Sensitivity. The application of activation analysis t o strontium-84 micro estimations allowed amounts of the order of gr:tm to be detected with ease. As the half life was relatively long, the samples could be processed in large batches and their activity detected later when all the samples were completed. Tests on solutions of known strontium-84 content gave experimental results which were in the range ztlyo of the (calculated value. Biological samples varying in known strontium-84 content from gram to 10-6 gram were malyzed by the above method and gave experimental results which were in the range *2Q/, of the calculated value.
CONCLUSION
This type of analysis was made possible by the use of activation analysis and made simple by the ability to add relatively large amounts of carrier strontium, thus removing the need for micro separation techniques. The mixed ferrocyanide was useful in the separation of calcium from barium and strontium.
ACKNOWLEDGMENT
The author thanks John Glaister, J.M.A. Lenihan, and Edgar Rentoul
for support and laboratory facilities during the investigation. LITERATURE CITED
(1) Feigl, F., “Spot Tests in Inorganic Analysis,” 5th Ed. p. 220, Elsevier,
London and New York, 1958.
(2) Nier, A. O., Phys. Rev. 54,275 (1938). (3) Sunderman, D. X., Townley, C. W.,
U. S. At. Energy Comm. Rept. NAS-NS 6, 1960. (4)Vogel, A. I., “Qualitative Inorganic :Analysis,” 4th Ed., p. 302, Longmans, .Green, and Co., London and New York, ,1954. 3010 p.
RECEIVED for review September 17, 1962. Accepted January 29, 1963. The work was supported by a grant from the Medical Research Council.
Identificatiion and Origin Determinations of Cannabis by Gas aind Paper Chromatography T. W. M. DAVIS and C. G. FARMILO Organic Chemistry and Narcotic Section, Food and Drug Directorate, Department of National Health and Welfare, Ottawa, Ontario
MIROSLAW OSADCHUK The Regional laboratory, Food and Drug Directorate, Toronto 5, Ontario, Canada
b The cannabinols present in the leaves and flowering tops ccin be characterized by relative retention times and Rf values. Pyrahexyl, a commercially available tetrahydrocannabinol homologue, may b e used as a reference. The Beam reaction diffcrentiatescannabidiol from the other ccinnabinols in gas chromatographic fralctions and on paper chromatograms. This test may now be considered ,characteristic of cannabis. A plot of peak areas of cannabidiol vs. cannabinol plus tetrahydrocannabinol permits differentiation between cannabis of different geog ra phica I origins.
T
FOLLOWING !cannabinols are contained in Cannabis sativa L.: HE
i=-0
CH/
‘CH3
Tetrahydrocannabinol (THC)
Cannabinol (CBN)
extract of cannabis. Crystalline T H C derivatives have been described (3, 16). Some doubt still exists regarding the position of the double bond in the cyclohexene ring of CBD, CBDA, and THC, although it is not conjugated ( 1 , 19) with the benzene ring. It would be useful for court purposes to present evidence particularly of the presence of T H C in cannabis samples. THC is assumed to be a mixture of isomers whose structures differ, both in the position of the alicyclic double bond and in optical and steric properties ( I S , 19, 20). T H C has paper and gas chromatographic values similar to those of the low-melting synthetic standard ( 5 ) . In reviews of the analytical chemistry of cannabis, it was shown (2,6) that further characterization of cannabinols was required. Results of recent work on the isolation, purification, and identification of cannabis extracts from plants of different origins by means of paper and gas chromatography are reported here. Their use in the determination of origin is discussed.
Cannabidiol (CBD)
Sample Preparation. Fresh cannabis samples of Canadian origin were stored a t - 30” C. for 15 months. T h e samples were air-dried just before analysis. The other samples were received as dry material. At the time of analysis, the ages of the samples in months were: Canadian Seizure and
Reviews of the chemistry and pharmacology of CBD, CBN, and T H C (14, 20), and of CBDA (17), show certain properties of the cannabinols which are important to the drug analyst faced with the problem of identification of large numbers of marihuana samples of different origins. CBD, CBDA, and CBN are solids with definite melting points, which suggests that they are homogeneous, pure, crystalline materials. THC, the physiologically active component (14, 21), however, was obtained until recently as a resin or viscous oil. Two synthetic T H C isomers, m.p. 62’ to 63’ C. and 125’ C., have been reported (11), along with a crystalline T H C from plants, (m.p. 120’ to 125’ C.). An earlier worker ( 9 ) had isolated a similar crystalline substance from an
c-0 C H j ‘CH3
EXPERIMENTAL
Cannabidiol acid (CBDA)
VOL. 35, NO. 6, MAY 1963
751
Greek, 39; German, Brazilian, and Swiss, 27; Moroccan, 15. All samples were crushed to -20 40 mesh. A small condenser of 60-mm. jacket length and 7-mm. i.d. was mounted vertically, tip down. Warm water (50' C.) was circulated through the jacket. A plug of cotton wool n-as placed in the lower end, its length and density adjusted to allow methanol to leak through a t the rate of about 1 ml. per minute. One-half gram of cannabis was placed in the inner tube and 7 5 ml. of methanol was allowed to flow through the sample in approximately 75 minutes. Complete extraction of cannabinols n-as determined by the absence of blue or purple color on application of the Duquenois test to the eluate. The eluate was caught in a 100-ml. centrifuge tube, and the tube was placed in a dry ice-acetone mixture for 10 minutes, then centrifuged for 3 minutes, and decanted. The residue was agitated with 50 ml. of cold (-75' C.) methanol, frozen again for 5 minutes, centrifuged, and decanted. The liquid was added to the first supernatant. Methanol was removed in a stream of nitrogen, the residue dissolved in benzene, and made up to 10 ml. in a volumetric flask. This solution was suitable for gas and paper chromatography, While for origin and quantitative determinations it was necessary to extract the material exhaustively, for simple identification of cannabis the sample vias extracted in a beaker with small portions of methanol and decanted through a filter paper. The methanol was evaporated, the sample taken up in benzene, and filtered through 6 grams of Florisil in a 25- X 50-mm. fritted glass funnel. This solution was suitable for color tests, and gas or paper chromatography. Florisil adsorbed polar constituents such as CBDA but CBD, CBN, and THC were eluted. Paper Chromatography. App.4RATUS. Museum jars, 24 X 23 X 11.5 cm., with three molded grooves to take glass rods for suspending the paper; Whatman S o . 1 chromatography paper cut in sheets 8 l / 2 X 91/* inches; Ultraviolet handlamp, Mineralight Model SL2537, Ultraviolet Products Inc., South Pasadena, Calif. REAGENTS. Standard solutions: CBDA diacetate; CBD; THC, m.p. 62" to 63' C. (11); CBN and Pyrahexyl (dbbott Laboratories), 1 mg. in 1 ml. of benzene, Chromatographic Solvent : Cyclohexane was shaken with excess dimethylformamide. The upper layer was used as the eluting solvent, the lower layer for impregnating the paper. Color Reagents: Diazotized p-nitroaniline: 0.3 gram of p-nitroaniline in 100 ml. of 1:3 hydrochloric acid-water solution. Five grams of sodium nitrite in 100 ml. of water. Five milliliters of the p-nitroaniline solution was put into a 50-ml. graduate, 0.3 ml. of the sodium nitrite solution added, and diluted to 50 ml. with distilled water. The mixedspray reagent was prepared just prior to use. Beam's Reagent: Five grams of potassium hydroxide in 100 ml. of
+
752
ANALYTICAL CHEMISTRY
methanol. CBD gives a purple color, while CBDA reacts very slowly. Duquenois' Reagent (Modified: not the test used in A.T.U. and customs and excise labs in the US.): Four grams of vanillin in 50 ml. of ethanol. To this solution, was added 2 ml. of acetaldehyde. Cannabinols give blue to purple colors with this reagent when concentrated hydrochloric acid is added. PROCEDURE. DeRopp's solvent system (3) modified by Osadchuk (15) was employed. Parallel to the longer edge of the paper, pencil lines m-ere drawn a t 12.5, 25, 30, and 145 mm. from the edge. The 25-mm. starting line accommodated eight equally spaced spots. The jar was filled with dimethylformamide-saturated cyclohexane to such a depth that the liquid level touched the 12.5-mm. line on the suspended paper. Twenty microliters of cannabis extract sufficed for each spot. Standards of CBD, THC, CBX, and Pyrahexyl were used in 5-pl. amounts. The following technique of impregnation and handling of the paper is important for good separation of the spots and reproducible R, values. Dimethylformamide, saturated with cyclohexane, m-as poured into a shallow tray. Starting from above the 145-mm. line, the paper was dipped into the solvent until the impregnation reached the 30-mm. line. The paper was withdrawn, blotted rapidly between two sheets of filter paper, and immediately hung in the chromatography tank. When all three papers were in the tank the cover was placed on it, and the solvent allowed to rise to the 145-mm. line. Development took about 13/4 hours. The paper mas dried in a current of air in the fume hood and examined under 2537 il.ultraviolet light. Absorbing and fluorescing spots and the solvent front were marked. For general identification the paper was sprayed with diazotized p-nitroaniline. Maximum colors varying from yellow to brown, on a white background, were developed within 5 minutes. The papers store well with minimum fading. CBD, which gave a purple spot when sprayed with Beam's reagent ( l a ) , was thus differentiated from other cannabinols. If only a very faint color was observed, the presence of CBD may be confirmed by observing the area with the ultraviolet lamp. A marked increase in absorption was apparent. Duquenois reagent was applied ab a coarse spray to wet the paper quickly and thoroughly. This was followed by a concentrated hydrochloric acid sprag on both sides of the paper. This treatment was repeated after 1 minute. The sensitivity of the reaction may be increased about 10 times by dipping the paper in concentrated hydrochloric acid instead of spraying. Although the sprayed papers faded within about two days, the dipped papers were useful only a t the time they were made. Gas Chromatography. APPARATUS. Research Specialties Co., Series 600 gas chromatography, with SrgObetaray argon ionization detector and katharometer in series; a 1/4-inch 0.d. stainless steel column 6 feet long;
Table 1.
Paper Chromatographic Data of Cannabinols
Behavior, Substance R, 2 5 3 7 8 . light Cannabidiol acid 0.05 fluorescence, blue-white Cannabidiol 0.15 absorbs Cannabinol 0 . 4 3 absorbs Tetrahydro0.65 absorbs cannabinol Pyrahexgl 0.84 absorbs
-1 t o + 5 m.v.. 1-second response, Minneapolis Honeyweli recorder, with Disc integrator. PREPARATIOX
OF
THE
COLUNN.
Three grams of silicone-gum rubber SE-30 (General Electric Co.) were dissolved in 130 ml. of methylene chloride in a short-necked, round-bottomed flask. To this solution were added 100 grams of Chromosorb-IT, 60- to 80-mesh. The solvent mas removed in a rotating flash evaporator. The powder was gently sieved to +60 -80 mesh size. The packed column n as placed in the chromatograph oven and 5 p.s.i. (argon) applied. The outlet of the column remained open. The oren temperature was gradually raised from 180' to 225' C. orer 8 hours, then brought to 325' C. and kept for 24 hours to complete the conditioning. OPER.4TlXG CONDITIOSS A S D TECHNIQUE. The following instrument settings were used: vaporizer 360' C.; column 212' C.; detector 240' C.; outlet line 290' C.; gas flow 125 ml. per minute; inlet pressure 27 p.s.i.; voltage on detector 1275 volts. The gas flow was determined at the outlet, using a soap film f l o r meter (18). At five times attenuation, the sample size varied from about 1 to 8 11. Areas under the curves were recorded by the integrator. Fractions were collected using helium as the carrier and the katharometer detector (10) ; filament current 300 ma. The material condensed at room temperature in 4-cm. lengths of 0.6-em. glass tubing. RESULTS AND DISCUSSION
The R, values and ultraviolet behavior of the four cannabinols and Pyrahexyl are shown in Table I. Colors of the spots with diazotized p nitroaniline and dibromo-p-benzoquinonechloroimide have been described (12). The authors' experience is that the color reaction with the diazo spray of both CBN and THC tends to the same yellow, with small concentrations. I n higher concentrations, the C B S spot is definitely more reddish. Extracts of marihuana frequently give a negative conventional-Beam reaction (4, 8, 22). However, the paper chromatographic technique described always shows a CBD spot. The alkaline Beam spray reagent gave positives in all cases. This is not unexpected since
TIME
(MINUTES1
Figure 1. Gas chrom,ztograms of standard substances (CBD, THC, CBN, and Pyrahexyl) and extracts of cannabis of different geographical origin
CBD is believed to lie a prccursor of T H C and CBN ( 6 ) . The reliability of the Beam test conducted in this manner is now improved and should be an important aid in marihuztna identification. The Beam reagent is approximately 10 times less sensitiw than the diazo spray reagent, which can detect 0.1 pg. of cannabinols. The Duquenois rea,gent was also investigated as a spray reagent, since it has been used as a ,sensitive test for cannabis for many pesrs. During this time, a claim has been made that -it is due to the presence of T H C ( 2 3 ) . This claim is unwarranted, since thc reagent reacts with all cannabinols. Its sensitivity as a ,;pray is abou;; equal to that of the Beam reagent sprz,y. The reaction was inlTestigated using a spot plate technique with a number of 1,3-dihydroxyplienols. Resorcinol, orcinol, and 4-chlororesorcinol gave deep blue and purple color:,; 1,3-dihydroxy4-benzoic acid turned dowly pink, then to purple and blue; 1,3-dihydroxy-5benzoic acid gave a pale green color. These results suggest that a t least the 6-carbon atom of a phenol must be unsubstituted for R reaci;ion to occur. If both the 4 and 6 positions are open, and not deactivated by a meta-orienting group in the 5 positio;i-e.g., orcinolthe compound should give a strong reaction. Both hydroxy groups need not be free, as C B S and T H C are 6H-dibenzo [b.d.] 1 q n n s and give strong reactioiv. During the early part of the investigation, the paper chroma tographic method of D('Ropp ( 3 ) was used. Difficulty
with crooked fronts, variable time of development, and varying R j values caused us to abandon it for the short-paper ascending system which is simpler and faster. Identification of the spot, R j 0.05, as CBDA was made as follows: Both CBDA and cannabidiol diacetate were observed to yield a blue-white fluorescence under the ultraviolet light. Their chromatographic behavior differed, howerer, the ester moving more slonly than the suspected CBDA. In the solvent system applied, migration of a substance is inversely proportional to its polarity. Cannabidiol acid diacetate is a stronger acid than CBDA (17) and the presumptive CBDA spot indicated its weaker acidity by a higher R,. I n addition to these observations, paper and gas chromatograph? gave further evidence. A benzene solution (4 nil.) of the leaf extract was analyzed by paper chromatography. THC, CBX, CBD, and the substance of R, 0.05 were found to be present. The benzene solution was extracted with 5% potassium hydrovide solution containing 2Y0 of sodium bisulfite. Paper chromatography of the extracted benzene solution gave no spot a t Rf 0.05. The alkaline solution was acidified and extracted with ether. The ether was washed with a small amount of water and brought to 4-ml. volume. Paper chromatography showed CBD, CBX, and T H C were absent but a distinct spot Rf 0.05 was obberved. Gas chromatographie analysis of the
original benzene extract shoned the presence of CBD, CBN, and THC. The CBD peak area was measured. The alkali-extracted benzene was chromatographed, injecting the same volume of sample. The CBD peak area was measured and n.as less than that of the original extract. The ether extracted was then analyzed, injecting the volume used previously for the benzene solutions. Only CBD was present and the peak area was measured. The combined peak areas of the alkali-extracted benzene and the ether extract were 957, of the CBD peak area of the original estract. These paper and gas chromatographic results showed that the spot Rf 0.05 was CBDA and that CBDA is decarboxylated to CBD in tlie gas chromatograph, Even a t a vaporizer temperature of 260" C., or a column temperature of 180" C. (using a 23-inch length column) , this decarboxylation occurred. Schultz and Haffner have reported decarboxylation of Cl3D.1 to CBD a t 100" C. and 0.1 mm. preswre (1'7). Gas chromatogram. of reference standardb and plant extract> are shown in Figure 1. The identity of the peaks was established by simultaneous injection of known compounds and samples, and collection of fractions for paper chromatographic examination and color tests. The criteria of identity thus established included relative retention times (RRT), R j values, and colors. The retention times relative to CBD, (RRTICBD) are THC, 1.36; CBN, 1.68; and Pyrahexyl, 1.89. The CBD peak appears in about 11 minutes. VOL. 35, NO. 6, M A Y 1963
753
Table 11.
Phenolic Content of Dry Cannabis Leaf by Gas Chromatography
Volume injected,
Origin& CBD, THC, % CBN, 70 Greece 0.68 3.3 2.81 0.10 0.44 Brazil 0.63 0.31 0.24 Morocco 0.48 0.10 0.36 Germany 4.7 0.09 0.11 1.6 Switzerland Mexico? (Seizure, Canada) 0.90 0.19 0.27 0.46 0.03 0.04 Canada, (Lyn, Ont.) 0.65 0.002 0.003 Canada, (Picton, Ont. 3 ) a Female plants except as noted. To give approximately full scale peak height for CBD peak. c Original CBD CBD from decarboxylation of CBDA.
M1.b
1.6 6.0 8.0 1.0 2.8 4.8 5.0 6.6
+
grams in Figure 1 shows that there are differences in the relative amounts of CBD, THC, and CBN between plants of different geographical origin. If the area under the CBD, THC, and CBN peaks is taken as loo%, and area of the CBD peak, expressed as a per cent of this total area, is plotted against the sum of areas of the T H C and CBN peaks expressed as a per cent of the total, the position of the points on the straight line so produced is related to the geographical origin of the sample (Figure 2). (It is obvious from earlier discussion that the CBD peak is the sum of the original CBD plus that formed by decarboxylation of CBDA) . Although the gas chromatograms of the Canada Seizure sample (believed to be of Mexican origin) and the authentic Brazilian sample are similar, the two origins may be distinguished in Figure 2. The chromatogram of the Seizure sample
Paper chromatographic examination of the three cannabinols separated by the gas chromatograph showed the presence of varying amounts of CBD in each, as follows: The intensities of the Beam reactions with fractions of CBD, THC, and CBN were, respectively, strong, weak but definite, and faint. In each fraction, however, the main chromatographic spot was identified by R j value as that of the peak from which it was collected. Assay of several samples, by measurement of the area under the respective peaks and comparison with the areas given by known weights of standards, is shown in Table 11. For weights of standards varying from 0.1 to 4 pg., the plot of integrator counts us. weight is a straight line. The slopes in counts per pg. were: CBD, 1700; THC, 2387; CBN, 2050. Examination of the gas chromato-
CLIMATIC FACTOR M . A C.,tenths
_____
60
T.OF
__.___ 2 - 3_ _ 6 0.- 7_ 0 4
50 /-
2 +
~
60-70
Rain, in. 10-25 25-50
\
40
I I-
LITERATURE CITED
Cain, C. U., Wearn, R. B., Baker, R. B., Wolff, H., J . Am Chern. SOC.6 2 ,
30
2ol
\
LYN, (MATURE) PICTON,ONT.
.\4
SWISS--
'e
IO
-
\,PlCTON.d
I
I
I
I
20
40
60
80
-
GERMAN. -
? - - -- -
- - -
- .-
5-6 6
lMMATURE-(77)
50-60 50-60
25-50 25-50
143)
132 6 )
io P ~ C T O NM,A~T U R E \I
100
C B D '10 Figure 2. Relative percentage of CBD, plotted against relative percentage of sum of THC CBN for cannabis of different geographical origins
+
Analyser by gas chromatography M.A.C. = mean annual cloudiness T = mean temperature Rain = annual rainfall, inches
754
ANALYTICAL CHEMISTRY
ACKNOWLEDGMENT
We thank Friedhelm Korte and Helmut Sieper of Bonn, Germany, and 0. E. Gchultz and Gert Haffner of E e l , Germany, for samples of the cannabi0. J. Braenden of the United nols. Nations, Geneva, Switzerland, and LjubiSa Grli6, Institute for Control of Drugs, Zagreb, Yugoslavia, kindly supplied some cannabis samples. R. Lane prepared the samples for assay and chromatography. (1) Adams, R., Loewe, S., Pease, D. C.,
0 0
is completely different from that of two samples of cannabis grown in Canada, namely the Lyn and Picton samples from the northeastern area bordering Lake Ontario. The latter show that the male Cannabis plant, at least in this area, has the same relative amount of phenols as does the female plant. Comparison of the chromatograms also shows that samples from northern regions-e.g., Germany and Canadacontain much less T H C and C B 9 than those produced in warmer climates such as Morocco, Brazil or Greece. Additional genuine samples are being investigated to determine the reliability and extend the scope of the method. The cause of variations in the amounts of cannabinols in plants from different countries is not known. The climatic factors, sunshine, temperature, and rainfall (see Figure 21, do show an apparent rough correlation with the amount of THC. A low mean-annual cloudiness, or a large numher of hours of sunshine, is found in Morocco and Greece where plants have the highest T H C content. Fulton ( 7 ) is of the opinion that plants from middleeastern countries have been specially selected by hashish producers for high resin content and potency. Further work on genetic and climatic factors is in progress.
2566 (1940). (2) Cheronis, N. D., Mzcrochern. J . 4,555 (1960). (3) De Ropp, R. S., J. Am. Pharm. Assoc., Sci. Ed. 49, 756 (1960). (4) Farmilo, C. G., United rations Document, ST/SOA/Ser.S/4, 1961. (5) Farmilo, C. G., Davis, T. IT'. M., J . Pharm. Pharmacol. 13, 767 (1961). (6) Farmilo, C. G., Davis, T. Wr.M., Vandenheuvel, F. A , , Lane, R., United
Kations Document, ST/SOA/Ser.S/7,
1962. (7) Fulton, C. C., 260-37 Union Turnpike, Glen Oaks, Queens, N.Y. private communication, April 8, 1962. (8) GrliE, L., Braenden, 0 . ,J., United
Xations Document, ST/SOA/Ser.S/l,
1960. (9) Haagen Smit, A. J., Wawra, C. Z., Koepfli, J. B., Alles, G. 4.,Feigen, G. A., Prater, A. K., Science 91, 602 (1941). (10) Kingston, C. R., Kirk, P. L., ANAL. CHEM. 33,1794 (1961). (11) Korte, F., Sieper, H., Ann. 630, 71 (1960).
12) Korte, F., Sieper, H., Tetrahedron 10, 153 (1960). (13) Leaf, G., Todd, .4. R., Wilkinson, S.,J . C‘hem. SOC.,1942, 185. (14) Loewe, S., Arc,h. Exp. Pathol. Phamaakol. 211, 175 (1950).
(15) Osadchuk, N., Relgional Office, Food and Drug Directorate, 55 St. Clair Ave. E., Toronto, Ont. private communication, June 7, 1962. (16) Powell, G., Salmm, IbI., Bembry, T. H., Walton, R. P., Science 93, 522 (1941)
(17) Schultz, 0. E., Haffner, G., Arch. Pharm. 291/63, 391 (1958). (18) Smith, D. M., Campbell, R. G., Chemist-dnalyst 50, 80 (i961). (19) Taylor, E. C., Strojny, E. J., J. Am. Chem. SOC.82, 5198 (1960). (20) Todd, A. R., Ezperientia 2 , 55 (1946). (21) Wollner, H. J., Matchett, J. R., Levine, J., Loewe S., J . Am. Chem. SOC.64, 26 (1942). (22) Wollner, H. J., Matchett, J. R., Levine, J., Valaer, P., J . Am. Pharm. Assoc. 27, 29 (1938).
(23) Young, J. R., “Tests for Marihuana,’’ Bureau of Internal Revenue, Alcohol Tax Unit, May 1, 1951. Cited in ref. I, p. 564. RECEIVED for review November 15, 1962. Accepted February 25, 1963. Division of Analytical Chemistry, 141st Meeting, ACS, Washingtpn, D. C., March 1962. Work reported in this paper was undertaken as part of the International Scientific Research on Cannabis, pursuant to Resolution 8 (XIV) of the Commission on Narcotic Drugs of the United Nations.
Microscale Identification of Several Sugar Phosphates by Paper Chromatography a nd Electrophoresis R O M A N O PIRAS ancl ENRICO CABIB lnstituto de lnvestigaciones Bioqurinicas “Fvndacibn Campomar” and Faculfad de Ciencias Exacfas y Naturales, Obligado 2490, Buen,x Aires, Argentina
b Severat sugar phosphates have been converted to cyclic phosphates or methyl esters by treatment with dicyclohexylcarbodiimide in methanolic solution. The producis obtained were then separated by paper chromatography with solvents containing cetyltrimethylammonium bromide or by paper electrophoresis in potassium or cetyltrimethylammoriium borate buff ers. The separations obtained were better than those previously described for the untreated esters and the complete procedure could be carried out with less than 0.5 pmole of substance. The compounds obioined in the dicyclohexylcarbodiimide reaction were studied in detail and general rules have been formulated for the prediction of the type of product to be expected in each case. Some correlations between chemical structure and chromatographic or electrophoretic behavior have been observed.
-
A
NUMBER
O F PAFERS
(11, 18, 28,
29, 33, 34) dealing with the appli-
cation of paper chromatography and electrophoresis to the separation of sugar phosphates and other phosphoric acid esters have appcsared. However, the resolution of diffeIent hexose phosphates achieved by the methods so far described is rather poor and is completely successful only in certain cases. Good results have bem obtained with exchange resin column chromatography (17), but this technique is somewhat cumbersome and is not easily adapted to microscale use. 11, is the purpose of this communication to describe an efficient method for the identification of several sugar monophosphates by
paper chromatography and electrophoresis. Rees has recently shown (27) that it is possible to increase the R, of sugar sulfates by adding a long-chain tetralkylammonium salt to the solvent. I n this way, solvents that are very selective for sugars can also be used for the separation of their sulfates. Attempts t o extend this method to sugar phosphates met with very limited success, apparently because sugar phosphates, unlike the sulfates, possess two dissociable anionic groups. To obtain compounds with a single acid group, phosphate esters were treated with dicyclohexylcarbodiimide (DCC) in methanolic solution, whereby the corresponding cyclic phosphates or methyl esters were formed (15, 16). The products could then be separated by paper chromatography with solvents containing cetyltrimethylammonium (CTA) bromide or by paper electrophoresis with potassium or CTA borate (25)* I n an attempt to ascertain the general applicability of the method, the structure of the derivatives obtained in the DCC reaction was studied in some detail, based on the findings of Khorana and his coworkers (16). EXPERIMENTAL
Materials. All sugar esters used belong t o the D-series. The l-phosphates of glucose, galactose, xylose, and mannose are t h e a-anomers. T h e phosphate group is abbreviated P , as in glucose 1-P. M o s t sugar phosphates used were commercial samples. A sample of galactose 1-P was prepared as described by Hansen, Rutter, and Krichevsky ( l a ) ,
and one of xylose 1-P was obtained according to Meagher and Hassid (23). The purity of the esters was investigated by submitting the corresponding sugars to paper electrophoresis after hydrolysis with dialyzed alkaline phosphatase (Armour Research Division, Chicago, Ill.). The phosphatase preparation was free from phosphohexoisomerases, as shown by the fact that crystalline glucoje 6-P gave rise to glucose only. Both fructose 6-P and mannose 6-P samples liberated some glucose, indicating a contamination, probably with glucose 6-P. I n addition, both mannose 6-P and galactose 6-P samples gave a fasterrunning, reducing spot in solvent A (see below). They were therefore purified by chromatography with this solvent on Whatman KO.3 paper, in the manner already described (6). Fructose 6-P was purified in the same fashion, but using solvent F (see below). Chromotropic acid (Eaqtman Organic Chemicals, Rochester, N. Y.) was purified by fractional precipitation with ethyl ether from an aqueous ethanolic solution and was stored under reduced pressure. Dowex 50 (H+) resin of 20- to 50mesh and 8% cross-linkage was used. General Procedure for t h e Treatment of Sugar Phosphates with DCC. The technique adopted was similar to that described by Khorana et al. (16). To prevent the formation of ,V-phosphoryl ureas the reaction was carried out with the addition of triethylamine and in the absence of water. Five micromoles of the ester (sodium salt) in 0.4 ml. of water were passed three times through a Dowex 50 (H+) resin column 5 em. long and 0.4 em. in diameter. The columns were flushed with 0.4 ml. of water. a 13- X 100-mm. test tube being used to collect the effluents. After adding 0.2 ml. of pyridine, the tube contents were brought to dryVOL. 35, NO. 6, MAY 1963
* 755
