&out equal to that obtained from polyIn-butyl methacrylate). dome organic substances such as isobutanol (0.570), butanol-1 (0.2Yc),and isobutyraldehyde (1.47,) are only tentatively identified tnd are thercfore listed under “unidentified." The chemical composition of 12.4‘30 of the total pyrolysis products could not be evaluatrd. The residue n as 5.i’TO. I t is erident that the acetic acid formed is R product of direct hydrolysis of the original acr,tatc,; acetaldehyde, on the other hand, could be formed both from the alcohol and acetate by thermal osidation and reduction, respectively. \Tater is a very probable product from
the easily detachable hydroxyl group of vinyl alcohol. LITERATURE CITED
(1) Doolittle, H. D., Ettre, H., Spurck,
R. F., S-Bradi. P. F.. IRE Trans. on Electron Devices 8. 390 (1961). ( 2 ) Ettre, L. S., Brenner. S . ,J . Chromatog. 4 , 169 (J960).
(3) Grassie, S . , “Chemistry of High Polymer Degradation Process,” Interscience. S e w l o r k , 1956. 14) JanBk. J.. S a t w e 185. 68-1 (1960). (5) Kokes, R. J., Tohin, H . , Jr., Emmett, P. H., J . =Lm. Chem. SOC.77, 5860 (1955). (6) Lehmann, F. +I.,Brauer, G. M., .\XAL. CHEM. 33, 6i3 (1961). ( 7 ) Radell, E. A , , Strutz, H. C., I h i d . , 31, 1890 (1959). (8) Strasehurger, J., Brauer, C;. 1I.,
Tyron, M., Forziati, -4. F., Zbid., 32, 454 (1960). (9) VBradi, P. F., 1961 International Gas Chromatography Symposium, Instrument Society of America, Michigan State University, East Lansing, Michigan, June 1961; I S A Proc. 3 , 133 (1961). (IO) S’Bradi, P. F., Doolittle, H. D., Ettre, K., in “Advances in Electron Tube Techniques,” Proc. 5th U. S. Sational Conference, Slater, D., ed., p. 114, Pergamon Press, S e n York, 1961. (11) Watson, E. S., Bresky, 11). R. (to Perkin-Elmer Corp.) U. S. Patent 2,757,541 (-4ug. 7, 1956). RECEIVED for review December 28, 1961. .kccepted April 2, 1962. Division of .\nal~-ticsl Chemistry, l4lst Meeting, A\CS. 11-ashington, D. C., March 1962.
Separation and Identification of Tranquilizers by Gas Chromatography KENNETH D. PARKER, CHARLES
R. FONTAN, and
PAUL L. KIRK
School o f Criminology, University o f California, Berkeley, Calif.
b Application of gas chromatography to the separation and identification of 50 drugs commonly known as “tranquilizers” was studied. The group included phenothiazine derivatives, Rauwolfia alkaloids, diphenylmethane derivatives, butanediols, propanediols, and others. Though chemically heterogeneous, their cornnon pharmacological use makes their separation and identification important TO the toxicologist. Nearly all are extractable from basic solution b y arganic solvents, such extraction being part of toxicological routine. All of the drugs were chromatographed in rhe free form, many of them also as salts. One- to 8-gg. samples, in aqanic solvent, were injected into the argon gas chromatograph equipped with a column of SE-30, 0.0570, coated m glass microbeads. Though some compounds failed to emerge, most of them exhibited characteristic retention times which assist in identification and cn the quick screening of samples for members of this group. Linear derector response, peak height, or area, with respect to weight of sample, was ijbserved. I,I(I:D T F S r b yvailahlp for cerY P l ? i n of thc popular individual tran1 1 1 l i 7 (~2 ~8.10) are of limited utilitv i ‘ t cning tissue evtracts in routine
010gy Various idrntification propof ?ne tranquilizing drugs (color
CI
1
r\ &tl ’7
tests, u-rsy
tve be
*
stiiclitvi
diffraction r?
%me
data on paper chroniatographic separation are available (9). The Curry and Powell method ( 1 ) for alkaloids, and the Mannering, Divon, Carroll, and Cope method (S) for opium alkaloids are of some value. Developing times of about 16 hours were necessary, ordinarily, for good separation. Spectrophotometric examination in the ultraviolet has also been found useful for screening the tranquilizing drugs. The phenothiazines absorb with two maxima and minima n-hich vary somewhat in range, depending on substitution; some diphenylmcthanes have one maximum and one minimum each. Certain of the tranquilizers have no ultraviolet absorption in 507, ethyl alcohol : rescinnamine, captodiamine, meprobamate, methylphenidate, betazole. Others which exhibit absorption have no curve inflections which are useful for identification (6, 7 ) . The application of gas chromatography to rapid screening for some 50 tranquilizing drugs and to their separation, identification, and quantitative estimation is described in this paper.
packed with 60- to 80-mesh microbeads coated 11-ith SE-30 0.057, b y weight. The flow rate of argon was kept a t 40.0 ml. per minute under an inlet pressure of 33.2 em. of Hg. Four column temperat’ures were used: condition A = 150’ C., B = 165’ C., C = 180’ C., D = 210’ C.. all 10.5’ C. Samples of free compound or its salt, in acetone or ethyl alcohol solution were injected into the heated portion of the column through the diaphragm of the sampling attachment (5) using a 1.O-pl. Hamilt,on microsyringe of capacity. The tranquilizers were obtained as pure solids (some in the free form and some as salts) from their manufacturers, and were used without furt,hcr purification. Standard solutions of the free compounds were made by dissolving 200 mg. of tranquilizer in acetone or ethyl alcohol to make 10.0 ml. of solution. When only the salt form was available, the free base was obt’ained by quantitative basic extraction with chloroform from a weight of salt equivalent to 200 mg. of the free base. The chloroform ivas carefully evaporat’ed, and the free base residue was dissolved 9s described. The resulting standards contained 20 p g . per pl.
EXPERIMENTAL
Apparatus and Reagents. T h e P y e argon chroniatograph (IT.G. Pye and Co., Ltd., Granta W o r k s , Cambridge, England) equipped nich a n ionization @-ray (strontiumgO) cietrctor, arid a 10mv nIinneapolis-Honr.ywell-~ro~~7n recorder n ith disk integmtor RIodel K 1-1, were emploved. The hromstographic column wa6 .i borosi1iL.de glass tube of *5 mm. i 4 , 4 feet in iength. It mas
QROCEDvW
The column v a s trniperature preconditioned :or 20 hour- a t ‘2.50’ C. This tie‘itmtrit presuman. resuited 111 some loss of !;ciuid phaar ,]nee d h r t m ing of --teiir>c ‘i times (.sultrt-l ‘Th cnhiinn Tras ’ :ice.’’ prtc.cnditir-Prl z t 16.5’ ( ’ for 2 to 4 h n i w by i v d m g injectioiis of 4 t o 3 pg. 2i promazinc or rhlorj)[omazint. I t boiit 4-rnin:ite I v t i -~
Q
Table
I.
Retention Times of Some Tranquilizer Compounds
Compound (Free Form) Phenothiazine Derivatives Chlorpromazine Ethopropazine Fluphenazine Mepazine Methoxypromazine Perphenazine Proclorperazine Promazine Promethazine Pyrathiazine Thiopropazate Thioridazine Trifluoperazine Triflupromazine Trimeprazine Rauwolfia Alkaloids Deserpidine Rescinnamine Reserpine Diphenylmethane Derivatives Adiphenine Azacyclonol Buclizine Captodiamine Chlorcyclizine Diphenylp yraline Hydroxyzine Meclizine Phenyltoloxamine Pipradol Piperilate Butanediols, Propanediols, and Related Compounds Carisoprodol Mephenesin Meprobamate Methocarbamol Phenaglycodol Ureides, Amides, Hydrazines a-Methylphenethylhydrazine
Dextroamphetamine Ectylurea Glutethimide Iproniazid Isocarboxazide Nialamide Phenelzine Acetylcarbromal Miscellaneous Betazole Chlormethazanone Chlorzoxazone Imipramine Methylphenidate Pyrrobutamine Thonzylamine a
*
Merck Index 1960 Page Reference
(4)
249 42 1 856 645 667 787 855 857 857 874 1040 643 1064 1065 1067
Retention Time, Minutes Chromatographic Conditionsa A B C D
b
5.2
5.2
2.7 4.2
5.0 3.2
18.8 2.7 1.5
1.8 3.8
41.0 0.9 2.1 2.3
b
b
3.3 1.3 5.4
68 1 33 1 426 488 565 638 716 792 10
2.8
881
1042
4.7 1.4
b b b
b
41.0 7.5
7.3 1.8
16.8
5.1 3 4
2.2
b b h
967 646 647 667 79 1
147 239 772 1051 908
b
22.7 5.3
3.1
203 899 899 35 114 174 292 236 383 546 640 806 824 1007
61.0 12.1
0.6
1.o
3.6 b
b
b
1.8
0.8 2.7 6.1
1.3
2.0 b
b
5.0 0.8 5.2
b
b
b
2.0 1.4 b b
b
b
b
b
b
b
b
b
b
b b
1.8 b
b b
3,. 6
b
2.5 b
5.0
2.7
2.3
Chromatographic conditions Temperatures: A = 150' C., B = 165' C., C = 180' C., D = 210" C.; Column: 4-foot packed with SE-30 0.057, on glass microbeads 60 to 80 mesh; Flow: (argon) 40.0 ml. per minute; Detector voltage: 1750 volts; Attenuation: x 1. Compound was injected but no response was observed.
vals. The latter procedure probably caused saturation of the liquid or liquidgaseous interphase with these compounds. This resulted in a greater apparent detector sensitivity-Le., greater response-area and height, with sharper peaks and less tailing. When
758
ANALYTICAL CHEMISTRY
the column was left a t the operating temperature and gas-flow over night, i t was found necessary to "use" precondition the column again to obtain the sharpest peaks and fullest responses. T o avoid the need for repeated ''use" preconditioning, one may remove the
column when not in use for periods of 4 hours or longer, and replace i t 10 minutes or so before intended use. The column used for the collection of the data given here was used almost daily for 2 months with no significant changes in performance. Various column Dackinps were tested before adopting t h e one described. All of the high-temperature liquids on firebrick support, at temperatures of 175" or 200" C., either held the compounds with no response registered, or yielded long and tailing peaks. Raising the temperature to 235" C. produced decomposition, as indicated b y the formation of numerous peaks from a single compound. Aside from the microbead column with SE-30 0.05%, only Carbowax 20M, 1% on firebrick 100/120, acid washed, coated with potassium hydroxide, 5%, gave any useful responses. The group of compounds reported here has also been successfully chromatographed, as a test only, using the hydrogen flame detector combined with the same type of column in the Aerograph Model 600 ("Hy-Fi," Wilkens Instrument and Research, Inc., Walnut Creek, Calif.). RESULTS
Tables I and I1 summarize the results of chromatographing the individual compounds for determination of retention times, in minutes, a t four specified temperatures. Table I lists data for the free compounds; Table 11, for salts. A few of the compounds, being neutral, form no salts. Horvever, being organicsoluble, they are obtained in the first organic extraction regardless of pH. For convenience in finding synonyms, structures, and properties of the compounds, Table I includes Merck Index (1960) page references (4). Figure 1, a reproduction of a chromatogram, illustrates the results obtained from a mixture of tranquilizers: two are diphenylmethane derivatives, and four are related to phenothiazine. Although the separations are in no instance complete, all the compounds show characteristic responses which allow presumptive identification. The magnitude of response (peak height and area) relative to the weight of sample injected was studied for a number of tranquilizers, in order to assess linearity. A plot of these two variables produced straight lines (between 1 and 20 fig.), each having a different slope. Figure 2 illustrates this for two compounds, using peak height (per cent of full-scale response) os. weight (1 to 8 IJg.1. DISCUSSION
Eleven of the 50 free-form drugs and five of the salts failed to give an observable response from an injection of as much as 8 pg. The Rauwolfia alkaloids
A
__-
.__
0. Chlorpromazine
Promazine
P
--
1
2
3
$9
Figure 2.
Figure 1 .
Separation of mixture
Temperature: 165' C.; column: SE-30 0.05% on glass microbeads 60/80; flow: 40.0 mb/minute; detector voltage: 1750 volts; attenuation: X 3 Peak
A B
C D E F G
Table II.
Compound Acetone Phenyltoloxamine Diphenylpyraline Triflupromazine Promazine Chlorpromazine Methoxypromazine
Retention Times of Some Tranquilizer Compounds
Retention Time, Minutes ChromatoeraDhic Conditions0
Compound (Salt Form) a Phenothiazine Derivatives Chlorpromazine hydrochloride 6.2 Ethopropazine hydrochloride 4.1 b Fluphenazine dihydrochloride Mepazine hydrochloride 7.7 hlethoxypromazine maleate 9.5 Proclorperazine dimaleate 37,. 0 Proclorperazine ethane disulfonate b Proclorperazine dihydrochloride Promazine hydrochloride 3.7 Promethazine hydrochloride 6.5 3.0 Pyrathiazine hydrochloride 7.6 b b Thiopropazate dihydrochloride Thioridazine hydrochloride 70.0 Trifluoperazine dihydrochloride 12.4 Trifluproniazine hydrochloride 2.6 5.3 Trimeprazine tartrate 3.2 Diphenylmethane Derivatives Adiphenine hydrochloride 5.3 3.0 Azacyclonol hydrochloride 6.5 b Buclizine dihydrochloride Captodiamine hydrochloride 17.9 Chlorocyclizine dihydrochloride 5.4 2.6 Diphenylpyraline hydrochloride 3.5 1.9 b Hydroxyzine dihydrochloride hleclizine dihydrochloride 49.0 Phenyltoloxamine dihydrogen citrate 1.9 1.o Pipradol hydrochloride 4.5 2.8 b Piperilate hydrochloride 2.4 Ureides, Amides, Hydrazines a-Methylphenethylhydrazine hydrob b chloride b Dextroamphetamine sulfate b Iproniazid phosphate 1.4 b Phenelzine dihydrogen sulfate Miscellaneous b Betazole dihydrochloride Imipramine hvdrochloride 5.3 2.6 b Methylphenidate hydrochloride 1.2 Pyrrobutamine diphosphate 5.8 Thonzylamine hydrochloride 5.8 2.9 Chromatographic conditions as described for Table I. b Compound was injected but no response was observed.
L
3.0 2.0 b
3.5 4.3 13.8
3.5 23.3 5.7
2,. 5 7.4 b
17.3
b
2.5
7.3 3.0 2.8 3.1
b
4.7 1.4
6.7 1.7 5.6 3.3
+
6
6
7
6
Compound
Linearity testing
were notable in that none of the conditions tested produced any response. With molecular weights above 500 and melting points above 230' C., this is not surprising. A t the other extreme, even a t the lowest temperatures used, dextroamphetamilie and betazole failed to be identified, their low molecular weights and boiling points suggesting that they emerged with the solvent. It is not to be expected that any single set of conditions would detect all members of a group so diverse as the tranquilizers. Of the four temperatures used in this study, 165' C. resulted in the greatest number of separations. Inasmuch as samples of from 1 to 8 pg, gave half-scale responses for many of the listed compounds, direct application of the method to extracts of small samples of blood should be possible, as indicated in connection v, ith the identification of barbiturates ( 5 ) . Combination with spectrophotometry for the determination of the chemical type of the compound should also be practical, by extraction from the cuvette samples after the absorption measurement. As stated earlier ( 5 ) , blanks from extracts of blood and tissue do not show peaks, or sufficient general response, to interfere seriously with the toxicological use of the gas chromatographic procedure. With regard to the imperfect separation shown in Figure 1, the toxicologist is rarely, if ever, confronted with so complex a mixture of these compounds in a practical situation. The linear responses shown in Figure 2 suggest the practical utilization of this technique in quantitative estimation. Occasionally, follon-ing storage of some of the tranquilizers, new small secondary peaks have been observed which gradually increased in height with the passage of time. This suggests a possible application of the method to quality control during manufacture, ACKNOWLEDGMENT
The authors gratefully acknowledge the technical assistance of H. R. Harvey and thank cooperating pharmaceutical supply houses for furnishing the pure compounds used in this study. VOL. 34, NO. 7, JUNE 1962
759
LITERATURE CITED
S.,Powell, H., .llature 173, 1143 (1954). 12’1 Hoffman. Allen J.. Ludwig. B. J.. ‘ J . 1 m e r . Phorvi. dssoc., Stir Ed. 48; 720 (1959). (3) hlannering, Gilbert J., Dixon, Arthur C., Carroll, Sicholas V., Cope, Ogle B., Lab. Clzn. M e d . 44,292 (1954). (4) “AIerck Index,” i t h ed., Merck and Co., Inc.. C-. S.A , , 1900. (1) Curry, A.
(5) Parker, Kenneth D., Kirk, Paul L., ANAL.CHEM.33, 1378 (1961). ( 6 j Rajesivaran, Ponnusamy, Kirk, Paul L., (Part 1). Bull. Narcotics, U . S., Dept. Social Aflairs XIII, KO. 3, 15-37 (1961). (7) Ibid.. Part 2 . in mess. (8) Ryan, James A,, J . Smer. Pharm. Assoc., Sci. Ed. 48, 241 (1959). (9) Stewart, C. P., Stolman, A., “Toxicology,” Vol. 2, pp. 549, 551, Academic Press, Xew York, 1961. (10) Walkenstein, ’ Sidney S., Knehel,
Cornelius ll., IIacmullen, Joyce A.. Seifter, Joseph, J . Pharmacol. E.rptl. Therap. 123,254 (1958). RECEIVED for review Xoveinber 27, 1961. Accepted March 2 , 1962. n’ork supported by grants from the rational Institutes of Health, U. S.Public Health Service (RG-4372 and RG-5802 I , and from the Research Committee, L-niversity of California. Reported at the Fall Meeting of the California .%ssociation of Criminxlists, San Francisco, Octolwr 1961.
Analysis of Zone Spreading in Paper Chromatography by a Detection Limit Method KANA1 LAL MALLIK and J. CALVIN GlDDlNGS Department o f Chemisfry, University o f Utah, Salt Lake City, Utah
F An experimental method i s proposed for determining the basic plate height parameters of paper chromatography in terms of the apparent spot length. This method corresponds with the usual operation of paper chromatography where zones are detected and evaluated by visual means. The theory relating the experimental observables and the basic molecular parameters is developed and applied to six chromatographic systems. The results are interpreted in terms of diffusion and adsorption phenomena within the paper. HE migration of a chromatographic zone is characterized a t the molecfilar level by a number of random proc’sses ( 3 ) . Foremost among these is ’he sorption-desorption process which makes the don nstream motion of solute molecules intermittent in nature. An important consequence of this random motion, aside from the influence on the Fate of zone migration, is the spreading ,f the zone in the flow direction. This cau‘es the overlap of neighboring zones. anti thus has a direct bearing on the rfficiencv of chromatographic ieparatiorl‘hesp factors have received a v a t w a 1 cri evperimental and throreti~ $ Pttcrition 1 in so far as gas chromatogr;rahg is wncerned, but little effort hren applied to correlate zone struc+ ~ ~with r p the niolecular processes occur1 ~ g in pRper chromatography. The i rTh in evperiinental evidence stems, r i l r t , :wm the conventional technique foi detecting zones in paper chroylatograuhy, i.. , a visual technique employing natural or induced light abi r 2 r ~ mpgr o p d i e s of the solute zones. quch 5 tecnnique is incapable of yielding ietaiied x a e structure, although it is po-sitile ro
