R. 1. Neman North Texas State Un~versitv Denton, 76203
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The Thin-Layer Chromatography of Drugs A laboratory experiment
Since 1969, an expansion has been taking place in drug screening, primarily in the methadone programs throughout the country. In New York City alone, 9900 addicts are on methadone maintenance today, more than a seven-fold increase from the 1300 addicts enrolled in 1969.' In these programs, methadone, an addictive narcotic, is administered to hardcore heroin addicts in an effort to curtail their intake of heroin and to help rehabilitate them. Taken orally, methadone does not cause the euphoria produced by heroin and blocks the craving for and effects of heroin, should heroin be taken. Urine samples from the addicts are analyzed for methadone, morphine (the metabolite of heroin), quinine, amphetamines, and barbiturates by the Dole2technique of organic qualitative trace analysis involving selective extraction of the various drugs from the urine samples at different pH values, concentration of the drug by cvaporat,ion, separation by tlc, and identification by both physical (uv light) and chemical means. In this day of concern over drug abuse and of incorporation of relevant experiments into the chemical curriculum, certain aspccts of the drug screening procedure described above lend themselves nicely to the undergraduate classroom. The experiment to be described requires neit,herheroin addicts nor urine samples, but involves the last two steps in the screening process, the separation of drugs by tlc and their identification by both physical and chemical means. The students are given a brief introduction to tlc, and are told that they will be given a sample known to contain certain drugs in various combinations. The students are to perform the separation by tlc, determine the components of t,heir unkno~r-n,and calculate R , values for the components. The entire experiment is designed for two laboratory periods, and is divided into two parts, alkaloids (morphine, quinine, and methadone), and barbiturates (phenobarbital, pentobarbital, and secobarbital). Alkaloids Appomfus
50 pl Hamilton syringe Thin-layer tank Thin-layer plate: Silica Gel, type F-2.54, Brinkman Corporation, Weatbury, New York 11590 Presented before the National Science Teachers Association, March 1971, Washington, D. C. ' "Screening far Abused Drugs," Laboratory Managemenl, November, 1971, p. 14. (No author, nor volume number given.) 'DOLE,V. P., ET AL., "Detection of Narcotic Drugs, Tranquilizers, Amphetamines, and Barbiturates in Urine," 3. Amer. Med. Assn., 198, 349 (October, 1966).
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Journal of Chemical Edumfion
Portable uv light source, either long or short wave Hot plate 3 reagent sprayers Reagents
Tank solvent system: 425 ml of ethyl acetate .50 ml of methyl alcohol 23 ml of concentrated ammonia 00 ml of solution Identification sprays for quinine: 0..57, H2S04solution for methadone: iodoplatinate solution: 5 ml of 5% K2PtC16.6H904.5, ml of 10Yo solution of KI, 100 ml of distilled water. This solution is acidified to 1 N by mixing with equal volumes of 2 N HC1. for morphine: ammonical AgNO*: mix 30 ml of 5 N NH8 with 30 ml of 50y0 AgNOa solution. Add 5 N NH3 until solution clears. Solutions to be chromatographed methadone hvdrochloride (1 melml in methvl alcohol) (these ;tre to he used in various combinations) Procedure 1) Rinse a clean Hamilton 50 pl syringe three times with methyl alcohol. 2) Draw into the syringe about 50 pL of a solution supplied by the instructor. This solution will contain methadone hydrochloride, quinine sulfate, and morphine sulfate in various combinations. of an inch from the hattom 3) Spot this solution about edge of a thin-layer plate, following the example of the instructor. Make sure that the tip of the syringe does not actually come in contact with t,he Silica Gel on the plate. Between drops, the spot may be air-dried if a stream of air is available in the laboratory. This will hasten the operation. If no air jet is avails,bl.ble, the process will be lengthened somewhat. 4) When the entire 50 pl of sample bas been spotted on the plate, place the thin-layer plate in a tank that has been previously lined with absorbant paper (to keep the atmosphere saturated wit,h solvent) and filled to a depth of about '1, in. with solvent. Be sure that the level of solvent will not come up above the spot on the plate when the plate is placed in the tank. the way up the plate. Remove 5) Allow the solvent to rise the plate from the tank, mark the leading edge of the solvent, a n d d l o w to dry. 6) When the plate is thoroughly dry, sqray it lightly with 0.5'% sulfuric acid solution and read i t immedmtelv under ultraviolet a blue fluorescent light. If quinine is present, i t will appear spot mid-way up the plate. With a pencil, mark the position of the spot (not visible without uv light) so that the RJ for quinine can he calculated. 7) Immediately spray the plate heavily (saturate) with iodoplatinate solution. Methadone will appear as a heavy brawncurole s ~ o with t a rather high Rt value, near the leading edge bf 6he solvent front. Morphine, if present, will appear as a dark blue spot (more readily seen if plate is air-dried a t this point) near the bottom of the plate. 8) Measure the distance the spots of methadone and morphine traveled, so that the RJ values can he calculated. Next saturate the area where the morphine spot is located with mmonical AgNOa and heat on a hot plate. The morphine will cause
reduction of the silver, and a hlack spot will form. I n actual drug situations, this final test with ammonicd AgNOa is proof t,hat the spat that gave a blue reaction with iodoplatinate was indeed morphine, and not some other drug or drugs that have a very similar RJ value to morphine. 9) Discard the plate, which will undergo a slow darkening as the final spray of ammonical silver nitrate reacts with light. At this point the students are asked to report. the results of their chromatography as well as the Rt values obtained.
6) When the plate is thoroughly dry, spray i t heavily with a. 1% solution of silver acetate. The various barbiturates will appear as chdky white spots against a grey hackground. Calculate the RJ values for the spots on your plate. Compare with the following table to determine what your unknown contained.
Questions
7) Discard the plate (which will undergo a slow darkening as the silver acetate reacts with light) and clean your work area. Again the students are asked to report the calculated Rt values and the contents of their unknown.
1 ) From a reference source, determine the structure of morphine, quinine, and methadone. 2) Determine the structure of hydromorphone from a reference source. Compare this with that of morphine and make a. prediction as to how you think these two might behave if a mixture of them was subjected to chromatographic treatment. 3) Rather than measure R, values to determine the identity of chromatographed drugs, what might one do in the case where large numbers of samples were placed on a single tlc plate and repetitive analyses had to he performed? 4) Morphine is the metabolite of heroin. In the body, heroin is broken dawn to morphine which is then excreted in the urine. How might such a procedure as the one you performed in the laboratory he used t o monitor the intake of drugs? 5) What is methadone? How is it used? 6) What function does quinine play in t,he problem of drug addiction? What medical use does quinine have?
In this laboratory exercise, which is similar to the one that you performed last week, you will he given a liquid sample known to contain certain drugs called barbiturates. Again, thru thinlayer chromatography, you will separat,e the sample into its various components to determine what it contains, and measure the Rt value for each component. I t might be well for you to reread the introductory remarks t o last week's experiment a t this time. Apparatus
50-p1 syringe (Hamilton) Thin-layer tank Thin-layer plates: type Silica Gel, 250 p, "Uniplate" by Analtech, Ine., 100 S. Justison, Wilmington, Delaware 19801 Reagent sprayer Reagents
Tank solvent system: 9: 1mixture of chloroform and acetone Identification sprays: lyOsolution of silver acetate Solutions to he chromatographed sodium secabarbital (1 mg/ml in methyl dcohol) sodium phenobarbital (1 mg/ml in methyl alcohol) sodium pentobarbital (1 mg/ml in methyl alcohol) (to be used in various combinations) Procedure
1) Rinse a clean Hamilton 50-pl syringe three times with methyl dcohol. 2) Draw into the syringe about 50 pl of s solution supplied by the instructor. This solution will contain various combinations of the three barbiturates. 3) Spot this solution about 3/s of an inch from the bottom edge of s thin-layer plate, following the example of the instructor. Note the difference in texture of this plate as compared with the thin-layer plate that you used last week. Air dry the spot between drops of the solution, and, as before, keep the size of the spot small-not larger than 1 cm in diameter. 4) When the entire 50 p1 of sample has been spotted on the plate, place the thin-layer plate in a. tank that has been previously lined with ahsorbant paper and filled to a depth of '/,-in. with a. 9 : l mixture of chloroform and acetone. Again make sure that the solvent in the tank is not so deep as to he above the spot on the plate when it is immersed. 5) Allow the solvent to rise 3/. the way up the plate. Remove and allow to dry. Be sure to mark the leading edge of the solvent.
Barbitwate phenopentoseco-
Questions 1) From a reference source, determine the structure of sodium secobarhital, sodium phenobarbital, and sodium pentobarbital. 2) What are the physiological effects of barbiturates? 3) Would you classify chromatography as an a r t or a. science? Discuss.
If the students wish, other drugs may be separated by tlc. Caffeine is an alkaloid, and a solution of No-Doze tablets (in methyl alcohol) separated according to the alkaloid procedure gives nice results. An asthma compound, Tedral, that may be purchased without prescription contains phenobarbital. A solution of this drug can easily be chromatographed to shorv the presence of phenobarbital. Not only are the laboratory exercises designed to introduce the students to tlc, they serve very well as a take-off point for the discussion of drugs, drug addiction, chemical structure, etc. Many sources of information pertaining to the drug problem are available. Some of the best information can be obtained from the National Clearinghouse for Mental Health Information, Chevy Chase, Md. 20015. Chromatography is, for the most part, an art rather than a science. Students may wish to try various plates and solvent systems of differing polarity to separate the components of APC tablets, or to tabulate R, values for other drugs known to contain certain ingredients. Darvon compound gives an interesting chromatoeram usine the alkaloid nlates: some amahet;mines can be separated on the'alkaldid plates'with ninhydrin used as the identifying spray. The references at the end of this paper give much information on the use of the technique indrug-addiction programs. The laboratory exercises discussed above have proved to be among the most popular of the entire course. The author (who once was employed as a chemist for a methadone clinic) uses commercially prepared plates for two reasons: to obtain decent results and to avoid the laborious task of preparing good tlc plates. Several of the questions posed to the students concern the application of the procedure to drug-abuse clinics. To answer some of these, first, the procedure will not separate morphine and hydromorphone. These two compounds give identical R, values and reactions to iodoplatinate reagent and ammonical silver nitrate. Repeated analyses (such as in a drug clinic) do not lend themselves to measurement of R, values. Rather, a reference standard containing all of the drugs to be identified is placed on each end of the 10 X 20 cm plate and chromatographed along vith the unknomms, ~5-hich arc spotted between the two reference spots. For identification purposes then, the chromatographed spots of the unknowns (addicts) are simply compared u
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Volume 49, Number 12, December 1 9 7 2
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with the spots given by the chromatographed references. In some drug societies, quinine is used as a dilueut when "cutting" heroin. Consequently, a positive identification of quinine suggests heroin is not far behind. I n some drug circles, however, the heroin is "cut" with lactose. Positive identification of quinine in itself is not significant; it may simply indicate that the addict had gin and quinine water the night before, or was taking medication for a cold. Schools and colleges may obtain permission to use drugs in the classroom for instructional purposes. Under section 301.22 of the Federal Registera ". . . conducting instructional activities with narcotic and nonna~cot~ic controlled substances listed in schedules I1 through V" is permissible. The school or college desiring to perform these analytical tests as part of their chemistry program should write for Form BND-224, Application for Registration Under Controlled Substances Act of 1970, obtainable from the United States Department of Justice, Bureau of Narcotics and Dangerous Drugs, P. 0. Box 28083 Central Station, Washington, D. C. 2000.5. There is no registration fee for State supported institutions; others are required a $5.00 fee annually.
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Journal of Chemical Education
For classification purposes, the federal government lists morphine and methadone under schedule 11, amphetamines under schedule 111, and barbiturates under schedule IV. The drugs may be ordered from supply houses under a federal registration number which will be issued upon clearance by the Bureau of Narcotics and Dangerous Drugs. Once obtained, the drugs are dissolved in methyl alcohol (1 mg/ml), thus rendering them unfit for consumption. Quinine sulfate is not a controlled substance, and may be purchased at any chemical supply house or from a pharmacy without prescription. Despite the small amount of legal red tape involved in performing these laboratory exercises, the procedures lend themselves nicely to situations \vhere incorporation of current social problems into the chemistry program is desirable. The procedures described herein have been used by the author in nonscience majors courses, majors courses, and organic courses with excellent results, both chemically and educationally. Federal Register, Val. 36, No. 80, April 24, 1071, Part 11, Department of Justice, Bureau of Narcotics and Dangerous Drugs, p. 7779.
