 minutes Chenopodium, sandalwood, turpentine. C Cloudy brown 5 minutes Yellow in 15 minutes F I X E DOILS. C a n d l e n u t , tung, Slow fading Light pink after 20 minutes D Cloudy pink Slow fading Light green after 20 minutes E Cloudy green coconut, linseed, mustard dilo, peanut, cottonseed, castor, sesame, cedarwood, Benzyl alcohol A Violet 3 minutes Fades t o slate in 5 minutes olive, hydnocarpus. B Green-blue green Slow fading Green after 20 minutes C Orange Slow fading Yellow after 20 minutes RESINS. Gurjun balsam, colophony, D Violet red Slow fading Pink after 20 minutes dammar, guiacum, Canada balsam, E Deep green Slow fading Pink after 20 minutes derris, kauri gum. A. Benzaldehyde. B. Vanillin. C. o-Nitrobenealdehyde. D . p-Dimethylaminobenzaldehyde. TERPENESAND SESQUITERPENES. E. Salicylaldehyde. Pinene, cedrene, carvene, camphene, vetivene, cadinene. SUGARS. Dextrose,levulose,sucrose, mannose, lactose, galactose. UNCLASSIFIED. Meconic acid, crude opium, citronellol, depending on the solvent used, gave light to brown-yellow citronellal, camphor, ethyl hydnocarpate, salicin, barbitone, colorations which persisted up to 20 minutes. With p nicotinic acid, phytosterol, carotene, ascorbic acid, abietic acid. dimethylaminobenzaldehyde, beautiful violet-red colorations It is considered, therefore, that the reactions recorded in were developed which faded slowly, the color however being Table I take place with a constituent or constituents present still perceptible after 20 minutes. With salicylaldehyde a in Cannabis sativa resin. The reactions have not been carried beautiful emerald green color was developed immediately out as yet with material of known physiological activity, so and persisted with slow fading for over 20 minutes. The that it is not possible to attribute the reaction to a physiologicolor faded to a yellow brown in 2 hours. The color changes cally active constituent or constituents. were not affected by the addition of acetaldehyde as recorded by Duquenois and Moustapha. Sensitivity The reaction did not take place if aliphatic aldehydes replaced the aromatic aldehydes. When experiments were Although the color is fleeting, the most sensitive reagent performed using acetaldehyde and formaldehyde in the is benzaldehyde in benzyl alcohol as solvent. A deep manner described, in no case was color developed. Similarly violet color is obtained immediately with amounts of the ketones such as acetone and acetophenone fail to react. order of 0.03 mg. Definite reactions can be obtained with That the reaction requires the presence of a free aromatic 0.1 mg. using vanillin, pdimethylaminobenzaldehyde, and aldehyde group is indicated with salicylaldoxime. When salicylaldehyde in benzyl alcohol. o-Nitrobenzaldehyde does this oxime is used in place of the free aldehydes recorded not appear to be as sensitive as the other aldehydes. The above, no reaction takes place. slow fading in color of the vanillin, p-dimethylaminobenzaldehyde, and salicylaldehyde reaction products suggest the Effect of Solvent possibility of colorimetric determination, but this point has not as yet been investigated. The best solvents appeared to be benzyl alcohol and, in most cases, isobutyl alcohol. Ethyl alcohol gave fair results Conclusion with all the aldehydes, but methyl alcohol and acetone were definitely inferior under the condition of test. This appeared A definite chemical reaction takes place between one or to be due to either the insolubility of the reaction products in more active constituents of Indian hemp and certain aromatic the solvents named or the cloudiness developed by the excess aldehydes dissolved in suitable solvents and in the presence acid required to develop the maximum color in these solvents. of concentrated hydrochloric acid. The color changes are characteristic of the aldehydes and Cannabis saliva resin. Effect of Acid Of the materials tested Cannabis indica resin alone reacted in the manner described. Neither sulfuric, nitric, nor acetic acid mas able to develop color under the conditions of the test. With methyl alcohol Acknowledgment and acetone as much as 1 cc. of acid was required to develop the color, whereas with isobutyl and benzyl alcohols 0.3 to The author is indebted to the Director of Agriculture for 0.4 cc. was sufficient. permission to publish these results. Ethyl alcohol
A B C D E
Violet Deep blue green Brown yellow Violet red Deep green
3 minutes Slow fading Slow fading Slow fading Slow fading
Fades in 5 minutes Deep green after 20 minutes Pale yellow after 20 minutes Pink after 20 minutes Green after 20 minutes
Specificity
Literature Cited
These reactions, which are similar in type, appear to be specific for Cannabis sativa resin. Ghamrawy has tested 60 drugs, Munch 107 drugs, and Duquenois and Moustapha have also recorded the specificity of their technique. I n this investigation several substances extractable from plant
Beam, W., “A Colour Reaction for Indian Hemp”, Wellcome Tropical Research Laboratories, Vol. B, pp. 25-6 (1911). . . Duqudnois, P., and Moustapha, H. N., J . E g y p t . Med. Assoc., 21, 224-7 (1938). (3) Ghamrawy, M.A.,Ibid., 20, 193-208 (1937). (4) Munch, J. C., Am. J . Phatm., 110,423(1938).
