Two-dimensional thin-layer chronmatography of ganja (Cannabis sativa L.)

Sections

ABSTRACT
Introduction
Experimental
Results and discussion
Acknowledgements

Details

Author: S. N. TEWARI and, J. D. SHARMA
Pages: 63 to 67
Creation Date: 1983/01/01

Two-dimensional thin-layer chronmatography of ganja (Cannabis sativa L.)

S. N. TEWARI and
J. D. SHARMA
Forensic Science Laboratory, Uttar Pradesh, Lucknow, India

ABSTRACT

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.

Introduction

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.

Experimental

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).

Results and discussion

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

Two-dimensional thin-layer chromatographic separation of ingredients of ganja

(Cannabis sativa L.)

 

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.

Figure

Full size image: 0 kB, Figure

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.

Acknowledgements

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.

References

01

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.

02

2. E. P. Chiesa, R. V. D. Rondina and J. D. Coussio, "Rapid thermomicrolthin layer chromatographic procedure for the identification of cannabinoids in marijuana", Journal of Chromatography, vol. 87, No. 1 (1973), pp. 298 - 299.

03

G. Bertulli, L. Mosca and G. Pedroni, "Rapid method for the detection and identification of cannabinoids in cannabis", Bolletino chimico farmaceutico, vol. 115, No. 10 (1976), pp. 714 - 719.

04

T. P. Hagg, "Detection of smoke poisons, especially hashish", Deutsche Apotheker Zeitung, vol. 110, No. 48 (1970), pp. 1874 - 1877.

05

J. M. Parker and H. L. Fiske, "Thin layer chromatography of marijuana", Journal of the Association of Official Analytical Chemists, vol. 55, No. 4 (1972), pp. 876 - 879.

06

J. E. Zabik and R. P. Maickel, "Relevance ofstreet drug analyses in the forensic laboratory to clinical toxicology of drug abuse", Drug Addiction, vol. 4, 1974, pp. 203 - 207.

07

S. N. Tewari, S. P. Harpalani and S. C. Sharma, "Separatiou and identification of the constituents of hashish (Cannabis indica) by thin layer chromatography and its application in forensic analysis", Chromatographia,vol. 7, No. 4 (1974), pp. 205 - 206.

08

K. Fonseka, M. Widmann and S. Agurell, "Chromatographic separation of cannabinoids and their mono-oxygenated derivatives", Journalof Chromatography,vol. 120, No. 2 (1976), pp. 343 - 348.

09

S. N. Tewari and J. D. Sharma, "Separation and identification of cannabinoids from Cannabis indica Linn. by thin layer chromatography", Pharmazie,vol. 34, 1979, p. 54.

10

S. N. Tewari and J. D. Sharma, "Detection of delta-9-tetrahydrocannabinol in the organs of a suspected case of cannabis poisoning", Toxicology Letters, vol. 5, Nos. 3 - 4 (1980), pp. 279 - 281.

11

Z. Mobarak, N. Zaki and D. Bieniek, "Chromatographic aspects of hashish analysis", Forensic Science, vol. 4, No. 2 (1974), pp. 161 - 169.

12

R. Fowler, R. A. Gilhooley and P. B. Baker, "Thin layer chromatography of cannabinoids", Journal of Chromatography, vol. 171, 1979, pp. 509 - 511.