ABSTRACT
Introduction
Experimental
Results and discussion
Acknowledgements
Author: S. N. TEWARI and, J. D. SHARMA
Pages: 63 to 67
Creation Date: 1983/01/01
An efficient and reliable two-dimensional thin-layer chromatographic technique for separation and identification of cannabinoids present in cannabis resin ( Cannabis sativa L.) is described. A total of 47 different cannabitnoids were successfully separated and 5 of these, cannabinol, cannabidiol, cannabichromene, trans-delta-8-tetrahydrocannabinol and delta-9-tetrahydrocannabinol, were identified. A 0.1 per cent solution of Fast blue salt B in 45 per cent ethanol was employed as chromogenic reagent.
The flowering tops of Cannabis Sativa L., popularly known as ganja, represent the most widely found cannabis preparation. Its illicit trafficking and consumption have markedly increased throughout the world in recent years. Many workers have reported their work on cannabis using chromatographic methods. Grlic [ 1] preferred amine-treated thin-layer chromatography (TLC) plates whereas Chiesa, Rondina and Coussio [ 2] described a thermomicro TLC procedure for the identification of cannabinoids. Using multiple development and impregnation, Bertulli, Mosca and Pedroni [ 3] identified delta-9-tetrahydrocannabinol (THC) and cannabinol and some other researchers [ 4] - [ 10] also utilized TLC for their purpose. Mobarak, Zaki and Bieniek [ 11] and Fowler, Gilhooley and Baker [ 12] reported twodimensional (2 D) TLC of cannabis. The available literature reveals, however, that most earlier workers confined their efforts mainly to unidimensional TLC and very few of them employed 2 D- TLC. The objective, therefore, was to develop a suitable 2 D -TLC system which would enable separation and identification of a larger number of cannabinoids.
An amount of 0.5 g of fresh ganja was taken in 50 ml chloroform with 10 drops of glacial acetic acid to make the medium faintly acidic. The mixture was kept for half an hour at room temperature and filtered. The filtrate was evaporated at temperatures below 50 o C to 0.5 ml and subsequently dried completely with a stream of hot air. The residue was then dissolved in 1 ml chloroform for spotting on TLC plates.
The glass plates (20 cm x 20 cm) were coated with a 0.25 mm thick layer of silica gel G slurry .(30 g gel + 65 ml water), dried at room temperature and ultimately activated at 110 o C for 40 min before use. The plates were spotted with 80 µg of cannabis resin in n-hexane at a common point 2.5 cm from the two sides of a plate on diagonal plane. A quantity of 5 µg each of 5 authenticated samples of cannabinol (6,6,9-trimethyl-3-pentyl-6 H-dibenzo-(b, d)-pyran-1-ol), cannabidiol (3-methyl-6-prop-2-enyl-4-pentyl-1,4,5,6-tetrahydrodiphenyl-2,6-diol), cannabichromene (5hydroxy-2-isohex-3-enyl-2-methyl-7-pentylchromene), trans-delta-8-tetrahydrocannabinol (6,6,9-trimethyl-3-pentyl-6a,7,10,10a-tetrahydrodibenzo(b, d)-pyran- 1-ol) and delta-9-tetrahydrocannabinol (6,6,9-trimethyl-3pentyl-6a,7, 8, 10a-tetrahydrodibenzo-(b, d)-pyran-1-ol) were also spotted separately at 2.5 cm above the plate edge near two adjacent corners but at points which were at least 12 cm from the point of sample application.
The TLC plates were first developed (by the ascending technique) in direction I, using a heptane/dichloromethane/butan-2-one (83/5/12 volume ratios) solvent system which was allowed to saturate the developing chamber for one hour. When the solvent front reached 12 cm, the plates were dried at room temperature, rotated 90 o and redeveloped in direction II using n-hexane/acetone (86/14 by volume). The ambient temperature was 25 o C.
These two-dimensionally developed plates were first viewed under short uv light (254 nm). The fluorescent colours of resolved spots were recorded and the plates were then sprayed with a 0.1 per cent solution of Fast blue salt b (3,3 -dimethoxybiphenyl-4-4 -bisdiazonium chloride) in 45 per cent ethanol as the chromogenic reagent. The different colours of resolved spots along with their respective migration distances in the two directions were recorded (see table and figure).
The 2D-TLC of ganja yielded clearly separated cannabinoid spots. The solvent systems used for development possessed excellent resolving properties and as many as 47 different cannabinoids were successfully separated (see table and figure). Of these, 5 major cannabis ingredients, i.e., cannabinol (CBN), cannabidiol (CBD), cannabichromene (CBC), transdelta-8-tetrahydrocannabinol (T-DELT 8THC) and delta-9-tetrahydrocannabinol (DELT9-THC) were identified using authentic controls.
The spray of 0.1 per cent solution of Fast blue salt b in 45 per cent ethanol was found to be an extremely effective and specific chromogenic reagent and it gave distinct colours with different cannabinoids. The colours
Spot migration (mm) |
Colour of spot b |
||||
---|---|---|---|---|---|
Refenence No. |
Cannabinoid a |
Direction I |
Direction II |
Under uv light (254 nm) |
With fast blue salt reagent |
1 | 3 | 3 |
Purple
|
||
2 | 6 | 6 |
Magenta
|
||
3 | 10 | 11 |
Red
|
Violet
|
|
4 | 10 | 24 |
Pale yellow
|
Faint violet
|
|
5 | 14 | 3 |
Pink .
|
||
6 | 14 | 18 |
Violet
|
||
7 | 16 | 11 |
Sky blue
|
Orange
|
|
8 | 20 | 10 |
Purple
|
||
9 | 20 | 20 |
Red
|
Purple
|
|
10 | 25 | 27 |
Pink-orange
|
||
11 | 26 | 16 |
Purple
|
||
12 | 27 | 6 |
Red
|
Violet
|
|
13 | 29 | 35 |
Violet
|
||
14 | 35 | 43 |
Yellow
|
||
15 | 36 | 37 |
Pink
|
||
16 | 39 | 50 |
Red
|
Magenta
|
|
17 | 41 | 30 |
Pink
|
||
18 | 43 | 35 |
Brown
|
||
19 | 43 | 38 |
Blue-violet
|
||
20 | 45 | 49 |
Sky blue
|
Blue-violet
|
|
21 | 46 | 13 |
Pink
|
||
22 | 51 | 75 |
Pink
|
||
23 | 52 | 64 |
Pink
|
||
24 | 53 | 55 |
Violet
|
||
25 | 55 | 20 |
Blue-violet
|
||
26 | 57 | 44 |
Pink
|
||
27 | 58 | 84 |
Pink
|
||
28 | 60 | 57 |
Pink
|
||
29 | 63 | 70 |
Purple
|
||
30 | 68 | 9 |
Purple
|
||
31 | 69 | 0 |
Pink
|
||
32 | 72 | 61 |
Pink
|
||
33 |
CBC
|
72 | 72 |
Yellow
|
|
34 | 73 | 43 |
Dark
|
Purple
|
|
35 | 73 | 99 |
Pink
|
||
36 | 81 | 83 |
Sky blue
|
Pink
|
|
37 |
DELT 9-THC
|
84 | 90 |
Dark
|
Deep violet
|
38 | 80 | 0 |
Purple
|
||
39 | 90 | 50 |
Purple
|
||
40 |
T-DELT8-THC
|
90 | 97 |
Dark
|
Magenta
|
41 | 91 | 14 |
Pink
|
||
42 | 91 | 44 |
Pink
|
||
43 |
CBD
|
91 | 88 |
Dark
|
Orange
|
44 | 96 | 0 |
Pink
|
||
45 | 96 | 56 |
Sky blue
|
Purple
|
|
46 | 101 | 106 |
Sky blue
|
Purple
|
|
47 | 111 | 117 |
Purple
|
aNo entry means ingredient was not identified.
bNo entry means spot was colorless.
of CBN, CBD and T-DELT 8-THC were so distinct, prominent and dense that these ingredients could be recognized by their colors alone: deep violet, orange and magenta.
The proposed technique, besides giving the largest number of cannabinoids resolved on a single chromatogram, has the added advantage that it completely avoids plate impregnation and makes use of readily available materials. It was found to be sensitive, reliable and reproducible. The best results were observed at 25 o C.
The authors wish to thank the United Nations Narcotics Laboratory of the Division of Narcotic Drugs, Vienna, Austria, which supplied them with the authenticated samples referred to in the text. Thanks are also due to the Bureau of Police Research and Development for the award of a fellowship and financial assistance to J. D. Sharma.
L. Grlic, "Simple thin layer chromatography of cannabinoids by means of silica gel sheets treated with amines", Journal of Chromatography, vol. 48, No. 3 (1970), pp. 562 - 564.
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