Rudolph M. Navaril Hollins College Hollins College, Virginia 24020
Laboratory Experiments Using Nicotine
"Warning: T h e Surgeon General H a s Determined T h a t Cigarette Smoking Is Dangerous t o Your Health." This statement a s well a s similar warnings from the Surgeon General, the American Cancer Society, and other organizations is very familiar to both smokers and nonsmokers of the United States. T h e effects of smoking cigarettes have been studied widely ( I d ) , and investigators have reported effects which range from cardiovascular (1, 4 ) and respiratory (3, 5-7) disturbances t o the severity of skin wrinkling (8). Although there is evidence t h a t the knowledge of the health dangers of smoking has resulted i n the termination of the use of tobacco by some individuals (3). there are estimates t h a t in 1970, the use of tobacco in the United States was 11 lbs/person over 15 yr of age (3, 9). This represents a 48.2% per capita increase in use since
1900. With the incentives of introducing undergraduates to the chemistrv of smokine and.. in . oarticular, nicotine, the chief toxic ingredient of tobacco smoke (1-3; 7, 9), and of continuine students with course material t h a t - t o orovide . relates to current interests, three laboratory experiments using nicotine were designed. T h e experiments provided a n introduction t o the fate of nicotine in t h e body and sewed a s a n excellent vehicle t o teach some analytical techniques. Prior t o performing the experiments, the students were given introductions t o both the physical and chemical properties of nicotine and the route and residence time of nicotine in various parts of the body (1-3, 7, 9). Nicotine (C10H14N2) is a volatile, weakly basic, liquid alkaloid. The amount of tobacco in regular or filter-tip cigaretes is approximately 1 glcigarette (10, 11), and the nicotine content of ordinary tobacco can vary from 1-8% with a normal value of -2-3% (3, 9-12). It is well documented t h a t the absorption of nicotine from mucous membranes, from the alveoli of the lungs, and from the skin is very rapid (3, 7, 9), and t h e distribution, duration, and excretion of nicotine in the body has been investigated widely (13, 9, 13). The introductory material provided the students with a n understanding of the basic chemistry of the experiments and served a s a foundation for possible further investieations into the general subject. I n addition to t h e che&try of nicotine, t h e analytical procedures employed in each exoeriment were introduced. T h e experiments included spkctrophotometry, distillation, extraction, and thin-layer and ion-exchange chromatography. T h e initial e x ~ e r i m e n tinvolved the evaluation of tohacco of various commercial cigarettes for the nicotine content. T h e nicotine was extracted by steam distillation and analyzed spectrophotometrically. This experiment was followed by a n exercise on the analysis of urine for nicotine which used extraction and thin-layer chromatography. T h e third experiment was a n estimation of the nicotine in a sample of blood using ion-exchange paper chromatography. Each of the three experiments was designed for one laboratory period (3% hr), although the actual total time t o complete any one of the experiments may 'Present Address: Division of Cardiopulmonary Laboratories and Research, Medical College of Virginia, P.O. Box 614, MCV Station, Richmond, Virginia 23298. 748
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vary with the previous laboratory experience of the students. Experimental Nicotine Content of Cigarette Tobacco Appomtus: Wiley cutting mill, Fisher Scientific Co; Distillation apparatus; and Beckman Model DU spedrophatometer. Reagents: 30% Sodium hydroxide (30 g NaOH diluted to 1W ml with distilled water); 0.05 N HC1; and 0.1 N HC1. Standard Solution: 1 I of an aqueous 0.05 N HCI solution cantaining 10 mg of nicotine; the nicotine was obtained from the Sigma Chemical Co. Procedure: Using a Wiley cutting mill, 0.5 g of tohacco from a regular or filter-tip cigarette was ground into powder form. The tobacco powder was placed into a conventional distillation flask with 30 ml of 30% sodium hydroxide. The distillation was started by bringing the mixture to boil end then continued with steam to distill the mixture for 30-60 min. It has heen shown that 98% of the nicotine alkaloids are distilled in the first 10-30 min of the steam distillation (14). A volumetric flask containing 10 ml of 0.1 N HCI was used to collect the distillate, and the tip of the candenser was immersed in the acid. The volume of the tobacco suspension in the distilling chamber gradually decreased to approximately one-third its original volume during the distillation. After the distillation was completed, the distillate was diluted with 0.05 NHCl to obtain a solution with afinal volume of 11. Using a Beekman Model DU spectraphotometer, the absorhance (Azse) of the diluted distillate and the absorbance (Am') of the standard solution were measured using the 0.05 N HCI solution as a blank. The concentration (c) of the nicotine in the 1 I distilled distillate is given by
a
where e' eouals the concentration of the nicotine in the standard solution. 10 mg/l. The amount of nicotine in the original 0.5 g of cigarette tohacco is the amount contained in the 1 1 of the diluted distillate. Discussion: The cigarette tohacco samples used in the experiment were the common commercial American regular or filter-tip cigarettes containing approximately 0.02 g nicotine/g tobacco. Students who regularly smoked cigarettes were encouraged to determine the nicotine content of their own brand. At the end of the experiments, comparisons were made among the various brands used by the individual students. In addition, the results were compared with published values of nicotine content of different cigarettes (15). and good comparisons with the literature were found. For cigarette tobacco with high nicotine content (greater than 2-3%). it was necessary to further dilute both the distillate and the standard. An additional dilution of ten with the 0.05 N HCI solution was adequate to obtain absorbance values in the range of the instrument. The 0.05 N HC1 was used as a solvent instead of water throughout the experiment since it enhances the ahsorptivity of nicotine (16). The analytical technique employed in this experiment has a sensitivity of 1-2 pg nicotine/ml of sample 19, 14, 1.51, which is well below the concentrations of the samples employed in the experiment. Some interfering alkaloids in the distillate are expected in this method, however. Nornicotine, if present, will be determined as nicotine since its spectrum is almost indistinguishable from nicotine (17). Nornicotine, however, is present in most ordinary tobaccos in concentrations much below the concentration of nicotine (16, 17). Other interfering substances may he alkaloid degradation products, lipids, and/or plant pigments. Although steam distillation minimizes the interfering alkaloids, it is possible to correct for the background absorption using the relationship
where A259ici is the corrected absorption at 259 nm, and AZJS. AZJS,and Am1 are the absorhances of the diluted distillate at the indicated wavelengths (16, 181. Urine Analysis for Nicotine Apparatus: Capillary pipets; Filter paper, Whatman No. 1; Thin-Layer tank; Thin-Layer plate (0.25 Silica Gel, type F-254, Brinkman Instruments, Inc.); and Reagent sprayers. Reagents: Indoplatinate (1 g of platinic chloride in 10 ml of distilled water is mixed with 10 g of potassium iodide in 200 ml of distilled water; this mixture is diluted to 500 ml with distilled water and stored in a refrigerator); Extracting solvent (chloroform: isopropoanol (24:1, vol %); Buffer (saturated aqueous NH&l solution, p E 9 . 5 ) ; Developing system: (ethyl acetatcmethanol:NH1OH (85:10:10, ~ 0 1 % ) )0.1 ; NHC1; and Methanol. Control: 0.20g-of nicatinein 100ml of ethanol. Procedure: Ten milliliters of urine and 1ml of buffer were added to 50 ml of the extracting solvent in a 125.ml glass stoppered flask, and the mixture was agitated by hand for 5 min. The aqueous layer was removed from the mixture by pipetting, and the organic layer was filtered through Whatman No. 1 filter paper. The organic filtrate was evaporated under a hood, and after approximately half of the solvent was evaporated, one drop of 0.1 N HCI was added. Following the complete evaporation of the filtrate, the remaining residue was dissolved in 0.25-0.50 ml of methanol. The sample and the control were spotted with a capillary pipet on Silica Gel thin-layer chromatography plates, allowed to dry, and then developed with the developing system in the thin-layer tank. The plates were dried in a horizontal position and then sprayed with cold iodoplatinate. The Rf value for the extracted nicotine was 0.31, and the R, value for the unentracted nicotine in the control sample was 0.91. Both the extracted and the unextrseted nicotine spots gave a color of blue-black with the iodoplatinate spray. In addition, a minor metabolite of nicotine was observed a t a Rr value of 0.68 and with a color of blue-black. Discussion: A number of studies have been performed on the urinary excretion of nicotine (131. In occasional smokers (1-4 cigarettes daily), moderate smokers (5-10 cigarettes daily), and heavy smokers (11-20 or mare cigarettes daily), the daily nicotine concentration of the urine was found to be 1.8-2.7 pg/ml urine, 2.8-3.6 pg/ml urine, and 4.9-6.6 pg/ml urine, respectively. There appears to be a definite correlation between the number of cigarettes smoked and the quantity of nicotine excreted in the urine. The urine specimens used in the laboratory were provided by the individual class members. Each student was asked to perform at least two determinations, one of a nonsmoker and one of a smoker. The urine specimens of the smokers were collected 2-3 hr after the smoker had smoked his first cigarette of the day; this was done in order to minimize the normal differences in the nicotine concentrations of the urine of smokers. The particular analytical technique employed was a basic screening test and not quantitative. However, the technique had a sensitivity of 0.1 pg nicatine/ml urine (19), and positive results were obtained for all the urine samples of the smokers, regardless of the number of cigarettes smoked each day by the individuals. The urine extraction and thin-layer chromatography analysis used in this experiment can be modified for use in screening urine for amphetamines, barbiturates, and opiates (19), and same interested students elected to perform some additional experiments in this area. Quantitative estimations of nicotine and drugs of abuse are best accomplished with fluorescent spectrophotometry and/or gas-liquid chromatography (20, 211. These analytical methnds can be used by advanced stude,nts. Determination of Nicotine in Blood Apparatus: Portable uv light source; Paper chromatography tank; Cellulose citrate ian-exchange chromatography paper, Type CTm, Whatman; Filter paper, Whatman No. 1; and Capillary pipets. Reagents: 10% Sodium tungstate solution (10 g of NaW0,.2Hz0 in 100 ml of an aqueous solution); 10% Sulfuric acid solution (100 ml of concentrated HzS04 in 1l of an aqueous solution); Diethyl ether; Concentrated ammonia solution (density: 0.88 g/ ml); Anhydrous sodium sulfate; Chloroform; and 0.1 M EDTA (37.2 g of the disodium salt of ethylenediaminetetraaeetic acid in 1I ofan aqueous solution. Controls: 20 mg of nicotine in 100 ml of ethanol; and 115 mg of nicotine in 100 ml of ethanol.
Procedure: Distilled water (31.5 ml), 10 ml of 10% sodium tungstate solution, and 3.5 ml of 10% sulfuric acid were slowly added with continuous stirring to a 5-ml whole hlood sample. This mixture was then placed in a boiling water bath for 15-20 min to allow the blood proteins in the mixture to precipitate. Next, the mixture was filtered through Whatman No. 1 filter paper, and the cooled filtrate was mixed with 30 ml of ether. The ether extract removed any remaining acidic compounds and was separated from the filtrate with a separatory funnel. The pH of the filtrate was then adjusted to an alkaline value of 9.0 with a concentrated ammonia solution, and ether was added to extract the basic compounds, including nicotine. The ether extract was passed aver solid sodium sulfate to remove any retained water, and then was evaporated to dryness at room temperature. The remaining residue was dissolved in 0.1 ml of chlorofom and spotted with a capillary pipet on ion-exchange chromatography paper. The nicotine control solution and any additional samples were spotted on the same chromatographic sheet. After the spotted samples dried in air, thg chromatography was carried out with the 0.1 M EDTA ascending solvent. Following ascent of approximately 90% of the paper by the 0.1 M EDTA solution, the paper was allowed to dry in air, and then was examined under uv light (259 nm). The nicotine in the hload samples and in the control showed a Rr value of 0.55 and gave a dark purple color under the uv light. Discussion: The majority of the bland samples used in the experiments were outdated normal human whole blond supplied by the American National Red Cross. Since the Red Cross does not test the blood it distributes for its nicotine content, there was no a priori method of concentration determination. As a result, each student was given two 5-ml samples of blood, one of which was unaltered and one which was mixed thoroughly with 0.1 ml of a 100 ml ethanol solution containing 175 mg of pure nicotine. With this procedure, the students were able to obtain at least one positive identification since this particular analysis has a sensitivity of a t least 0.01 mg nicotine/ml blood (22). There were, however, some positives with the unaltered American Red Cross blood samples as well as with some pooled hload samples obtained hy finger pricks of some members of the class who were heavy smokers (aver 20 cigarettes/day). Some investigators have reported that the hload of smokers who had smoked 20 or more cigarettes during the day contained 0.1-0.3 pg nicotine/ml of hlood (1, 23). Since the concentration of nicotine in the blood varies with the type and amount of tobacco smoked, the varying residence time of nicotine in the hlood, and s number of other factors (13), it is difficult to state a "normal" nicotine content of the hlood of smokers. For very low levels of nicotine in blood, a spectmphotametrie estimation has been reported (241. It is more time consuming than the ion-exchange paper chromatography method described above, but it detects lower levels of nicotine in the blood, and it could he attempted by advanced undergraduates. The ion-exchange paper chromatography method is basically a screening test, while the spectrophotometric technique should give good quantitative results. Both methods can be employed far the detection of other alkaloids in the blood. Discussion
T h e reaction of t h e s t u d e n t s t o t h e experiments using nicotine was very enthusiastic. A high level of interest a n d a large incentive to go beyond t h e suggested laboratory exercises were co~itinuouslydisplayed b y t h e students. In fact, m a n y of t h e individual m e m b e r s of t h e class requeste d laboratory t i m e t o perform additional quantitative determinations of t h e a m o u n t of nicotine in samples of blood a n d urine from themselves, classmates, a n d friends who were n o t m e m b e r s of t h e class. T h e laboratory exercises with nicotine not only t a u g h t t h e s t u d e n t s a wide variety of analytical procedures a n d techniques, b u t they also pointed o u t a large n u m b e r of distinct and practical applications of analytical chemistry. The applications of t h e subject m a t t e r of t h e course a p peared t o b e very i m p o r t a n t a n d often essential t o t h e l e a m i n e Drocesses of most of t h e m e m b e r s of t h e class. T h e ap&ations initiated questions concerning t h e applicability a n d sensitivity of a particular analytical techlogical a p nique,-and, in general,. oriented t h e class t o ; proach to choosing a particular analytical method. Volume 51. Number 11. November 1974
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The experiments described in this communication have been used in the latter stages of a general chemistry course. as well as in first and second level analvtical chemistry courses. The success of the exercises was not d e ~ e n d e n ton the level. and small adiustments in preparaiion were made in order to account for the different backgrounds of the students a t each level. Literature Cited (1) Laraon, P. S., Hmg, H. B., and Silvett.. H.. "Toham. Experimental and Clinical Studies; The Williamaand Wilkina Company. Baltimam, 1981. (2) Laraon, P. S., and Siluette. H.. '"Tohaem. Experimental and Cliniesl Studies; Supplement I, The Williama and Wilrinscompsny, Baltimaa. 1968. (3) L a m n , P. S..'and Silvetfe, H.. "Tabacm. Experimental and Clinical Studics," wi~iiams and wiikin~company, 1971. ~ s ~n. ~ ~ I ~ ~ (4) Roth. G.M.. andShiek,R.M.,Ann.N.Y. A c d . S e i , PO,308(19M)l. (5) Gager, F.L.,Nedloek, J. W.,andMartin, W. J., Corbohyd. R s s , 17,327i1971). ( 6 ) Gagrr, F.L.. N d o c k . J. W.. andMartin, W. J., Carbohyd. Roa., 17.335(1971l. (7) Polson. C. J., and Tafbrasll. R. N., "Clinical Torieolw: 2nd Ed.. J. B. Lippinmtt Company. Philadelphia, 1969. pp. 522-534. (81 Dsniell, H. W..Ann. Intern. Mod., 75.873(1971). (9) Thiones. C. H.. and Haley, T. J., "Clinical Torirolw." 5th Ed., h a and Fobiger
750 1 Journal of Chemical Education
Company, Philadelphia. 1972, pp. 19-22. 280-284.386387. (10) Kenhhaum, A., B e b f . S.. Hirahayashi, M., and Foinherg. L. J., J Amen M d Assoc., 201,135(19671. o Tabacm Smoke." Academic (11) Wyndor, E. L.. and Honman, D.." ~ ~ b a e eand Press, Ine.. NewYark. 1967, pp. 371-381. (12) Pieme.1. H . J . Lob. Clin Msd.. 26.1322IILMIl. (13) Fennegan. J. K.. Lam". P. S., end Haag. H. 8..J . Phonnool. Exp. T k l . 91.
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2%" ,>O.",
(14) Robinson, R.H.,Anal. Chem., 27,1351 119551. (15) Lipp, G.,Beilr Tab&forseh, 3,1(1965). (16) W i l l i ~ ,C. 0.. Swain, M. L., Connolly, J. A,, and Brim. B. A.. Anal. Chem.. 22. 430 (19501. (17) Swain, M. L., Eisncr. A . Wmdruard, C. F.. and Brice, B. A,. J. Arne,. Chem. Soc.. 71.1341 (1949). (18) Brice, B. A . and Swain, M. L., J. Opt. Sue. Arne,., 35,532 (1945). (19) Chin. J. M.. and N a v k , R. M., "Detection af Drugs of Abuse in Human Urine," AmericanInstituteofChcmical Engineer~NationalMeeting. Detmit. 1973. (20)~Sunshine. ~ I., "Handbmk of Andytical Toxieolaey." Chemical Rubber Company. Cleveland, 1969. (21) Sunshine, I.. "Manual of Analytical to rim lo&^." Chemical Rubber Compsny, Clevelsnd. 1971. (22) Street. H . V . Clin Chim. A d o , 7.226(19621. (23) Wolff, W. A,. Hawkins. M. A,. and Gila. W. E.. J. Phormool. Elp. Tkr.. 95,
