A combined spectrophotometric differentiation of samples of cannabis


Characteristics of parameters used
Practical differentiation of samples


Author: Ljubiša GRLIC
Pages: 25 to 30
Creation Date: 1968/01/01

A combined spectrophotometric differentiation of samples of cannabis

Ljubisa GRLIC Yugoslav Lexicographical Institute, Zagreb

Cannabis is the most widespread narcotic drug in the world. The hemp plant ( Cannabis sativa L.) can be cultivated almost anywhere, and it also grows as a weed under quite different climates in various parts of the globe.

The conglomerate of types and races of both cultivated and wild hemp, differing in their morphological characteristics, have accompanied man in his wanderings and in his colonization of the world [ 1] . Such botanical diversity is reflected in variations in the chemical' composition of physiologically active resin; in addition, various ecologic factors may also give rise to a multitude of chemical types of cannabis.

The drug cannabis is essentially derived from the dry flowering and fruiting tops of the female hemp plant, its strongest form being the pure resin; other parts of the plant (larger leaves, stalks, male tops) which, are weaker or negative in drug effect, may often be presented, in a mixture of cannabis.

The great diversity of forms of the drug encountered in illicit traffic, which vary in their organoleptic properties, chemical composition and physiological potency, is also due to the fact that much cannabis is produced on a small scale, by local cultivators, and that the mode of preparing the drug depends on different local habits and circumstances. Possible adulterations, and also chemical changes occurring during the storage and aging, may also affect the heterogeneity of the drug.

On account of such great possibility of variations, the development of methods which might make it possible to discriminate and to identify various types of seized cannabis would be of importance in the campaign against the illicit traffic of the drug.

Variations in the chemical composition of the resin extracted from various forms of cannabis have been examined by several authors [ 2] - [ 6] and the differences found have been mostly attributed to climate and other ecologic factors. However, little has been done till now on methods or systems which might be of practical use in distinguishing cannabis on the basis of the characteristic chemical composition of the resin. Several chromatographic methods for quantitative analysis of the main constituents of cannabis resin have been proposed in the course of the last few years [ 7] - [ 9] , but no systematic comparative data have been made available on the quantitative composition of a greater number of samples which might enable the classification and the differentiation of various chemical types of cannabis on this basis.

In a series of our previous studies carried out within the United Nations joint programme of cannabis research, the main differences in the chemical composition of the resin from various samples of cannabis have been explained by the progress of the phytochemical process (called "ripening" of the resin), by which cannabidiolic acid (CBDA) is gradually converted to cannabidiol (CBD), tetrahydrocarmabinols (THC) and finally to cannabinol (CBN). According to the progress of this process in the resin, the samples of cannabis have been classified into five types as "unripe" (predominance of CBDA), "intermediate" (CBD), "ripe" (THC) and "overripe" type (CBN), while a separate group consists of cannabis which has been decomposed or altered by unfavourable treatment or storage ("altered" type). For the purpose of such classification, several procedures have been proposed, and comparative results of various samples of cannabis have been made available as obtained by means of 9 different methods: the reaction by Beam [ 10] , [ 11] , peroxide-sulphuric acid test [ 12] , ferric chloride reaction [ 13] , indophenol reaction [ 14] , determination of the acid fraction [ 6] , of ultraviolet [ 15] and infrared [ 16] absorption characteristics, microbiological assay on antibiotic potency [ 17] and corneal arettexia test in rabbits [ 18] , [ 11] . The results obtained by all these methods have been in fairly good agreement and have proved that an unknown sample of cannabis may be easily characterized as belonging to one of the five types if it is parallelly examined by means of two (or more) of the methods proposed. In the course of the cited work it was generally confirmed that the process of "ripening" is more advanced in cannabis from f tropical areas ("ripe" and "overripe" type), than in the resin from the hemp produced under temperate climate (" unripe" and "intermediate" type). How ever, as the above classification is based on a simplified explanation of interconversion of cannabinols (which probably covers only one part of the complex phyto-chemical processes occurring in cannabis), it may only give a general picture of the chemical composition of the drug and will not prove satisfactory when more detailed differentiation or specific information on chemical constituents, physiological potency and the provenance of the drug are needed.

In an attempt to develop a practical system for a more detailed differentiation of various chemical types of cannabis resin, as compared to techniques previously described, only those methods have been considered which give easily reproducible results, and which can be accurately and numerically recorded.

Some colour reactions (such as Beam, FeCl 3 and H 2O 2-H 2SO 4 tests), although attributable to certain defined constituents of cannabis, could not meet this requirement on account of instability of colours and the factor subjective judgment in classifying them.

The best results have been obtained by a combined application of absorption spectrophotometry over the ultra-violet, the visible and the infra-red region of the spectrum. Measurements over the ultra-violet and the infra-red region are carried out directly with the resin under examination, whereas the absorption over the visible part of the spectrum is measured after a preliminary treatment of the dissolved resin with chlorimide reagent. Nevertheless, the combined method as a whole can be regarded as a direct spectrophotometric technique, as it does not include any preliminary separation or isolation of various constituents or fractions. Therefore, the results are interpreted here in accordance with the practice applied earlier in direct spectrophotometric examinations of various plant extracts [ 19] - [ 21] . A particular advantage of this technique is that the results can be given in terms of relative values (extinction quotients and transmittance differences); this excludes possible errors which might arise from inaccurate concentrations of the measured, solutions. Altogether six parameters (two in each spectral region) are applied and on the basis of the dispersion of the values obtained for the examined samples of cannabis, a coding system is worked out for the separation and characterisation of groups of-samples with close chemical composition.


A quantity of 2 g of cannabis is macerated for 24 hours in 40 ml of petrol ether, the mixture is filtered, the solvent evaporated and the residue dried in vacuum. A small part of thc residue is dissolved in 95 % ethanol to obtain a 0.05% solution of the dry extract; this solution is further used for spectrophotometry in the ultra-violet and visible region. The remaining part of he dry residue is used for infra-red analysis.

Ultra-violet region. Ethanolic solution is further diluted with the same solvent until at 260 m an extinction between 0.3 and 0.5 is reached (using 1 cm cells and ethanol as a blank). The extinction is then measured at 260, 280, 300 and 310 m, and the ratios E 260/E 280 and E 300/E 310 are calculated.

Visible region. To 2 ml of ethanolic solution 0.5 ml of chlorimide reagent (a daily prepared 0.03 % ethanolic solution of recrystalized 2,6 dichloroquinonechlorimide "Merck ") are added. The mixture is rapidly transferred into 5 ml of a 0.6% solution of crystaline Na 2SO 3 in 0.1 N NaOH and shaken immediately thereafter. The extinction of the coloured product is measured at 420, 520 and 630 m (using 1 cm cells and ethanol as a blank) and the ratios E 630/E 520 and E 420/E 520 are calculated.

Infra-red region. The dry residue is deposited in the form of a thin film on a rock salt plate and examined in an infra-red spectrophotometer by usual absorption technique. From the obtained spectral curves the transmittance values at 1130, 890 and 815 cm-1are noted and the difference values T 890- T 1130 and T 815- T 890 are calculated.

Characteristics of parameters used

E 260 /E 280 . All cannabinolic compounds in the ultraviolet region show an absorption maximum between 270 and 280 m (22). The peak of CBDA is at lowest wavelength. CBN, having 6 double bonds in the molecule, exhibits the highest absorptivity and also shows a bathochromic shift of the peak if compared with its precursors. The value E 260/E 280 will be consequently in reverse relation to the progress of phytochemical conversion of cannabinols, and the "over-ripe" samples, containing predominantly CBN, will be characterized by particularly low values in this ratio (even 0.7). On the other side, high values (> 1.40) are characteristic for cannabis grown for industrial purposes and also for some other very fresh samples of cannabis grown under moderate climate. The latter (" unripe ") group of samples exhibited gradual decrease in this ratio in the course of the first few years of storage at normal temperature; for example, the values 1.50-1.60 obtained for fresh cannabis experimentally grown in central Europe have decreased after two years of storage to approximately 1.25-1.35; however, even such decreased values dearly differ from the results which are characteristic for highly potent cannabis from tropical countries and from the eastern Mediterranean area; the ratio in such samples was usually between 0.7 and 1.0,

A combined spectrophotometric differentiation of samples of cannabis 27 and remained quite constant, without significant change even after 3-4 years of storage. Extremely high values of this ratio (> 1.60) are exhibited by cannabis originating from tropical or sub-tropical areas, if grown under an unfavourable climate, for example in central Europe or in northern countries [ 23] .

E 300 /E 310 . From the constituents examined only CBDA exhibits an increase of the absorption curve over the region 300-310 m [ 22] . Therefore, this ratio is in reverse relation with the content of CBDA when compared to the amount of other chromogenic constituents. By means of this value the "unripe" type of cannabis may be easily separated. Samples grown for fibre, experimental samples grown under conditions different from those in their original area as well as wild cannabis from moderate regions exhibit values lower than 1.3. It is of interest to note that such low values of these groups of samples show even a tendency to decrease during storage, whereas the high values characteristic for the "ripe" type (>1.55) may still slightly increase in the stored drug. It means that the differences in this parameter between the two types, existing in fresh cannabis, may become even more characteristic after longer storage. This may be explained by a supposition that in such samples a small additional amount of CBDA is formed during storage, even more rapidly than it becomes decarboxylized and converted to other cannabinols.

E 630 /E 520. This ratio is mostly affected by the proportion THC/CBD in the resin. Pure CBD itself gives a pink colour with the described reaction, having a peak at approximately 520 m, whereas THCs yield a blue colour with a peak nearly 630 m; the E 630/E 520 value of pure CBD is 0.40, while that of THC is 2.4 (14). (The presence of CBDA apparently also influences the reaction as this acid is easily decarboxylized to CBD.) Absorption maxima of cannabis samples with various THC/CBD proportion will consequently range between 520 and 630 m, and the colour of the reaction products ranges from pink through violet and indigo to blue (or even bluish green in "over-ripe" samples containing some decomposition products, having a marked absorption at 420 m). Cannabis resin containing mostly CBDA and CBD exhibits a pink reaction product with an absorption maximum approaching 520 m. Such resin has the lowest values of this parameter, ranging from 0.4 to approximately 0.8. This group includes mostly samples from northern countries, from central Europe and generally cannabis developed under unfavourable climatic conditions, without a marked hashish effect; analytical results obtained for hemp plant grown for industrial purposes also correspond to this group. In samples ranging in this ratio from 0.8 to approximately 1.0 a certain amount of CBD has already been converted to THC. This group of "intermediate" resin mostly belongs to can nabis (both wild and cultivated) from temperate regions. Somewhat higher ratio (1.0-1.5) indicates more potent resin, mostly from Mediterranean areas. Values higher than 1.5 indicate the predominance of THC. In such ("ripe") cannabis the phytochemical process of interconversion of cannabinols is developed in a way to contain the greatest quantities of physiologically active constituents; the exceptions are "over-ripe" samples in which this ratio is higher than 1.9; they mostly originate from the same (tropical) areas, but contain predominantly CBN, the final inactive conversion product. It should be noted that this ratio seems to be connected with some genetical properties of the hemp plant more than other parameters used here; it does not change essentially if, for example, tropical varieties of hemp are grown in a moderate climate [ 23] .

E 420 /E 520 . In the resin obtained from female flowering (or fruiting) tops of the hemp plant this ratio mostly exhibits the values lower than 1.15; the exception is "over-ripe" cannabis in which this ratio reaches as high as 1.40. The petrol ether extract exhibiting this value higher than 1.40 may be considered as originating from decomposed or adulterated cannabis, from that obtained from male plants or containing large leaves (instead of tops). The resin with this ratio lower than 1.15 can be divided into various groups. For example, extremely low values (0.65) are characteristic for tops of female hemp cultivated for fibre. This parameter seems to be correlated with certain ecologic factors and has a tendency to increase if a given variety of hemp is grown under unfavourable conditions (e.g. tropical cannabis when cultivated in a moderate climate).

T 890 - T 1130. The absorption band at 890 cm -1is attributed to the group Full size image: 1 kB present only in CBDA and CBD, while the band at 1160 cm -1 is skeletal frequency for the group Full size image: 2 kB , present in THC and CBN (16). The latter group is supposed to be formed by reduction of the former one in the course of the process of "ripening ". Therefore, the difference between the transmittance values at these two frequencies may be used as a rough indication of the ratio between the constituents after and before this reduction has occured, i.e. as an indication of the ratio (THC + CBN) / (CBDA + CBD). Negative values of this parameter (from -25 to 0) are characteristic for cannabis from central-European countries grown for fibre and, also for other samples belonging to the "unripe" type. The "intermediate" type exhibits values from approximately 0 to 25, while in the "ripe" and "over-ripe" cannabis this parameter was usually higher than 25.

T 815 -T 890 . The absorption band at 815 cm-1is characteristic for 1,2,4-trisubstituted benzene ring present in CNB (16). Therefore, this parameter will be correlated to the ratio (CBDA + CBD) / CBN. Although the content of THCs, constituents responsible for the physiological activity of the drug, does not directly influence the results, this parameter will be also connected with the "ripening" of the resin, the highest values (10-30) being typical for "unripe" samples. "Ripe" and "over-ripe" cannabis show negative values (up to -16). Consequently, this parameter is in reverse relation to the difference T890-T1130. However, this will not be the case in some altered samples, so that the disagreement in the results of the two infra-red parameters may indicate the presence of an altered, decomposed cannabis.

Practical differentiation of samples

Although the values of the parameters used mostly and generally depend upon the way and the progress of the gradual conversion of cannabinols to each other, a parallel examination of all the values obtained for the samples examined indicate that each of the parameters is at the same time affected by certain specific and particular properties of the resin. Therefore, by an alternate, cross application of the six parameters proposed, a more detailed differentiation of various types of cannabis will be possible. For practical purposes, on the basis of the dispersion of analytical values obtained for 104 samples of cannabis from 20 countries, the above coding system is proposed, containing 32 coded numbers, each of them corresponding to a given range of one of the parameters used.


Key number


Key number


Key number

E 260/E 280
E 300/E 310
E 630/E 520
1.15- 1.30
0.85- 1.15
1.30- 1.55
0.80- 1.00
1.15- 1.40
1.55- 1.70
1.00- 1.50
1.40- 1.60
1.50- 1.90
E 420/E 520
T 890/T 1130
T 815/T 890
- 12
> - 10
0.75- 1.00
- 2- 0
- 10- 0
1.00- 1.15
0- 15
0- 10
1.15- 1.60
15- 25
10- 20

By using this key, the results of various samples (together with other known characteristics such as type of the drug, organoleptic and morphological properties, origin, age etc.) may be transferred on punched cards, so that each card bears the coded results for one examined sample. In this way, the selection and comparison of samples with close chemical composition will be possible. Groups of samples which are in certain parameters identical or similar may be often separated by means of other parameters. For example, cannabis from the western Mediterranean area (Spain, Morocco) have been similar to samples from Greece in E 630/E 520 ratio; however, it has a much higher E 260/E 280 value than the samples from Greece. If the results of all existing types of cannabis are included in a punched card system, an unknown sample, analysed by the method described, will be easily separated together with other samples belonging to the same chemical type. The possibilities of such a system may be illustrated by the coded results of some characteristic types of cannabis:

Type of cannabis

Major constituent

Coded characteristics

Tropical, maconha, highly potent (Brazil)
2, 11 (10), 16, 19, 27, 29
Tropical, Africa (Ghana, Kenya, Nigeria)
1, 10 (11), 17, 21, 27, 29
Western Mediterranean, kif (Morocco, Spain)
4 (5), 8 (9), 15, 19, 26, 30
Eastern Mediterranean, hashish (Lebanon)
5 (4), 7 (8), 14, 18 (19), 25, 30
USA, marijuana, wild (Kentucky)
3, 8 (9), 13, 19, 25, 30 (31)
Industrial hemp (Europe)
5 (4), 7, 13, 18, 24, 32

A combined spectrophotometric differentiation of samples of cannabis 29

The technique described should be regarded only as a means for the separation and characterisation of cannabis samples as belonging to a given chemical type. As the chemical composition of cannabis, owing to the instability and variability of its major constituents, may not depend only on the variety of the hemp plant and on its geographical provenance, but also on a number of other factors, the proposed method might be of limited value when applied to determine the exact geographical origin of a seized sample. For the same reason, the problem of origin determination of cannabis will still encounter great difficulties.



N. I. Vavilov, Studies on the origin of cultivated plants, Leningrad 1926.


C. C. Fulton, lndustr. eng. Chem. Analyt. Ed. 14, 407 (1942).


R. A. Todd, Endeavour 2, 69 (1943).


H. Asahina, Y. Siuchi, Bull. Natl. Hyg. Lab. (Japan), No. 76, 115 (1958).


O.-E. Schultz, G. Haffner, Arch. Pharm. 293, 1 (1960).


Lj. Grlic, A. Andrec, Experientia 17, 325 (1961).


M. Lerner, Science 140, 175 (1963).


F. Korte, H. Sieper, J. Chromatog. 14, 178 (1964).


United Nations Secretariat, Document ST/SOA/SER.S/13 (1965).


W. Beam, 4th Report, Wellcome Trop. Research Lab., Rep. Sudan Gov. 25 (1911).


Lj. Grilc, Bull. Narcotics 14, No. 3, 37 (1962).


Lj. Grlic, J. Pharm. Pharmacol. 13, 637 (1961).


Lj. Grlic, N. Tomic, Experientia 19, 267 (1963).


Lj. Grlic, Acta Pharm. Jug. 11, 129 (1961).


Lj. Grlic, Farm. Glas. 17, 424 (1961).


Lj. Grlic, Planta Medica 13, 291 (1965).


A. Radosevic, M. Kupinic, Lj. Grlic, Nature 195, 1007 (1962).


H. Gayer, Arch. exper. Path. Pharmacol. 129, 312 (1928).


Lj. Grlic, J. Pharm. Belg. 1959, 45.


Lj. Grlic, Farm. Glas. 19 , 163 (1963).


Lj. Grlic, Farm. Glas. 23, 311 (1967).


United Nations Secretariat, Document ST/SOA/SER.S/2 (1960).


Lj. Grlic, United Nations Document ST/SOA/SER.S/10 (1964).



F. C. Ball, of the Addiction Research Centre in Lexing-ton, Kentucky, made a follow-up study of 242 Puerto-Rican patients discharged from the Public Health Service Hospital in Lexington.

Heroin use among subjects of this study began as a part of recreational or street activities. The juvenile initiate usually had smoked marihuana before his first experience with heroin and in both instances he secured or was given the narcotic by neighbourhood friends. The common sequence of events was commence-ment of marihuana smoking at age sixteen or seventeen, heroin use at eighteen and arrest for possession or sale of drugs at age twenty. The mean age at which marihuana smoking began was 17.3 years for the males and 17.4 for the females. The youngest age at which marihuana use occurred was eleven years and the oldest age was thirty. Although fifteen of the 119 opiates addicts reported that they had never used marihuana, of those who had smoked marihuana 91 per cent. reported that marihuana use preceded opiate use. Thus, although marihuana smoking commonly preceded heroin addiction among the Puerto-Rican youth of this study, this was not invariably the case.

Of the 107 male addicts, almost two-thirds had started use of opiates by age nineteen. The youngest age at onset was twelve years. After this first opiate use, addiction commonly followed within three months. Heroin was the predominant drug first used (by 86 per cent of the males), followed by morphine, demerol (pethidine), dilaudid (hydromorphone) and opium. ( The British Journal of Criminology, 407, 1967.)


In view of situation about the abuse of drugs in the country, and taking into account the stress laid by the World Health Organization, the Commission on Narcotic Drugs and the International Narcotics Control Board on epidemiological studies of the sociological, legal and other factors underlying the phenomenon of drug abuse, the Institute for the Study of Drug Dependence was incorporated in the United Kingdom last January, with premises at Chandos House, 2 Queen Anne St., London W.l.

The Institute's principal functions are:

  1. to collect, collate, interpret and disseminate the results of research work, both in the country and elsewhere;

  2. to promote further research and to undertake research directly where necessary;

  3. to establish a reference library and an information retrieval system in conjunction with other bodies at home and abroad;

  4. to provide facilities for meetings, discussions and seminars, including international conferences;

  5. to provide objective information, especially to those who help to form public opinion.

The Institute intends to put in hand as soon as possible a number of studies judged likely to yield useful results over a fairly short period. The following topics have been provisionally selected:

  1. a survey of the risks involved in medical treatment with LSD in Britain (short-term study, six to nine months).

  2. an annotated bibliography of the medical and scientific information on cannabis (short-term study, six months).

  3. a comparative study of prosecutions and sentencing policies for drug offences. This would be a pilot study conducted in selected Police Divisions typical of urban areas where drug taking is an established phenomenon (medium-term study, up to two years).0

  4. a controlled study of the value of films in changing adolescent attitudes to the use of drugs (medium-term study, up to two years).


A Bureau of Narcotics and Dangerous Drugs was formed on 8 April 1968 in a major step to strengthen the Federal Government's effort against the illegal sale or use of narcotics and dangerous drugs.

The Bureau resulted from the merger and transfer into the Department of Justice of the Treasury Department's Bureau of Narcotics and the Department of Health and Welfare's Bureau of Drug Abuse Control.

The number of agents in the new bureau will be increased from about 600 to more than 800.

The Bureau of Narcotics and Dangerous Drugs will be headed by John E. Ingersoll as Director with two Associate Directors: Henry L. Giordano, who headed the Bureau of Narcotics, and John Finlator, who headed the Bureau of Drug Abuse Control.