Improved Spectrophotometric Method for Determination of Nicotine in Tobacco Smoke ANDERS H. LAURENE and
T. GIBSON HARRELL
R. J. Reynolds Tobacco Co., Winston-Salem, N. C.
b The spectrophotometric method devised for the determination of nicotine by Willits et a / . may be used with good results for the determination of nicotine in tobacco smoke, if the acidic smoke concentrate is first steam-distilled for a short time to reduce the background in the ultraviolet spectrum. Results obtained b y this modified procedure were in good agreement with values obtained b x the gravimetric silicotungstate method, and good reproducibility was indicated in duplicate determinations.
T
AOAC modification ( I ) of Jensen and Haley's (10) gravimetric method for the determination of nicotine has been the accepted means for analysis of tobacco and tobacco smoke for many years. This method employs precipitation of nicotine silicotungstate from hydrochloric acid solution, with subsequent ignition of the precipitate. The method is subject to coprecipitation errors from other basic constituents in smoke (8) and to solubiIity loss of the nicotine silicotungstate (8). Also, it is tedious and time-consuming. Chromatographic techniques (8, 9, 12) and colorimetric methods (S, 6, 6, 1S, 14, 16) have been devised in attempts to improve the specificity and reduce the time in the determination of nicotine. None of these methods is satisfactory for smoke analysis. Rillits, Swain, Connelly, and Brice (16) used the ultraviolet absorption band of nicotine hydrochloride a t 259 mp for the quantitative determination of nicotine in tobacco. The use of this method for tobacco smoke analysis has been reported (4, 7). However, the procedure has gained only limited acceptance, probably because of background interference. This background, which is not present in the ultraviolet spectrum of nicotine steam-distilled from tobacco, is a serious interference in the determination of nicotine in tobacco smoke. This interference can be removed easily and simply, as outlined in the experimental section. Thereafter, the procedure becomes equivalent to that of Willits et al. (15). A procedure similar t o that proposed has been reported recently by Keith and Newsome (11). However, these HE
1800
ANALYTICAL CHEMISTRY
authors gave no analytical data in support of their method. REAGENTS, SOLUTIONS, AND APPARATUS
Calcium hydroxide, ACS grade. Ethyl alcohol, 95%, benzene-free. Ethanolic hydrochloric acid solution, 0.lN. Dilute 8 5 ml. of concentrated hvdrochloric acid to 1000 ml. with 95% ,ethyl alcohol. Hvdrochloric acid solution. 0.1N. DilGte 8.5 ml. of concentrated hydrochloric acid t o 1000 ml. with water. Hydrochloric acid solution, 1 5. To 1 part of concentrated hydrochloric acid add 5 parts of water. Nicotine test solution. Mix equal parts of the 1 5 hydrochloric acid solution and a 12% solution of silicotungstic acid. Xcotine standard, stock solution. Accurately weigh 0.5 gram of freshly distilled nicotine into a 1-liter volumetric flask and dilute to volume with 0.1N hydrochloric acid solution. Automatic smoking machine.
+
+
B
I
Absorption train, for collecting - smoke (see Figire 1). ' SDectroDhotonieter. Beckman Model DL" or Gmilar instrument, equipped with ultraviolet lamp and silica cells. PROCEDURE
Preparation of Calibration Curve. From the standard nicotine solution prepare a series of dilute standards containing 2.5, 5, 10, 15, and 20 mg. of nicotine per 500 ml. of 0.1N hydrochloric acid solution. Measure the absorbance of these standards a t 259 mp, using O.1N hydrochloric acid as the blank, and plot absorbance against concentration. Preparation of Smoke Absorption Train. Measure 40 ml. of the ethanolic hydrochloric acid solution into trap A (Figure l ) , 35 ml. of 0.1N aqueous hydrochloric acid solution into trap B , and 20 ml. of ethyl alcohol into t r a p C. Analysis. Smoke three representative cigarettes to the desired butt length. After smoking, allow 20 minutes for the smoke to become completely absorbed. Discard the contents of trap C, and transfer the contents of trap B into trap A (Kjeldah1 flask). It is not necessary to rinse trap B . Rinse the entry tube n-ith ethyl alcohol and pour rinsings into the Kjeldahl flask. Distill the contents of the flask until the alcohol is removed. Then admit steam and continue distillation for 10 minutes. Discard the distillate. Cool the contents of the flask, dilute to approximately 50 ml., and add 2.0 to 2.5 grams of calcium hydroxide. Steamdistill a t a moderate rate into 25 mI. of 5 hydrochloric acid solution the 1 until 300 t o 400 ml. of distillate are collected, and the distillate gives a negative test with the nicotine test solution. Distillation usually requires 35 t o 50 minutes. Dilute the distillate to 300 ml. Transfer a portion of the distillate to a silica cell and measure the absorbance at 236, 259, and 282 mp against a blank of 0.111' hydrochloric acid solution. Calculation. Make correction for background contribution using the equation of Willits et al.:
+
(cor.)
=
1.059 [ A m
Figure 1 . Absorption train for collection of cigarette smoke
-
(A236
+
A282)I
Read the nicotine from the calibration curve.
EXPERIMENTAL
Analysis of Pure Nicotine Solutions. Freshly distilled nicotine was used in t h e preparation of standard solutions. T h e absorbances of solutions containing known concentrations of nicotine in 0 . 1 X hydrochloric acid were measured with a Beckman DU spectrophotometer at 259 mp. -4calibration curve of absorbance us. concentration of nicotine m-as constructed from these data. Solutions of known concentrations of nicotine were analyzed by the silicotungstate method, and the results were compared with readings taken from the calibration curve after measurement of absorbances in the spectrophotometer (Table I). The solubility loss of nicotine silicotungstate is evident from the results shown. This error, inherent in gravimetric procedure, becomes very serious a t low nicotine concentrations. Analysis of Tobacco Smoke. Saniples of nicotine from smoke Tvere obtained in t h e following manner. Cigarettes w r e puffed in a machine similar t o t h a t described by Bradford, Harlan, and Hanmer ( 2 ) . The smoke was trapped in ethanolic and aqueous hydrogen chloride (Figure 1). The alcohol was removed from the smoke concentrate by distillation, calcium hydroxide was added t o the aqueous solution, and the nicotine wis steamdistilled into aqueous hydrochloric acid. The nicotine hydrochloride samples mere diluted, adjusted t o the proper acidity, and measured in a Beckman DU spectrophotometer a t 236, 259, and 282 nip. Calculation of the nicotine content involved the equation established by Willits et al. This equation, which corrects for background contribution to the 259-mp peak, awunes that the background absorbance betn-een 236 and 282 m p !s linear. The background absorbance for cigarette smoke is usually concave upwards, but varies with the type of tobacco smoked. Aliquots of the nicotine hydrochloride sample were analyzed also by the silicotungstate method for comparison. Each value shown in Tables I1 to V represents the smoke of three 70-mm. cigarettes where a 50-mm. portion of each \vas burned. The lack of agreement in results by the two methods appeared to be due t o high and inconsistent backgrounds in the ultraviolet spectra and was particularly apparent with the burley samples. Modification of Spectrophotometric Method. I t was found t h a t t h e background could be reduced considerably and made more consistent by steam-distilling t h e acidic smoke solution for 10 minutes following removal of t h e alcohol. No loss in nicotine occurs even after 30 minutes
Table I. Determination of Nicotine Nicotine SpectroPresent, Gravimetric, photometric, i\f g. 11.56 8 78
bfg. 11.44 11.41 8.60 8 63
RIg. 11.54 11.57 8.79 8.77
Table II. Nicotine Content of Smoke from Cigarertes Made from a Single Type of Tobacco GraviSpectroTobacco metric, photometric, Mg. Mg. TJ pe Burley A 12 19 16 14 Burley B 9 32 10 41 Flue-cured -4 9 49 9 15 Burley C 11 63 12 22 Burley D 9 18 9 77 Flue-cured B 9 65 9 93
of such distillation, b u t 10 minutes is sufficient for removal of interferences. Duplicate samples were taken from smoke solutions to be analyzed. One sample was carried through the analysis, omitting the 10-minute steam distillation following alcohol removal, and the other sample was analyzed with inclusion of this step.
Table 111.
Effect of Impurity Removal on Nicotine Values Spectrophotometric Detn., Mg. Acid Acid Tobacco distillation distillation included Type omitted Burley A 9.24 9.81 Flue-cured h 7.61 8.30 Burley B 10.62 9.69 Blend 7.46 7.60 Burley C 10 68 11.92 Burley D 10 82 9 85 Flue-cured B 8 90 9.60
Table IV. Comparison of Silicotungstate and Modified Spectrophotometric Methods SpectroTobacco Gravimetric, photometric, Type 11g. hfg. Burley A 13.54 13.81 Flue-cured -4 9.72 9.64 Burley B 12 34 12 70 Burley C 9 71 9 83 Flue-cured B 8 92 9 18 Turkish 5 22 5 32 Blend A 7 48 7 64 Blend B 7.39 7.44 Blend C 6.89 7.03
Table V. Precision of Duplicate Spectrophotometric Determinations DifferTobacco Sicotine Content, ence, Type Mg. Mg. Blend A 8.63 8.71 0.08 Flue-cured A 9.14 9.20 0.06 Burley A 9 76 9 54 0 22 Blend B 9 42 9 37 0 05 Burley B 9 22 9 25 0 03 Flue-cured B 9 65 9 72 0 07 Burlev C 9 81 9 74 0 07 Turkhh 5.14 5.25 0.11 Burley D 11.97 12.20 0.23 Burley E 12.24 12.56 0.32
ods. Table IV gives results from a comparison of nicotine determination in smoke from different tobacco types by t h e gravimetric and modified spectrophotometric methods. Recovery of Nicotine. The reliability of t h e two methods was also Figure 2. Ultraviolet absorbance of compared by determination of niconicotine in hydrochloric acid solution tine added t o t n o residues from distillation of smoke solutions. The amount of nicotine added to each Figure 2 s h o w the ultraviolet spectra residue !vas 14.62 mg.; 14.60 mg. of pure nicotine in hydrochloric acid, 9, nere recovered using t h e spectroof nicotine hydrochloride from smoke photometric method a n d 14.31 and with no steam distillation of the acidic 14.34 mg., respectively, using t h e smoke concentrate following alcohol gravimetric method. removal, B, and of nicotine hydrochloPrecision of Modified Spectroride which had been subjected t o 10 photometric Method. To determine minutes of steam distillation follon-ing t h e reproducibility of t h e spectrothe removal of alcohol, C. photometric method, analyses were Comparison of Gravimetric and made x i t h duplicate cigarette samModified Spectrophotometric Methples. These cigarettes were chosen VOL. 30, NO. 1 1, NOVEMBER 1958
1 801
to be within a narrow range of weight a n d draft resistance (Table V). Because a n y variation in the physical properties of the cigarette sample would contribute greatly to the variations exhibited in Table V, determinations mere made on five aliquots of the same smoke concentrate, thus eliminating this variable. The results were 11.64, 11.68, 11.66, 11.63, and 11.64 mg., respectively, of nicotine recovered. DISCUSSION
Steam distillation of the acidic smoke concentrate prior to distillation of the nicotine eliminates a considerable portion of the interferences which contribute high and inconsistent backgrounds t o the ultraviolet spectra, and which cause inconsistent results. If freshly distilled nicotine is used in preparation of the caJibration curve, there will be no appreciable background in the ultraviolet spectrum. and the absorption at 259 mp will correspond directly to the nicotine concentration. However, there will be some background
if the nicotine has not been freshly distilled. The interfererdes which cause high and inconsistent backgrounds in the ultraviolet spectra of nicotine derived from tobacco smoke are not evident in nicotine, which is steam-distilled from tobacco itself. For this reason, the preliminary steam distillation of acidic solution found neceseary in the analysis of smoke is not a required step in the determination of nicotine in tobacco. Khen the smoke of ten or more cigarettes is contained in the solution t o be analyzed, or when the smoke has an abnormally high nicotine content, the dilution technique of Willets et al. will probably have to be employed. LITERATURE CITED
(1) Assoc. Offic. Agr., Ckernists, "Official Methods of -4nalysis, 8th ed., p. 66, 1955. (2) Bradford, J. A., Harlan, W. R., Hanmer, H. R., Ind. Eng. Chem. 28, 836-9 (1936). (3) Fabre, R., Truhaut, R., Boudene, C., Ann. pharm.franc. 10, 579-94 (1952).
(4) Filk, H., Sturmer, E., Loeser, A., drznezmztfel-Forsch. 4 (No. 6), 367-8 (1954). (5) Freeman, F. M., Analvst 80, 520-2 (19t5). (6) Giovanni, G., I1 Tabacco 57, 103-21 (1953). (7) Greenberg, L. A., Lester, D., Haggard, H. W.,J . Pharmacol. Exptl. Therap. 104, 162-7 (1952). (8) Houston, F. G., ANAL. CHEM. 24, 1831-2 (1952). (9) Jeffrey, R. Tu'., Eoff, W. H., Ibid., 27, 1903 (1956). (10) Jensen, C. O., Haley, D. E., J . Agr. Research 51,267-76 (1935). (11) Keith, C. H., Newsome, J. R., Tobacco Sei. 2, 14-19; Tobacco 146, Yo. 9,22-7 (Feb. 28,1958). (12) Leiserson, L., Walker, T. B., ANAL. CHEW ...~ . 27.1129-30 f 1955). (13) hlarkn-ood, L. N.,j. Assoc. O@c. Agr. Chemists 22, 427-36 (1939). (14) hforani, V., Giovannini, E., Chim. e ind. (Milan) 37, 109-12 (1955). (15) Willits, C. o., Swain, M. L.,Con~
nellv. J. A.. Brice. B. A.. ASAL. CHEM.
22, i30-3 (1950). ' (16) Wolff, W. A., Hawkins, 31. A., Giles, TI-. E., J . Biol. Chem. 175, 825-31 (1948).
RECEIVEDfor review March 22, 1958. Accepted July 2, 1958.
Determination of Chlorine and Bromine by the High-Temperature Combustion Method S. W. NICKSIC
and L. L. FARLEY
California Research Corp., Richmond, Calif.
b High-temperature combustion apparatus normally used for sulfur determination has been a d a p t e d to samples containing chlorine and bromine. The procedure for determining halogens i s similar to that used in determining sulfur in petroleum fractions. A weighed sample in a ceramic boat is slowly moved into the combustion zone while a stream of oxygen i s passed over it. The sample burns, and the exit gases are scrubbed with a solution of 5% aqueous sodium bisulfite. When the sample has burned completely, the absorbing solution i s drained out and titrated potentiometrically with silver nitrate. The method i s suitable for materials that are difficult to decompose by other methods, and it i s especially useful for determining the chloride content of refractory inorganic catalysts.
C
methods of analysis have distinct advantages. They consume less time than other methods because of the rapid sample decomposition which is peculiar to these procedures and they are not usually restricted to OMBUSTION
1802
ANALYTICAL CHEMISTRY
any one type of sample or concentration range. Such methods for the determination of halogens date back to the days of Pregl (6). Considerable interest in them has been reawakened intermittently (1-3, 6, 7, 8) and today combustion methods are common in petroleum laboratories. The high-temperature combustion apparatus has long been used in the steel industry for the determination of sulfur and carbon in ferrous metals. I t s usefulness has been extended to petroleum products and related materials, and it is being studied by ASTM Committee D-2 on Petroleum Products and Lubricants (6)as a possible ASThI standard for sulfur determination. This paper describes the adaptation of the high-temperature combustion apparatus to compounds containing chlorine and bromine. Sodium bisulfite solution is used in the absorbers to absorb chlorine and bromine and t o convert them quantitatively to the ionic form for subsequent titration. Iodine compositions were not studied. B y using the high-temperature combustion apparatus where it is available, not only is a separate furnace for halogens eliminated,
but also the scope of analysis is extended to samples that cannot otherwise be easily handled. APPARATUS A N D REAGENTS
The apparatus has been described (2). The furnace used is a dual, resistance-type unit operating at 2400" to 2600" F. The combustion tubes are ceramic. The absorbers consist of extra coarse gas dispersion tubes assembled in a glass container with a glass stopcock for draining out the absorbing solution. The quartz wool plug in the hot part of the combustion zone is essential, and the position of this plug, as well as the position of the combustion tube in the furnace, is an operating variable. Brief experiments were made with an induction furnace, but the first results were not as good as those obtained with the resistance unit. The halogen-containing solutions were titrated on a Precision-Dow Recordomatic Titrator. This versatile instrument shortened the analysis time. However, the halogens may be titrated potentiometrically by any standard method. The reagents required are a 5% aqueous solution of sodium bisulfite and
