Summary
Introduction
Methods
Acknowledgement
Author: M. REPETTO, , M. LOPEZ-ARTIGUEZ, , D. MARTINEZ,
Pages: 69 to 74
Creation Date: 1976/01/01
The three main cannabinoids - cannabidiol (CBD), tetrahydrocannabinol (THC) and cannabinol (CBN) - can be isolated from purified extract of cannabis by preparative gas chromatography in satisfactory amounts and with a degree of purity similar to that of the sample substances provided by the United Nations Narcotics Laboratory.
One problem encountered in any toxicological study on cannabis is the difficulty of isolating cannabinoids which are pure enough to be used for an experimental study. It has been clearly demonstrated that the different cannabinoids have certain synergic and certain antagonistic effects on one another, so that work with plant extracts of highly variable composition leads to results which are not reproducible and may even be contradictory.
A great many techniques have been tried out for isolating the active substances of cannabis. The procedures used include silica gel or alumina column chromatography with innumerable combinations of eluents, and preparative paper or thin-layer chromatography; but none of these techniques has proved to be altogether satisfactory, since the amounts obtained are small and the slowness of the process leads to deterioration of the substances separated.
After a number of experiments, we succeeded in isolating the three main cannabinoids - CBD, THC and CBN - in sufficient amounts and with an acceptable degree of purity, using a preparative gas chromatography technique.
Although preparative gas chromatography is less widely used than analytical chromatography, it is a technique which is often very useful for separating and purifying certain substances. The functional diagram for the process is based on the possibility of separating a number of fractions of the eluate which collects at the end of the chromatograph column (fig. 1).
The yield is improved by the possibility of repeating the cycle automatically. The solution of the substance to be chromatographed is placed in an injection chamber from which predetermined amounts pass into the column. At the end of the column, the eluate is separated into two parts; the smaller amount goes to the detector, while the larger amount passes into the traps. The substances are collected in one or another of the traps in response to signals given by the detector in accordance with a previously established programme.
Programming is effected in the optimum conditions for obtaining the chromatogram, which are determined experimentally; and the programme is such that only the upper part of the peaks on the chromatogram - i.e. the part which indicates the presence of the purest fractions - is used for isolating substances of interest. Fractions of no interest are routed to a residual product trap.
The apparatus we used, a Perkin-Elmer F-21, consists essentially of three modular units: an electronic and pressure regulation unit; a central unit with the oven, the column and the injection chamber; and a third unit comprising a detector connected to a recorder and the fraction collector, with the traps submerged in a cooling bath (figs. 2 and 3).
The basic substance used was cannabis resin oil obtained from the plant and purified in the following manner:
The plant extract obtained by reflux heating with n-hexane is purified by silica gel column chromatography, using a mixture of three parts benzene to seven parts chloroform to elute an initial 300 ml fraction. The eluates are analysed by thin-layer chromatography.
The fraction collected is then concentrated in a vapour centrifuge and dissolved with n-hexane. This solution is then analysed by preparative gas chromatography.
The apparatus has metal columns filled with SE-30 on chromosorb G (length of each column - 90 cm; external diameter 94.6 mm).
We experimented with various types of columns and operating conditions with a view to achieving optimum separation, particularly of the three main cannabinoids - cannabidiol, tetrahydrocannabinol and cannabinol.
The substances separated in the traps are collected in n-hexane and analysed by thin-layer chromatography and by chromatography in an F-20-FE until completely pure substances are obtained, comparable to the sample substances supplied by the United Nations.
The operating conditions for the F-21 were finally established as follows:
Number of columns: 3; total length: 2.70 m;
Carrier gas: N 2at 2.6 kp/cm 2;
Injection unit temperature: 300° C;
Oven temperature: 200° C;
Distributor temperature: 280° C;
Operating line temperature: 280° C;
Refrigeration of traps: ice-salt;
Retention time: CBD: 83'6 min; THC: 118 min; CBN: 149 min;
Temperature programming: T = 200°,T 2 = 250°;
Heating time: 5 min;
Injection time: 3 seconds;
Residual component collection time: 15 min;
Cooling time: 3 seconds.
The operating conditions for analytical chromatography in the F-20 FE were as follows:
Column: FS 3 per cent Chrom. G.80/100;
Oven temperature: 200° C;
Inlet temperature: 250°;
Detector temperature: 250°;
Carrier gas: N 2 at kp/cm2.
In this way, we succeeded in separating the main cannabinoids. It is also possible to isolate the most important metabolites. We are now in the process of doing this in order to obtain metabolic derivatives in sufficient amounts for pharmacological study.
The authors wish to thank the United Nations Narcotics Laboratory, which supplied them with the sample substances referred to in the text.
