species, as previously shown (IO),the elution order is largely affected by temperature; at high temperature there is a n inverse isotope effect and the deuterated species are eluted first. At low temperature (as in our experiments at liquid nitrogen) there is a normal isotope effect and the retention volumes increase by increasing the number of deuterium atoms on the molecules. The separation between a consecutive pair of deuterium- or tritium-substituted molecules decreases as the mass ratio between each pair approaches unity. There is a linear relationship between the logarithm of the relative retention volumes of consecutive pairs cs. their mass ratio, as shown in Figure 2. Comparing the elution order of the deuterated and tritiated species, one can see that the tritium substitution has a larger effect on the retention volumes than expected from the simple mass effect. In fact, molecules such as CH3T and CH2TZ merge from the column later than the corresponding CH2D2 and CD4of the same mass. Many interesting applications can be found from the gas chromatographic separation of isotopic methanes. One is the purity control of various isotopic molecules, another is the possibility of studying the kinetics of isotope exchange reactions, For instance, the mixture of partially deuterated methanes was prepared by exchange of Dz with CH4 induced by X-rays. By means of consecutive Chromatographic analyses one can follow the relative abundances of the deuterated molecules at different irradiation times (Figure 3).
Another interesting application is the determination of the monodeuterated methane in natural methane (Figure 4). In the upper part of the chromatogram the separation of CHID/ C H 4 in the same amount is shown; in the lower part, by injecting a large amount of CH4, one can observe the small peak of CH,D and measure the isotopic abundance (CHID = 0.06
(10) A. Liberti, G. P. Cartoni, and F. Bruner, “Gas Chromatography, 1964,” A. Goldup, Ed., The Institute of Petroleum, London, 1965, p 312.
RECEIVED for review January 14, 1969. Accepted February 28, 1969. The authors thank the Consiglio Nazionale delle Ricerche for providing funds to support this investigation.
C-------a-alOO
257
259
-p
261
263
Figure 4.
265
267
269
271
273
I
275 TlME(min)
Separation CHd-CH3D
Upper chromatogram: same amount Lower chromatogram : natural isotope abundance
x).
-
Assay for Heroin in Illicit Preparations Using Partition Chromatography George R. Nakamura’ and Herman J. Meuron Alcohol, Tobacco and Firearms Laboratory, Internal Revenue Service, Box 36075, San Francisco, Calif 94102 THEQUANTITATIVE ESTIMATION OF HEROIN (diacetylmorphine) in most illicit preparations by the classical solvent extraction methods ( I ) proved to be tedious and time-consuming while that by direct ultraviolet absorption measurement was inapplicable because of the effects of adulterants and dark colored resinous impurities. Since the end of World War 11, a large number of adulerants have been added to heroin powder and the heroin itself often times has not been carefully purified by the clandestine laboratory. Thus, the Mexican, or brown colored heroin, which appears on the West Coast seems to contain dark resinous matter, possibly some residual constituent of opium. In addition, this brown heroin is generally very intimately mixed with procaine, and then coarsely mixed with other adulterants or diluents such as lactose and starch. 1 Present address, Bureau of Narcotics and Dangerous Drugs, San Francisco Regional Laboratory, Box 36075, 450 Golden Gate Ave., San Francisco, Calif. 94102
(1) “Methods of Analysis,” Internal Revenue Service Publication No. 341, (Rev 6-67), Internal Revenue Service, U. S. Treasury Dept., Washingion, D.C., June 1967. 1124
ANALYTICAL CHEMISTRY
The presence of the dark resinous matter as well as procaine, also interferes with a number of identification tests, such as the formation of microcrystals with platinic chloride ( I ) and with quantitative procedures. The assay method employing gas-liquid chromatography developed by Grooms (2) in this laboratory was not affected by the common adulterants except quinine. However, it was considered desirable to develop an alternate method for use by laboratories not having a gas chromatograph and the choice was some foim of rapid column chromatography procedure. Levine (3) showed that many alkoids, such as heroin, can be extracted as ion pairs by chloroform and other chlorinated hydrocarbons. The principles underlying the phenomenon are discussed by Doyle and Levine (4) and by Higuchi e[ al., (5) and are used as a basis for the separation of a wide range of pharmaceutical amines under various conditions. The application of ion pair elution presented here affords ~(2) (3) (4) (5)
J. 0. Grooms, J. Assoc. Off: Anal. Chem., 51, 1010 (1968). J. Levine, J. Pharm. Sci., 54, 485 (1965). T. D. Doyle and J. Levine, ANAL.CHEM., 39, 1282 (1967). T. Higuchi, A. Michaelis, T. Tan, and A. Hurwitz, ibid., 39,
974 (1967).
a simple and rapid procedure for separating heroin from illicit mixtures and determining its amount of UV absorption. The clean-up, as well as the assay of heroin, is effected from a single column. A number of identification tests (1) can be made for forensic purposes after use of the clean-up step. EXPERIMENTAL Apparatus and Reagents. A Cary 15 recording spectrophotometer was used for UV measurement. A manually operated spectrophotometer may be used. Glass columns were those made by Kontes Glass Company, Cat. No. K-420300, and also those made in the laboratory according to Levine (3). Heroin HC1 and acetylcodeine were prepared according to Wright (6) while 6-monoacetylmorphine was prepared by the method of L. S. Small as described by Wright (7). Other chemicals and reagents were commercial products. Ethylidene chloride (1,I-dichloroethane), pract. Eastman or J. T. Baker was redistilled for use. Further purification is recommended by treating ethylidene chloride with K M n 0 4 and N a H C 0 3 with intermittent washing with water prior to distillation (according to J. T. Baker Chemical Co.). Spectrograde solvents are preferred wherever applicable. For elution purposes, all solvents were water-washed. Celite 545 was obtained from Johns Manville Corp. Determination of White Heroin. Mix three grams of Celite 545 and two ml of 0.1N HCl in a small beaker, transfer to column having glass wool plug, and tamp. (Pack just enough fine glass wool to hold the packing in place). Introduce 50 mg of heroin dissolved in one ml of 0.1N HCl by means of a pipet onto the surface of the packing and cover with a small pad of glass wool (if caffeine is present, wash column with 50 ml ethylidene chloride). Pass cu. 40 ml of CHC13 through the column and collect the eluate in a 50-ml volumetric flask containing four drops of concentrated HCl and five ml methanol and bring to volume with CHC13. Prepare reagent blank of CHC13 using the same proportions of HC1 and MeOH. Scan spectrum of eluate and of standard heroin HCl solution, 5 mg/50 ml in CHCl, containing HC1 and MeOH, from 340 to 250 mp against the blank. Determine heroin concentration at maximum of 280 mp. A :?, value obtained for heroin HCl monohydrate in this laboratory was 50 at this wavelength. Determination of Brown Heroin. Prepare a suspension of 50 mg of brown heroin sample per ml of 0.1N HCl and filter or centrifuge. One-ml aliquot of clear supernatant is introduced to a column prepared as above. Wash the column with 50 ml of ethylidene chloride. Discard the washing. Then pass 45 ml of CHC18 through the column, collect the eluant, and determine the spectrum in the manner described previously for noncolored heroin. Preparatory Clean-up of Brown Heroin for Qualitative Tests. Dissolve cu. 100 mg of brown heroin in three ml of 0.1N HCI, centrifuge if solution is not clear. In a small beaker, mix the supernatant with three grams of Celite, pack the resulting mixture into a column, and cover with a pad of glass wool. Pass 100 ml of ethylidene chloride or 40 ml of ethylene chloride (1,2-dichloroethane) through the column and discard washing. Then pass 40 ml of CHCls through column, collect the eluant into a small beaker, and evaporate to dryness. Dissolve residue in appropriate reagents for microcrystalline tests such as with platinic chloride or with mercuric chloride
Table I. Specificity of Procedure as to Some Constituents of Illicit Heroin Preparations Elution solvents arranged in seauence used 1. Ethylidene 3. Triethylamine Compound chloride 2. Chloroform in CHClp Caffeine Heroin Acetylcodeine Procaine Quinine Morphine 6-Monoacetylmorphine Methapyrilene -
+
a
+ +
5 ml2Oz EtaN in CHC13 followed by 33 ml 1
+ ++ ++
zEt3N in CHC18
(IO).
Table 11. Recoveries of Heroin Standard per se and Added to Brown Heroin Sample Heroin HC1
Heroin HC1 added to brown heroin (20 mg)
Mg added Mg recovered
9.22 7.37 5.53 3.69 1.84
9.25 7.36
8.56 6.42 4.28 2.14
8.40
zRecovery
5.50
3.63 1.72 6.32 4.22 2.10
loo. 1
99.9 99.5 99.7 98.8 98.1 98.4 98.6 98.3
(1).
can be detected by a thin-layer chromatography procedure (8) and has been shown to be devoid of UV absorbance at 297 mp in alkaline media, in which heroin is deacetylated to morphine and exhibits a maximum at the wavelength (9). Evaporate the chloroform eluant from the standard assay procedure to dryness and quantitatively transfer the residue to a 50-1111 volumetric flask in 0.1N NaOH. Compare sample against a standard heroin, 1 mg/lO ml, in 0.1N NaOH, at the maximum peak, 297 mp, as described in (9). Specificity. The assay of illicit heroin preparations must in all cases be preceded with a careful qualitative screening by spot tests and/or chromatography methods (1). Table I shows the disposition of a number of probable constituents in the present Celite column system. Common diluents and impurities, including the degradation products of heroin, namely, monoacetylmorphine and morphine, are removed from or retained in the column under standard assay conditions. Lactose, mannitol, and sucrose are commonly found in illicit preparations as diluents but they are virtually insoluble in the elution solvents and are therefore retained in the column. Samples from a number of commercial brands of these sugars were introduced in the column and eluted with chloroform and ethylidene chloride under standard conditions; the eluants obtained were transparent in the spectral range used in the method. Behavior of a number of other pharmaceutical amines in the Celite column under different conditions is described by Levine and Ottes (11) and Doyle and Levine (12).
(6) C. R. Wright, J. Chem. Soc., 27, 1031 (1874). (7) C. I. Wright, J. Pharmacol. Exp. Ther., 71,164 (1941).
(8) G. R. Nakamura, J . Assoc. O&c. Agr. Chem., 46,769 (1963). (9) G. R. Nakarnura, ibid., 49, 1086 (1966). (10) J. Levine, ibid., 44, 285 (1961). (11) J. Levine and R. T. Ottes, ibid., 44,291 (1961). (12) T. D. Doyle and J. Levine, ibid., 51, 192 (1968).
Determination of Heroin in Presence of Acetylcodeine. Table I indicates that if acetylcodeine is present in the illicit heroin sample, it also is eluted with CHC13. Acetylcodeine
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1125
Recovery Tests. Results of recovery tests of standard compounds introduced into the column are presented in Table 11. To provide for a more rigorous basis of recovery, measured amounts of standard heroin were added to brown heroin. Data indicated that the method was sufficiently accurate for routine analysis. The present method of analysis was compared with that by gas-liquid chromatography (2) using random selection of seized heroin samples containing procaine as the chief adulterant; all of the samples were tan to brown in color, Table 111 indicates that the agreement is good for the two diverse methods of analysis. DISCUSSION
Blank correction due to base line elevation is negligible in most brown heroin samples while others exhibit a small rise of not more than 0.01 A at 310 mp after a thorough washing with the specified amount of ethylidene chloride. Unusually dark samples may require twice the amount of solvent for clean-up. Because of variances in instruments, individual analysts should plot their own standard curve for the assay. In illicit samples containing less than 1% heroin, 500 mg (of samples) dissolved in dilute HCl may be loaded on the column and assayed under standard conditions. Such samples of low percentages examined in this laboratory contained mostly lactose and/or starch as diluents. This laboratory does not routinely assay the amounts of adulterants-e.g., caffeine, procaine, and quinine-as they occur in illicit heroin preparations. However, it is possible to estimate their amounts by separate elutions from the same
Table 111. Comparison of Assay of Heroin in Illicit Mixtures by Two Procedures Sample No. Present method, % GLC method (2), 295-1 295-6B 295-6C 372 389-1 389-2 403-5 403-7 412 42 1 6745
34.8 22.1 20.5 8.0 12.1 12.2 5.6 5.3 3.5 5.8 8.3
36.0 21.8 20.0 8.5 12.0 10.3 6.4 6.5 3.8 5.0 8.1
column used for heroin assay. Preliminary studies have shown that caffeine, procaine, and quinine obey Beer's law up to at least 50 mg in their respective elution solvents in Table I. Further studies are being conducted for their determinations in the presence of a number of interfering substances. ACKNOWLEDGMENT
The authors are indebted to Joseph Levine, Food and Drug Administration, Washington, D. C., for his consultation in the study and to Julian 0. Grooms, Internal Revenue Service, Philadelphia, Pa., for GLC analysis of heroin samples. RECEIVED for review March 17, 1969. Accepted April 28, 1969.
Determination of the Hydrocarbon Type Composition of Petroleum Distillates Boiling up to 185 "C Using Type X MoIecuIar Sieves J. V. Brunnock and L. A. Luke BP Research Centre, The British Petroleum Co., Ltd., Chertsey Rd., Sunbury-on-Thames, Middlesex, England A PREVIOUS communication ( I ) has discussed the use of Type 13X molecular sieve in a high temperature gas-solid chromatographic process to determine the naphthenes and paraffins of a saturate petroleum distillate at each carbon number up to and including Go. The technique, however, because of interference from aromatics, is not directly applicable to the analysis of a straight run distillate of wide boiling range. The methods previously used to reduce this interferencee.g., prior dearomatization or fractionation of the distillateare tedious and time consuming. Consequently a new approach, whereby the separation of the aromatics from the saturate hydrocarbons is achieved by the use of Type 1OX molecular sieve as the adsorbent in a short precolumn, has been developed. The separation is rapid and quantitative and is demonstrated by the results obtained on a number of petroleum distillates. EXPERIMENTAL
Materials. Pelleted Type 1OX Molecular Sieve (Union Carbide and Carbon Corp.) was ground and screened to 4C-60 mesh. The screened sieve prior to packing was treated by soxhlet extraction with distilled water for 4 hours. 1126
0
ANALYTICAL CHEMISTRY
The sieve was then dried and heated to 450 "C for 3 hours. Pelleted Type 13X Powder 40-60 mesh was prepared and treated with aqueous caustic soda in the manner previously described ( I ) . Columns. Glass columns 7 inches X 6/32-inch i d . and 26 inches X 6/32-inchi.d., respectively, were used. The first precolumn was packed with 3 inches of Type 1OX sieve and the larger column with 22 inches of Type 13X sieve. Both columns were packed in such a way that the sieve packing terminated 2 inches from the inlet and outlet of each column. The dead space was packed with 1.5 inches of Embacel (May and Baker Ltd.). This arrangement tends to prevent holdup of high molecular weight material in the samples occurring at cold spots at the end of the column. Apparatus. The columns were contained in separate ovens, and the precolumn (Type lox), fitted with a backflushing valve, was connected via a two-way valve to the main column (Type 13X). The need to backflush was dictated by the excessive elution times of CS and higher aromatics from the precolumn. Both valves and all connecting lines (1) J. V. Brunnock and L. A. Luke, ANALCHEM,40, 2158 (1968).
