Anal. Chem. 1984, 56, 7944-7947
1944
searching for such a probe. The luminescence method is quick, easy, and reliable. Acid-base titrations of polyelectrolytes can also be quick, easy, and reliable. We have found, however, that sulfonated polymers are often contaminated with monomeric acids. Because of this, acid-base titrations frequently produce spuriously low EWs. The luminescence procedure described here is not affected by this contamination. For example, acid-base titration of our 1100 EW Nafion gave an EW of 1026 before dialysis and an EW of 1093 after dialysis. The luminescence procedure produced the same EW both before and after dialysis. Freedom from interference from monomeric acid is a major advantage of the luminescence procedure. Registry No. Ru(bpy)3(C12),14323-06-9; DA, 33423-98-2; NaPAS, 91178-70-0; NaPMM, 26950-79-8; NaCl, 7647-14-5; HC104, 7601-90-3; Nafion, 39464-59-0; sodium polystyrene (homopolymer),9080-79-9. LITERATURE CITED Schwoyer, W. L. K., Ed. “Polyelectrolytes for Water and Wastewater Treatment”; CRC Press: Boca Raton, FL, 1981. Wang, L. K.; Shuster, W. W. Ind. Eng. Chem. Prod. Res. Develop. 1975 14, 312. Buttry, D. A,; Anson, F. C. J . Am. Chem. SOC. 1983, 105, 685. Martin, C. R.; Rubinstein, I.;Bard, A. J. J . Am. Chem. SOC. 1982, 104, 4817,and references therein. Martin, C. R.; Rhoades, T. A.; Ferguson, J. A. Anal. Chem. 1982, 5 4 ,
1639. Johnson, B. C.; Tran, C.; Yllgor, I.; Iqbal, M.; Wightman, J. P.; Lloyd, D. R.; McGrath, J. E. Polym. Prepr., Am. Chem. SOC.,Dlv. Polym. Chem. 1983, 2 4 , 31. Mandel, M.; Stork, W. H. J. Biophys. Chem. 1974, 2 , 137. Stork, W. H. J.; Van Boxsel, J. A. M.; DeGoelj, A. F. P. M.; Dettaseth, P. L.; Mandel, M. Biophys. Chem. 1974, 2 , 127.
(9) Fenyo, J. C.; Mognol, L.; Delben, F.; Paoletti, S.; Crescenz, V. J . Polym. Sci., Polym. Chem. Ed. 1979, 17, 4069. (IO) Turro, N. J.; Okubo, T. J . Phys. Chem. 1982, 62. 1535. (11) Melsel, D.; Rabanl, J.; Meyerstein, P.; Matheson, M. S. J . Phys. Chem. 1978, 8 2 , 985. (12) Jonah, C. D.; Matheson, M. S.; Melsel, D. J . Phys. Chem. 1979, 83, 257
(13) Mirawetz, H.; Vogel, B. J . Am. Chem. SOC. 1969, 91, 563. (14) Takaglshl, T.; Naol, Y.; Kurokl, N. J . Polym. Scl., Polym. Chem. Ed. 1979, 17, 1953,and references therein. (15) Kurlmura, Y.; Yokota, H.; Shlgehara, K.; Tsuchida, E. Bull. Chem. SOC.Jpn. 1982, 55, 55.
(16) Prleto, N.; Martin, C. R. J . Nectrochem. SOC. 1984, 131, 751. (17)Turro, N. J.; Okubo, T. J . Am. Chem. SOC. 1982, 104, 2985. (18) Nagata, I.;Okamoto, Y. Macromolecules 1983, 16, 749. (19) Chen, R. F. NBS Spec. Publ. (US.)1973, No. 378, 183. (20) Miller, D. 8.; Graflus, M. A. Anal. Lett. 1978, 9 , 125. (21) Iglol, G. L.; Penzer, G. R. Anal. Blochem. 1975, 6 4 , 239. (22) Weber, G.; Borris, D. P. Molec. Pharmacol. 1971, 7 , 530. (23) Metzler, D. E. “Blochemistry”; Academic Press: New York, 1977;pp 187-190,and references therein. (24) Kelnfleld, A. M.; Pandisclo, A. A.; Solomon, A. K. Anal. Blochem. 1979, 94, 65. (25) Rubalcara, B.; Martinez de Munoz, D.; Gltler, C. Biochemistry 1969, 8 , 2742. (26) Scatchard, G. Ann. N . Y . Acad. Scl. 1949, 51, 660. (27) Willard, H. H.; Merrltt. L. L., Jr.; Dean, J. A.; Settle, F. A., Jr. “Instrumental Methods of Analysis”, 6th ed.; D. Van Nostrand Co.: New York, 1981 109. (28) Willard, H. H.; %rrltt, L. L., Jr.; Dean, J. A,; Settle, F. A,, Jr. “Instrumental Methods of Analysis”, 6th ed.; D. Van Nostrand Co.: New York, 1981;p 84. (29) Melsel, D.;Matheson, M. S. J . Am. Chem. SOC. 1977, 9 9 , 6577. (30) Martin, C. R. Trends Anal. Chem. 1982, 1 , 175. (31) Kalyanasundaram, K. Coord. Chem. Rev. 1982, 46, 159.
RECEIVED for review September 6, 1983. Resubmitted April 26,1984. Accepted May 1,1984. This work was supported in part by the Office of Naval Research.
Simultaneous Reaction Rate Spectrophotometric Determination of Cyanide and Thiocyanate by Use of the Pyridine-Barbituric Acid Method Shigeru Nagashima
Department of Chemistry, Tokyo Metropolitan Industrial Technical Institute, Nishigaoka, Kita-Ku, Tokyo 115, Japan
The rate of reaction between thiocyanate and chloramine-T varied wlth the pH of the soiutlon, which gave a complicated pH dependence; Le., It was rapid in the acidic and weak aikailne regions (around pH 5 and 8) and was slow in the neutral and alkaline reglons (around pH 7 and 9). The effect of thiocyanate on the pyrldlne-barbituric acld method was interpreted on the basis of the results and pH condltlons at the determination of cyanide. The reaction between cyanide and chioramlne-T was fast and Independent of the pH of the solution over a wide pH range. The difference between both reaction rates was applied to the simultaneous determination of cyanide and thiocyanate.
Recently, this author pointed out that thiocyanate caused a less positive error in the former method than in the latter (4) and also found that the rate of reaction between thiocyanate and chloramine-?‘ varied greatly with the pH of the solution (Le., an acid-base catalyzed reaction). Results are available for the interpretation of the effect of thiocyanate on the pyridine-pyrazolone method (5). In the present study using the pyridine-barbituric acid reagent, almost the same pH dependence was observed in the reaction of thiocyanate with chloramine-T. On the other hand, the reaction of cyanide with chloramine-T was fast and independent of the pH of the solution over a wide pH range. The difference between both reaction rates was then applied to the simultaneous determination of cyanide and thiocyanate by the procedure as described in the previous paper (5).
It is well-known that thiocyanate causes a significant positive error in the spectrophotometric determination of cyanide based on the Konig reaction ( I ) , such as the pyridine-pyrazolone method (2) and the pyridine-barbituric acid method (3). The reason is that thiocyanate, as well as cyanide, reacts with chloramine-T used as the oxidant in the methods to produce the intermediate compound cyanogen chloride.
EXPERIMENTAL SECTION Reagents. Standard Cyanide Solution. Dissolve 2.51 g of KCN in water and dilute to 1 L (1000 mg of CN-/L). Prepare working solutions by dilution with water. Standard Thiocyanate Solution. Dissolve 1.68 g of KSCN in water and dilute to 1L (1000 mg of SCN-/L). Prepare working solutions by dilution with water.
0003-2700/84/0356-1944$01.50/00 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984 0.81
I
1
I
I
I
I
I
I
,
1945
I
0.4
0.2 PI U
t
Figure 2. Effect of pH on the reaction between thiocyanate (or cyanide)and chloramine-T (25 "C): (0)SCN- taken, 16.0 pg/20 mL; (e)CN- taken, 7.0 pg/20 mL.
I
0
10
20 30 T i m e (min)
Figure 1. Absorbance-time curves in the reaction between thiocyanate and chloramine-T at various pH (25 "C): SCN- taken, 16.0 pg/20 mL; pH (1) 5.5, ( 2 ) 5.8, (3) 6.1, (4) 6.4, (5) 6.7, (6) 6.9, ( 7 ) 7.2, (8) 7 . 5 , (9) 7.8, (10) 8.1, (11) 9.1.
Buffer Solutions. Prepare them by mixing 0.10 M KHzP04 and 0.10 M Na2HP04in various ratios. Pyridine-Barbituric Acid Reagent. Wet 5.0 g of barbituric acid and a small quantity of water, add 75 mL of pyridine and 15 mL of concentrated hydrochloric acid, and mix. After the mixture was cooled to room temperature, dilute to 250 mL with water and mix (6). Prepare daily. Chloramine-T Solution (1% w / v ) . Prepare daily. General Procedure. In a dried volumetric flask (50 A)place , 20.0 mL of sample solution containing cyanide and/or thiocyanate. Add 4.0 mL of buffer solution and mix thoroughly. Add 0.5 mL of chloramine-?' solution, stopper the flask, and shake gently. Keep the solution at 25 0.1 O C for a fixed time (1-30 min). Add 5.0 mL of pyridinebarbituric acid reagent, dilute to 50 mL with water, stopper the flask again, and mix. Keep the mixture at 25 O C for 10-20 min. Measure the absorbance at 578 nm against a reagent blank with a 10-mm cell.
RESULTS AND DISCUSSION Effect of pH on the Reactions of Cyanide and Thiocyanate with Chloramine-T. The effect of pH was examined by varying the buffer solution (0.10 M phosphate solution) and by varying the standing time after the addition of chloramine-T solution, where the pH was denoted as the value for the mixed solution containing the sample and the buffer solution. (The pH hardly changed by addition of the chloramine-T solution.) Results in Figure 1were obtained for 16.0 fig of SCN-/20 mL. The rate of reaction between thiocyanate and chloramine-T varied greatly with the pH of the solution. The plot of absorbance vs. pH as for the data in Figure 1 revealed a diagram having a minimum and a maximum (Figure 2); where the pH of the final solutions was 5.8-5.9. On the other hand, the reaction between cyanide and chloramine-T was fast and independent of the pH of the solution over the range of pH 4.7-9.1 (Figure 2). Pyridine-Barbituric Acid Reagent. The color developed with the usual reagent (3, 7) being unstable, the reagent preparation was then improved as described in the previous paper (6).In the present study, the improved reagent was used. The preparation is noted in the Experimental Section. Effect of Concentration of the Phosphate Buffer Solution. The reaction of thiocyanate with chloramine-T was
much dependent on the pH of the solution, and the effect of concentration of the buffer solution was then examined (0.10-0.25 M). With the 0.25 M solutions, almost the same diagram as shown in Figure 2 was obtained for thiocyanate, but the absorbance for cyanide was found to decrease slightly, especially in the alkaline region. In the present study, the 0.10 M buffer solutions were then used. (In the case of pyridine-pyrazolone reagent using the 0.25 M buffer solutions, the absorbance for cyanide tended to decrease in the acidic region (5).) Reaction of Thiocyanate with Chloramine-T. In Figure 2, the diagram exhibits pH-rate profiles as for cyanide and thiocyanate, and the profile of thiocyanate has a minimum and a maximum. Almost the same profile was obtained by use of the pyridine-pyrazolone reagent in the previous paper (5),where it was discussed that the reaction was a complicated acid-base catalysis. Effect of Thiocyanate on the Methods. In the pyridine-pyrazolone method, cyanide is usually determined at the neutral region (pH 6-7 in ref 8). On the other hand, in the pyridine-barbituric acid method, cyanide is usually determined at the weak acidic region (around pH 5 in ref 7). As shown in Figure 2, it was then indicated that thiocyanate caused a less positive error in the former method than in the latter. In the following, the alkaline, neutral, and acidic regions were used in procedures for the simultaneous and differential determinations of cyanide and thiocyanate. Procedure I for the Simultaneous Determination of Cyanide and Thiocyanate at the Alkaline Region (pH 9.1). As Figure 1shows, in the early stage of the reaction of thiocyanate ( < l o min) at pH 9.1, the absorbance increased linearly with time. For 10-40 pg of SCN-/20 mL, the slope of the straight line, which exhibited the initial reaction rate, increased in proportion to the amount of thiocyanate. For the solutions containing cyanide and/or thiocyanate, the results indicated that the thiocyanate and cyanide were determined by use of the slope of the straight line and the absorbance at the intercept ( t = 0), respectively (Figure 3). The procedure was available for the solutions containing around 5-40 wg of SCN-/20 mL. Procedure I1 for the Differential Determination of Cyanide and Thiocyanate at the Acidic and Neutral Regions (pH 4.7 and 6.9). As Figure 2 shows, the reaction of thiocyanate, as well as cyanide, with chloramine-Twas rapid in the acidic region (pH 4.7), which gave linear calibration curves as for cyanide and thiocyanate (A and B in Figure 5). (The standing time after the addition of chloramine-Tsolution was fixed at 1-2 min.) On the other hand, the reaction of thiocyanate with chloramine-T was slow in the neutral region
1946
ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984 I
I
b l
0.6 I
U
0
Y
5
A
I
10
Time (min) Flgure 3. Absorbance-time curves in the reactlon of thiocyanate andlor cyanide with chloramine-T at pH 9.1 (25 O C ) : amount taken, (1) 20.0 pg of SCN-, (2) 20.0 pg of SCN- 4- 3.0 pg of CN-, (3) 10.0 pg of SCN- 4- 5.0 pg of CN-, (4) 7.0 pg of CN-120 mL.
0.6
, 0
/
,
I
xi
I
20 pg/20rnl
Flgure 5. Calibration curves for the differential determination of cyanide and thiocyanate by procedure 11: (A) CN- (at pH 4.7), (6)SCN- (at pH 4.7), (C) SCN- (prepared by using the absorbance at the intercept in Figure 4), (D) SCN- (prepared by using the difference between calibration curves, B - C).
1
Table I. Determination of Cyanide and Thiocyanate by Procedures I and I1 (pg/20 mL)
amt taken
I I1 I11 IV 0 " 0
,
Xr 10
1
5
I
V
amt found procedure I procedure I1
CN-
SCN-
CN-
SCN-
CN-
SCN-
2.0 3.0
30.0 20.0
5.0 5.0 7.0
10.0
1.9 2.8 5.0 5.2
30.4 20.0 10.2
5.2
10.1
5.1
5.0
4.8 1.9
5.0 2.0
7.1
10
Tirne(rnin)
Absorbance-time curves in the reaction between thiocyanate and chloramine-T at pH 6.9 (25 "C): SCN- taken, (1) 10.0, Flgure 4.
(2) 20.0, (3) 30.0, (4) 40.0 pg/20 mL.
(pH 6.9). Thus, when the solution containing cyanide and thiocyanate was analyzed at the acidic region and also at the neutral region, the difference between the absorbances was expected to increase with increasing amount of thiocyanate. For 10-40 pg of SCN-/20 mL, the absorbance-time curves in Figure 4 were obtained at pH 6.9. The absorbance in the time of 2-10 min increased linearly with time, and the absorbance at the intercept ( t = 0) increased in proportion to the amount of thiocyanate. A calibration curve of thiocyanate was then prepared by using the absorbance at the intercept (C in Figure 5). Figure 5 represents the procedure for the differential determination of cyanide and thiocyanate. Calibration curves A and B were prepared at pH 4.7 for cyanide and thiocyanate, respectively. Calibration curve C of thiocyanate was prepared by using the absorbance at the intercept in Figure 4. Calibration curve D of thiocyanate was prepared by using the difference between calibration curves (B - C). When the solution containing cyanide and thiocyanate was analyzed at pH 4.7 and 6.9, which gave absorbance A , (at pH 4.7) and absorbance A2 (of the intercept at pH 6.9), the procedure was as follows: The amount of thiocyanate X1 was obtained by using the difference between absorbances ( A , - Ag) and calibration curve D. Absorbance B1was read by using X1 and calibration curve B. The amount of cyanide X 2 was obtained by using the difference between absorbances ( A , - B,) and calibration curve A.
Determination of Cyanide and Thiocyanate by Procedures I and 11. The procedures were applied to the determination of cyanide and thiocyanate by use of several mixed standard solutions. As Table I shows, good results were obtained. Effect of Iron(II1) on the Reaction of Thiocyanate with Chloramine-T. Epstein (2), who proposed the pyridine-pyrazolone method, found that the reaction between thiocyanate and chloramine-T was slow and that it was accelerated by adding iron(II1) chloride. Asmus and Garschagen (3) determined thiocyanate by use of iron(II1) chloride solution, chloramine-T solution, and the pyridinebarbituric acid reagent. However, it seems that the reaction is not catalyzed by iron(III), but the acceleration is rather due to pH change, because the pH of the solution is shifted to the acidic region by adding iron(II1) chloride solution. Cyanide and thiocyanate were determined simultaneously by the present procedure using the pyridine-barbituric acid reagent without iron(II1) chloride solution. Use of Chloramine-B. Chloramine-B is an oxidizing agent similar to chloramine-T. In the present study, it was also found that almost the same pH-rate profile of thiocyanate as that in Figure 2 was obtained by using chloramine-Binstead of chloramine-T in the pyridine-pyrazolone method and the pyridine-barbituric acid method. However, the use of chloramine-T was preferable to that of chloramine-B for the simultaneous determination of cyanide and thiocyanate, because the absorbance for cyanide tended to decrease in the case of chloramine-B. Registry No. SCN-, 302-04-5; CN-, 57-12-5;Chloramine-T, 127-65-1; pyridine, 110-86-1;barbituric acid, 67-52-7.
Anal. Chem. 1084, 56, 1947-1950
LITERATURE CITED Kbnig, W. J . Prakt. Chem. 1904, 8 9 , 105-137. Epsteln, J. Anal. Chem. 1947, 19, 272-274. Asmus, E.; Qarschagen, H. Fresenius’ Z . Anal. Chem. 1953, 138, 414-422. (4) Nagashima, S.; Ozawa, T. Int. J . Environ. Anal. Chem. 1981, 10, 99-106. (5) Nagashlma, S. Anal. Chem. 1983, 55, 2088-2089. (6) Nagashlma, S. Water Res. 1983, 17, 833-834.
1947
(7) “Standard Methods for the Examlnatlon of Water and Wastewater”. 15th ed.;American Water Works Association: Washington, DC, 1980; Part 412. (8) “Standard Methods for the Examination of Water and Wastewater”, 13th ed.; American Water Works Assoclatlon: New York, 1971; Part 207.
RECEIVED for review February 21,1984. Accepted April 23, 1984.
Determination of Nicotine in Tobacco by Circular Dichroism Spectropolarimetry W.Marc Atkinson, Soon M. Han, and Neil Purdie* Chemistry Department, Oklahoma State University, Stillwater, Oklahoma 74078
A circular dlchrolsm (CD) spectropolarimeter has been used as the detector for the determination of (S )-(-)-nlcotlne In chopped tobacco leaves, after a straightforward single extraction of the analyte Into methanollc KOH. Other compounds extracted from the leaf absorb In the UV range, but none is CD actlve, eilminatlng all possible Interferences. Results are reported lor the nicotine contents in a few smokeless tobaccos and clgareltes.
Circular dichroism (CD) spectropolarimetry has been successfully applied to the identification and determination of alkaloids in a variety of complex matrices (1-8). Since two prerequisites must be satisfied for CD activity, namely, the analyte must simultaneously absorb electromagneticradiation and be optically active, determinations can be made quickly and directly, after simple solvent extraction and without separation from the other components in the mixture. Previous work was devoted to the determination of opiates, Lcocaine, and D-LSD in confiscated specimens and of tetracycline in human urine (9). In d of these instances the analyte was either the only CD active or the predominant CD active component in the mixture. In this work we have characterized the CD spectrum of @)-(-)-nicotine in aqueous, 2-propanol, and methanolic KOH solutions and have determined the total nicotine alkaloid content of several cigarette brands and smokeless tobaccos. These experiments are preliminary to later studies of the nicotine contents in tobacco smoke, in pesticides, and in biological fluids and to a full analogous study of the cannabinols in marijuana. Nicotine determinations are of particular importance to the tobacco industry and in the area of toxicology. As expected, the analytical techniques applied to this purpose are many and varied. A fairly thorough review of these methods (10) pointa up the significant contributions from chromatographic procedures: thin-layer, liquid, and gas chromatography, often times coupled with mass spectrometry. Other techniques include potentiometry (1I), absorption spectrophotometry (121,and polarimetry (13). Many of the methods involve a prolonged extraction and work-up procedure prior to determination, but the comparisons among the resulta for nicotine in leaf, in smoke, and in biological fluids are excellent. We have found no reference in the literature to the determination of nicotine in smokeless tobaccos. 0003-2700/84/0356-1947$01.50/0
The gas chromatographic study of Severson et al. (14)describes a simple extraction procedure followed by a rapid determination of the relative distribution of the nicotine alkaloids in cured tobacco leaves. This is a significant contribution in that in most other methods the analogues were determined as total nicotine, The important minor nicotine-type alkaloids constitute about 5% of the total alkaloid fraction. A knowledge of their distribution is important to product development, but not necessarily to quality control. An earlier CD study of nicotine-type alkaloids was done for the purposes of a preferred conformation study (15). Spectra were not included for reference, but molecular ellipticity data are included for nicotine, nornicotine, anabasine, and methylanabasine, the last three being minor constituents of tobacco. The evidence suggested that the analogues are indistinguishable from nicotine by CD. This was confirmed in our laboratory for anabasine. Results are reported, therefore, as total nicotine. EXPERIMENTAL SECTION (SI-(-)-Nicotine, as the liquid free base, was obtained from Eastman Kodak and used without further purification. CD spectra were measured for the analyte dissolved in aqueous HCl, aqueous NaOH, and in a variety of buffers over the pH range 6.2-8.6. Extractions from tobacco leaves were made using either analytical grade 2-propanol or 0.05 M methanolic KOH (Baker Chemical Co.). Samples of smokeless tobaccos and cigarettes were chosen at random from local stores. UV absorption measurements were made on a Perkin-Elmer Model 552 spectrophotometer. CD measurements were made on a JASCO-5OOAautomatic recording spectropolarimeter fitted with a DP-500N data processor. Daily calibration of the ellipticity scale was made against a standard solution of androsterone in dioxane as recommended. Measurements were made over the wavelength range 230-320 nm, with base line correction made for the blank by spectral subtraction on the data processor. Sensitivity,scan rate, and repeat functions were selected which optimized the signal to noise ratio. Sample weights were chosen both for nicotine and for the tobacco extractions such that the nicotine concentration was in the range of lo4 to M. Cell sizes were either 1 mm or 1 cm path length. For the extraction experiments, the sample weights for the smokeless tobaccos were on the order of 200 mg, while individual, randomly chosen, cigarettes were used with average net weights (filters removed where appropriate) in the range of 550-850 mg. All determinations were done in duplicate. For 2-propanol extractions, the samples were agitated with either 5-mL or 10-mL aliquots of solvent for 1h on a mechanical shaker. For methanolic KOH, extractions were made during a 1 h ultrasonication pro@ 1984 American Chemical Society
