of In [(ip-i)1/2/i] us. E is linear over a major fraction (70%) of the rising portion of the wave for planar electrodes, and yields as slope RT/nF for Nernstian charge transfer (even with kinetic complication) or RT/anF for irreversible charge transfer. The log plot criterion of reversibility is approximately obeyed a t spherical electrodes as well, provided that the radius of curvature is not too small. Derivations of the equations herein presented and further discussion of their implications will be given in papers now in preparation. NOMENCLATURE
a
=
C
=
(nF/RT) times rate of potential scan initial concentration of the reactive species
D E Ei E"'
diffusion coefficient potential = initial potential of scan = formal standard potential of electroactive couple exp = natural exponential function F = Faraday's constant i = current density j = running index in infinite series k = rate constant of homogeneous first-order chemical reaction k , = rate constant of heterogeneous charge-transfer reaction a t Eo' In = natural logarithmic function n = charges per molecule transferred in electrode process p = coefficient of term in infinite series r = radius of spherical electrode R = gas constant t = time from start of scan T = absolute temperature cy = charge-transfer coefficient =
=
$ p
4
= 6
exp [(nF/RT)(E"'- .Ei)-at]
=d/Dlrl/cUa =
x
=
6
=
k,/a (ka/d(Yao)(iC./6)a m
z LITERATURE CITED
(1) DeMars, A. D., Shain, I., J . Am. Chem. SOC. 81,2654 (1959). (2) Matsuda, H., Ayabe, Y., 2. Elektrochem. 59,494 (1955). (3) Reinmuth, W. H., ANAL. CHEM. 32,1891 (1960). (4) ZU.,33, 185 (1961). (5) Reinmuth, W. H., J. Am. Chem. Soc. 79,6358 (1957). W. H. REINMUTH De artment of Chemistry
CoPumbia University New York 27, N. Y . RECEIVEDfor review August 4, 1961. Accepted September 18, 1961.
Separation of Components of Marijuana by Gas-Liquid Chromatog raphy SIR: In a recent study, in this laboratory, on the chemical identification of Cannabis sativa (marijuana), it was necessary to find a quick and efficient method for the separation of the components found in the resin produced by the plant. Satisfactory separations of the high boiling resinous components were possible with gas-liquid chromatography, using a silicone rubber (SE-30) liquid phase. The instrument used was an Aerograph A-904 gas chromatograph equipped with the standard katharometer and a Varian G-10 (10-mv. span) recorder. The column was a &foot length of 1/4 inch 0.d. copper tubing filled with SE-30 ('2%) on Chromosorb W (SO/lOO). The initial separations were made directly with a petroleum ether extract of the leaves and flowering tops of the Cannabis plant, concentrated to a 10 to 20% solution. Six fractions could be detected with an injection of 20 pl. of the extract, a t 250' C. with a flow rate of 40 cc. per minute of helium. Twelve fractions could be detected by collecting each fraction from several runs and rechromatographing them a t 225' C. Figure 1 is a composite representation of the twelve peaks; the areas denoted by the numbered lines are the initially detected fractions. Ultraviolet absorption curves, using a Beckman DB recording spectrophotometer, were determined for seven of the fractions; a summary of the data is listed in Table I. Since fraction j was probably cannabinol, a small quantity was acetylated 1794
ANALYTICAL CHEMISTRY
with acetic anhydride by refluxing in a sealed tube. The product crystallized from ethyl alcohol after standing 2 days in the refrigerator, The colorless crystals melted a t 75" C., and, with ethyl alcohol as the solvent, showed an ultraviolet curve essentially the same as that reported for cannabinol acetate by Adams, Cain, and Baker (1). From the ultraviolet data, it would appear that fractions d and/or e might be tetrahydrocannabinol isomers. The main product (assumed to be the methyl homolog of tetrahydrocannabinol) of a pulegone-orcinol condensation, synthesized as described by Adams, Smith, and Loewe (S), had a retention time of 6.85 minutes with the same conditions
Table I. Summary of Ultraviolet Data
Per Per Maxi- Cent Mini- Cent Frac- mum Trans- mum, TransMp mittance Mp mittance tion b
c d
e
{
279 268 258 226 280 261 280 276 281 276 281 278 260 254 284 218
66.5 64.0 65.8 33.0 71.3 72.0 56.6 57.5 80.0 79.8 61.2 42.0 20.0 20.7 65.2 37.0
275 263 254 221 268 252 252
67.0 66.5 66.0 33.8 75.0 73.3 75.8
253
86.5
249 273 240
84.2 44.0 34.0
250
89.0
shown in Figure 1. This is consistent with fractions d and e being tetrahydrocannabinol isomers. The particular resin used apparently had very little or no cannabidiol present, since none of the fractions showed a positive reaction with the alkaline Beam test (alcoholic KOH) (2). In view of the variation in composition of the resin of the Cannabis plant depending upon the variety (6), the age of the material] and perhaps the area in which the plant was grown (4, it is possible that gas-liquid chromatography will enable a determination of the origin of the plant material, and it is hoped that samples will be available soon for an investigation of this. With the above separation it was possible to study the reaction of the test described by Duquenois and Moustapha (6) on some of the individual components of the resin. This test is the most commonly used color test for marijuana in narcotic and toxicological laboratories, and utilizes two solutions: A, vanillin and acetaldehyde in ethyl alcohol (9573, and B, concentrated HCl. Solution A is added to the material to be tested, then an equal amount of B is added. Fractions c, d, e, and g produced a violet color with the reagent within a few seconds; a fleeting initial orange or pinkish color was sometimes noticed. Fraction j was slow to react, and passed through the color sequence of green to blue-green to an intense blue. Fraction b did not react with the reagent. Fractions f, k, and 1 showed positive results, but it is not certain
a t this time whether this was due to traces of other fractions trailing over; fraction 1from the initial separation also showed positive results. The main product of the pulegone-orcinol condensation previously mentioned produced a violet color which turned to blue-violet after standing 15 to 20 minutes; after dehydrogenation of the main product with sulfur (which should result in the methyl homolog of cannabinol) a blue color was produced. The acetylated cannabinol did not react with the reagent. The color sequences observed when an extract from the plant material is tested with the reagent are obviously results of the interaction of the various components. Similar colors can be obtained with other compounds. Several resorcinol derivatives produce a violet color-orcinol, for instance, goes through the sequence of orange to pink to violet-and a blue color can be obtained with thymol, which goes through the sequence of pink to violet to blue. The mechanism of the reactions is probably a condensation of the type experienced between aldehydes and phenols; the increased reactivity of the resorcinol-type structure produces a rapid condensation under the acidic conditions of the test. Although they are useful such color tests cannot be considered as rigorous proof for the presence of a complex botanical material. The use of gas-liquid chromatography provides an identifica-
34
20
6
5
b
io
I5
-----t
rnin
I
3
4
Figure 1 .
2
I
Fractions detected in Cannabis sativa resins
Temperature 225' C.; flow rate 40 cc./mln. Numbered lines indicate the six initial fractions
tion based on the retention patterns which is superior to that of color tests and allows separation of a complex mixture, as in the case of Cannabis resin, and an identification to be made by establishing the presence of some particular substance. ACKNOWLEDGMENT
The authors express appreciation to the State of California Department of Justice, Bureau of Karcotic Enforcement, for material used. LITERATURE CITED
(1) Adams, Roger, Cain, C. K., Baker, B. R.. J. Am. (:hem. SOC. 62, 2201 (1940): (2) Adams, Roger, Hunt, Madison, Clark, J. H., Ibid., 62, 196 (1940).
(3) Adam, Roger, Smith, C. M., Loewe S. Ibid., 63, 1973 (1941). (4) hou uet, R. J., U. N. Bull. on Narcotics 4 , 1 4 (1950). (5) Duquenois, P., Moustapha, H. N., J. Egypt. Med. Assoc. 21,224 (1938). (6) Matchett, J. R., Levme, J., Benjamin, L., Robinson, B. B., Pope, 0. A., J. Am. Pharm. ASSOC., Sci. Ed. 29, 399 (1940).
a:
CHARLES R. KINGSTON
PAULL. KIRK School of Criminology University of California Berkeley, Calif. RECEIVEDfor review July 31, 1961. Accepted September 21, 1961. Taken in part from the Master's thesis of Charles R. Kingston, University of California, 1961. Work supported by grants from the National Institutes of Health, U. S. Public Health Service (RG-4372 and RG5802), and from the Research Committee, University of California.
Experimenta I Eva I ua ti o n of Liquid-J unctio n PotentiaI SIR: The increasing use of nonaqueous solvents for investigating the nature of ion-solvent interactions and mechanism of organic electrode reactions has prompted us to submit the following communication. Although a large number of electrochemical investigations have been conducted in various solvents, most of the studies are of little value in furnishing information fundamental to understanding better the nature of ion-solvent interactions and organic electrode reactions, because of our inability to ascertain the magnitude of the liquidjunction potentials included in the potentials obtained. The complicated nature of junctions betu-een reference electrode systems and solutions being examined excludes any, but probably highly complicated, theoretical means of evaluating junction potentials. A number of attempts have been made to compare the electromotive force series in various solvents (5). Pleskov (5) has suggested the use of the rubidium couple as the standard
reference electrode, because of the low and nearly identical solvation energy of the large, slightly polarizable, noncomplex-forming rubidium ion in all solvents. Because of the limited number of solvents in which the potential of the rubidium couple can be evaluated and not constant nature of the solvation energy of rubidium ion in all solvents, use of the rubidium couple for the evaluation of liquid-junction potential, however, has ,attracted little support. The reversible nature of the 4,7dimethyl-1, 10-phenanthroline ferric4,7-dimethyl-I, 10-phenanthroline ferrous [DMFe(III), DMFe(II)] couple, its more accessible potential, and the nearly identical free energies of solvation of the two species in various solvents-because of their large size, slight polarizability, and noncomplexforming properties-make the DMFe(111), DMFe(I1) couple an eminently more superior couple for the experimental evaluation of liquid-junction potentials than the rubidium couple. Table I summarizes the half-wave
potentials (us. S.C.E.) observed for the oxidation of DMFe(I1) at a rotating platinum electrode in a number of solvents. The uncertainty of the potentials reported is less than =kt5 mv. The electrode, a 1-cm. length of 22-gage platinum wire extending from the tip of a soft glass tubing, was rotated a t ca. 600 r.p.m. DMFe(II)(ClOd)z was prepared by mixing 4,7-dimethyl-1, 10-phenanthroline and ferrous perchlorate-hexahydrate in a 3:1 mole ratio in acetone. The product was air-dried, and analysis showed it contained 6.36% of Fe(I1). The theoretical value is 6.36QjO0. The close to 0.06 value of the slopes indicates that the couple is reversible in practically all the solvents. In solvents in which DMFe(I1) perchlorate is not sufficiently soluble to give a measurable wave, the halfwave potentials were evaluated by extrapolating the El 2 values in several solvent-acetonitrile mixtures to zero acetonitrile concentration. The validity of this extrapolation technique has been checked with a number of systems in VOL. 33, NO. 12, NOVEMBER 1961
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