(7) R. Alexander, E. C. F. KO, A . J. Parker, and T. J. Broxton, J . Am. Chem. Soc., 89, 3703 (1967). (8) P. S. Buckley and H. Hartley, Phil. Mag., 8, 320 (1929). (9) D.C. Luehrs, R. T. Iwamoto, and J. Kleinberg, J. Inorg. Chem., 5 , 201 (1966). (10) E. J. Meehan and Grace Chiu, J . Pbys. Chem., 70, 1384 (1966). ( 11) M.B. Hugh. "Light Scattering from Polymer Solutions", Academic Press,
London, 1972, Chapter 4. (12) E. J. Meehan and W.H. Beattie, J . Colloid Sci., 15, 183 (1960).
RECEIVED for review August 11, 1977. Accepted September 19, 1977.
Determination of Nitrite Ion Using Differential Pulse Polarography Shaw-Kong Chang, Raymond Kozeniauskas, and George W. Harrington" Department of Chemistty, Temple University, Philadelphia, Pennsylvania
19 122
Nitrite ion can be determined with a high degree of accuracy and sensitivity by differential pulse polarography utilizing the rapid and quantitative reaction between nitrite ion and diphenylamine at low pH with thiocyanate ion as catalyst. The calculated detection limit is shown to be 0.3 (as NO2-) ppb in simple aqueous solution. The method is further used to determine nitrite in processed meats and human saliva. The results for processed meat are compared to those obtained by the AOAC method. The agreement is excellent.
of this paper is t o demonstrate that the formation of D P N can serve as the basis for a useful analytical method.
Because of the role of nitrite ion as an important precursor in the formation of N-nitrosamines, many of which have been shown t o be potent carcinogens (1-3), and it widespread occurrence in our environment, either naturally ( I ) or by its use as a food preservative ( 2 ) ,it is important t h a t sensitive and accurate methods be available for the determination of this ion. Such methods should also be simple and quick and capable of determining nitrite ion in various types of real samples. I t is the purpose of this paper t o demonstrate that differential pulse polarography (DPP) coupled with an appropriate chemical reaction satisfies these requirements. A large number of methods for the determination of nitrite have been reported, principally involving colorimetric methods. These methods have been summarized and compared by Szekely ( 4 ) and Sawicki e t al. ( 5 ) . The methods all have limited sensitivity and dynamic range. They frequently depend on unstable colors and involve long reaction times. Determination of nitrite by differential pulse polarography has been previously reported. In the first instance (6) nitrite was determined as nitrous acid in a citrate buffer. However, since nitrous acid is unstable and its reduction potential is close t o t h a t of oxygen, the sensitivity of the method was limited t o 0.5 ppm. A second report appeared by O'Laughlin e t al. (7),in which nitrite and nitrate were determined by D P P using the enhancement of the ytterbium peak current. A detection limit of 14 ppb nitrate, or nitrite, nitrogen was achieved under ideal conditions. As part of a continuing study of N-nitrosamines (8-11) the authors have discovered t h a t diphenylamine (DPA) reacts instantaneously and quantitatively with nitrite under appropriate conditions to yield diphenylnitrosamine (DPN). This nitrosamine, which has been found not to be a carcinogen (12-I5),yields a very sharp differential pulse polarogram with E, = -0.55 to -0.60 V vs. SCE at p H 1-2. The polarographic behavior of D P N has been well studied (16,17). The purpose 2272
ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
EXPERIMENTAL Apparatus. The instrumentation, cells, etc. were previously reported ( 1 1 ) . The drop time was 1 s, pulse height, 50 mV; and scan rate, 2 mV/s. Hg flow rate was 1.26 mg/s. Hg column height was 82.4 cm. All microliter pipets were calibrated in terms of precision and accuracy. pH was measured with a Leeds and Northup Expanded Scale pH meter. Reagents. All inorganic chemicals used were reagent grade and used without further purification. A-1-Napthylenediamine used for modified Griess Reagent was obtained from the Eastern Regional Research Laboratory of the U S . Department of Agriculture. Diphenylamine (Aldrich Chemical Co.) was recrystallized from water and methanol. All distilled water used was triple-distilled. Dilute solutions of nitrite were prepared from stock solutions, which were 1.00 X lo-* M in NaN02, by appropriate quantitative dilution or by direct pipetting into samples. High purity nitrogen further purified by passage through a chromous scrubber was used for degassing solutions having concentrations of 0.1 pM or higher. Helium was used for lower concentrations. Irreproducibile results were obtained when nitrogen was used for the more dilute solutions, possibly because of trace amounts of nitrogen oxides present in the gas. Saliva was collected by expectoration into clean vials. Approximately 1 mL was obtained from each subject. Filtration, if necessary, was performed using nitrite-free No. 1 filter paper; the filtrate, or clear saliva, was used directly as the sample. Frankfurters were prepared for analysis using the AOAC method (18, 19). Procedure. A differential pulse polarogram was run on a degassed solution containing diphenylamine, percholoric acid, and NaSCN, as a catalyst. Details are given below. The background current was recorded. To this solution was added an appropriate aliquot of sample and the current-voltage curve recorded again. The background current was subtracted from the current observed after addition of sample, yielding the desired current signal. Nitrite was then determined by the method of standard addition. For the analysis using the AOAC method, a working curve was established.
RESULTS
In order to determine those solution conditions yielding the fastest reaction time, several solutions of varying composition were examined. T h e DME was potentiostated a t the peak of D P N and the current recorded as a function of time. A voltage integrator was used to drive the X-axis of the recorder. T h e reaction rate was followed from time of addition of the known amount of nitrite solution. After the desired interval of reaction time had passed, a large concentration of SCN-
Table I. Rate of Formation of Diphenylnitrosamine (DPA) for Various Solutions Conditions ( t = 25.0 "C) DPA, Nitrite SCNSolution concn, concn, concn, pM t , /,, min-' No. pH !.lM PM 0 0.777 1 1.00 200 25.0 2 3 4 5 6 7 8 9 10 11 12 13 14
1.50 2.00 2.50 1.50 1.50 1.00 1.00 1.00 2.00 2.00 2.00 2.00 2.00
200 200 200 200 200
100 300 400 200 200 200 200 200
25.0 25.0 25.0 10.0 5.00 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0
0 0 0 0 0 0 0 0 125 250 375 500 1000
2.42 7.03 21.3 2.35 2.40 1.01 0.694 0.648 3.52 2.44 1.80 1.47 0.811
was added to the solution. This was done simply to accelerate the reactions. When there was no further increase in current, t h e value was recorded as the 100% current This current was further checked by determining the peak of an equimolar amount of DPN under the same solution conditions. In all cases plots of In (1- Z p / I ~ ~vs. ~ time J were linear; hence, t l l S could be calculated. Results are shown in Table I. NaC104 was added in appropriate amounts to maintain constant ionic strength. Acid was added as HC104. From the results for solutions 1, 2, 3, and 4 in Table I is seen that the reaction is fastest at low pH. This is in contrast to the rate of formation of other nitrosamines which have maximum rates at pH 3 (20-22). This may be associated with the fact that DPA is a very weak base. From the results for solutions 2, 5, and 6 it is obvious the t l l z is independent of nitrite concentration over the range studied. This observation agrees with previous reports (23,24) that the rate of formation of aromatic nitrosamines is frequently first order with respect to nitrite ion. The results for solutions 1,7, 8, and 9 yield the expected result that the rate increases with increasing concentration of DPA. The results for solutions 3, 10, 11, 12, 13. and 14 confirm the previously reported observation (20, 25) that thiocyanate ion greatly enhances the rate of the reaction. It has been suggested that this effect is due to the conversion of the H N 0 2 to NOSCN (26), thus increasing the concentration of the nitrosyl ion which is the actual nitrosating agent. From these experments we can expect that the half-time of' the reaction will be less than 3 s if DPA concentration is greater than 100 wM, thiocyanate concentration is greater than 7000 wM, and the p H = 1.5. Using these conditions, the reaction rate was too fast to measure by monitoring the increase of peak current. Addition of NO2- to this solution after 10-s stirring resulted in a peak current that did not change with time, indicating t h a t the reaction was complete before the measurement could be made. The rapid rate of reaction for DPA agrees with predictions based on kinetic studies of other secondary amines by Mirvish (21). It should be noted that neither DPA nor thiocyanate is electroactive; hence, the presence of excesses of these compounds had no effect on the determination. The reagent solution was thus prepared from the following stock solutions: Solution A: 2.60 X M diphenylamine in 4070 methanol (methanol is required to ensure solubility of the amine). Solution B: 0.100 M NaSCN. Solution C : 0.40 M HC104. T h e reagent solution was prepared by adding 5.00 mL of A and 10.00 mL each of B and C to 100.0 mL distilled water. The reagent solution prepared in this fashion yielded the best background i-E curves in terms of slope and noise level and also provided the necessary constant ionic strength buffer.
Table 11. Nitrite Determination in Aqueous Solution NO,- found NO,Colorimetry concn, (n = 4) PM (Griess reagent) DDP ( n = 4 ) 4.00 4.05 i 0.19 4.00 i 0.02 8.00 12.00
8.05 12.20
i i
0.33 0.20
8.00 12.12
f i
0.05 0.06
Table 111. Nitrite Determination in Aqueous Solution NO, concn ( X 10-7 M ) I , ( c o r r . ) IIA n 0.200 0.400 0.600 0.800
1.00 1.20 2.00 3.00 4.00 5 .OO 6.00 7 .OO 8.00 a
10.0 Standard deviation.
*
3.3 6.3 i 9.3 i 12.2 i 15.4 t 18.4 f 30.8 i 46.0 5 61.0 i 76.2 i 91.6 i 106.2 i 121.0 i 151.0 f
0 'La 04 0.4 0.5 0.3
0.4 0.4 0.3
0.5 0.6 0.4 0.6 0.6 1.4
5 5 5 5 5 5 4 3 3 3 3 2 2 4
The solution was prepared fresh each day and stored in a dark bottle containing a small amount (ca. 1 g) of zinc metal shot to destroy any DPN that might form simply on standing after exposure to air. This solution was used as described above in the Experimental section. Aqueous Solutions. Some typical results for simple aqueous solutions are shown in Tables 11 and 111. In Table I1 and others, n values refer to the number of independent determinations. Table I1 shows the results of comparisons between the usual colorimetric method using Griess reagent and the differential pulse method. [t will be noted that the agreement is excellent. Table I11 shows the results of the differential pulse method for a series of known nitrite solutions. These solutions were prepared by adding a n appropriate aliquot of nitrite stock solution t o 10.00 m L of reagent solution described above. The data appear to be quite good. Peak current is linearly related to concentration with a correlation coefficient (r2)= 0.999. Using the data in Table M to the lowest I11 for the concentration range 1.20 X value determined, the detection limit calculated by the method of Skogerboe and Grant (27) is 6 X M (0.3 ppb as NOz-). The calculation uses the formula, d.1. = tbx/m where 6 x is the pooled standard deviation, m = slope (151 nA/pM), and t is the one-tailed statistic constant a t 99% confidence level for 30 independent determinations. A solution 6.3 x lo-' M NO2- was run to check the reasonableness of the calculated detection limit. A corrected current of 0.8 f 0.3 nA was observed. The error of approximately 4070 a t this concentration agrees with the meaning of detection limit according to Ingle (28). At the lowest concentration in Table 111, 2.00 X M, the standard deviation is about 1270. If a standard deviation of 270 or better is desired, 1.00 X lo-'' M (4.6 ppb) should be considered the practical working limit. T h e samples to be discussed below were all examined at concentrations above 0.1 wM. In the potential region used in this study, -0.4 to -0.7 V , a noise level of about 3 nA or higher is not uncommon. The presence of DPA, however, a t the recommended level, has a marked smoothing effect on the i-e curve. Essentially no noise was observed in background even at 50-nA full scale deflection. ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
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Table IV. Stability of Diphenylnitrosamine Under Conditions of Analysis Time NO; found as DPN' 20 min 4.97 pM 23 h 5.03 pM 4.92 pM
46 h = 5.00 pM
NO;
added as NaNO,.
This is most likely a surfactant effect due to the DPA which apparently, from our data, does not negatively effect the analytical method. Table IV shows data that indicate the stability of the nitrosated product as a function of time. It can be seen from Table IV that D P N is quite stable under these conditions. Saliva. Fifty to two-hundred microliters of either clear saliva or saliva filtrate were added to 10.00 mL of the reagent solution. Results are shown in Table V. In samples C and D, known amounts of nitrite were added to check recovery. The original nitrite found in each sample was within the range previously reported for human saliva (29). Total analysis time per 5 samples was approximately 20 min. Processed Meat (Frankfurters). Frankfurters from three different manufacturers were examined. The suspensions prepared by the AOAC method were divided into two portions. One was analyzed by the DDP method and the other by the AOAC method using N-1-naphthylenediamine as reagent. Fifty to five-hundred microliter aliquots were used for the D P P method. Exactly 10.00 mL of reagent solution were used. In addition to determination of residual nitrite, a known amount was added to each sample and recovery checked. The results are shown in Table VI. It is apparent that the agreement is quite good and that the small amounts of miscellaneous material present in the frankfurther extract do not affect t h e electoanalytical determination under the conditions used. Table VI1 contains data comparing the results for t h e D P P method a t two different dilutions. The AOAC method of sample preparation requires a dilution of 1:lOO. T h e D P P method, however, can be used a t 1:lO dilution. At the lower dilution, the D P P method yields better accuracy as seen from data in Table VII. I n t e r f e r e n c e s . There are relatively few interfering substances in the type of samples studied in this investigation. Since the method basically determines the amount of nitrosating agent present, any NO in the air will also give rise to a peak. The N-1-naphthyldiamine used for Griess reagent
will also react with nitrosating agents. I t was observed that prolonged exposure of the reagent solution to laboratory air resulted in development of a small peak as seen during the background scan if the solution was not stored over zinc. Nitrate is not electroactive under the conditions used in these determinations, and hence does not interfere. In fact, additions of nitrate up to 500 times the concentration of nitrite did not affect the observed currents. The peak potential of D P N is sufficiently different from that of oxygen a t this p H that oxygen is not a serious interference. The usual degassing and blanketing procedures are sufficient except as noted in the Experimental section concerning very dilute solutions. Diphenylnitrosamine already present in the sample could, of course, yield erroneous results. Its presence is unlikely in the type of samples studied since it has not been found to be a naturally occuring nitrosamine (30). If there is reason to suspect the presence of this nitrosamine, its presence and concentration could be checked by a variety of methods. A portion of the sample could be treated with urea to destroy the nitrite and a n aliquot then run in the reagent solution. Alternatively, one could simply add a n aliquot of the sample to the reagent solution prepared without the DPA and then scan the potential region. If necessary, corrections could then be made in the final results. This check was performed in the samples used in this study and no electroactive species were observed in the potential range -0.4 to -0.7 V.
DISCUSS I ON From the results given in Tables I-VII, it seems clear that utilization of the nitrosation of diphenylamine provides the basis for a sensitive and rapid analytical method for determining nitrite ion using differential pulse polarography. Analysis times are short and the reagents required are readily available and relatively non-toxic. DPN, although not a carcinogen, may under certain conditions act as a nitrosating agent via transnitrosation. D P N solutions should, therefore, be disposed of properly using a denitrosating solution such as 10% NaOH (aqueous) containing aluminum metal. In the case of saliva, very minor sample pretreatment is required. In some instances, raw saliva could be used directly. This method should be especially useful for studies involving the effect of diet and other parameters on nitrite concentrations in human saliva or for monitoring nitrite levels in processed meats. The method is currently being extended to include nitrate determination subsequent to nitrite determination. Results of these studies will be reported later.
Table V . Nitrite in Saliva Nitrite found, ppm Sample A (1114177) (S.K.C.) B (1118177) (S.K.C.) C (G.W.H.) C + 1.74 ppm Recovery = 95 i 3% D (B.W.L.)
D + 5.00ppm Recovery = 99.0
i
1
2
3
4
5
3.20 3.52 0.25 1.87
3.24 3.57 0.23 1.95
3.15 3.61 0.28 1.90
3.23 3.55
3.26 3.66
5.18 10.12
5.30 10.31
5.34 10.24
*
i 5
0.04 0.05 0.03 0.04 0.08 0.10
3.0%
6.73 -f 0.20 Sample A 2.25 % 0.08 Sample B 5.46 % 0.11 Sample C 5 p M NO,- was added in all cases.
2274
3.22 r 3.58 0.25 i 1.91 i 5.27 10.22
Table VI. Residual Nitrite in Frankfurter: Comparison of AOAC Methods vs. DPP Method Concentration, @mol DPP ( n = 4) AOAC ( n = 5 ) Recovery of added NO,-"
a
Average
95.7 i 6.1% 98.4 i 2.4% 98.8 % 4.0%
A N A L Y T I C A L CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
6.55 2.37 5.39
%
i i
0.07 0.03 0.07
Recovery of added NO,-' 101.7 i 2.4% 98.8 i 1.6% 99.0 i 2.4%
(12) R. Druckery, S. Preussmann, D. Ivankovic and 2. Schmahl, Krebsforsch.. 69, 103 (1967). (13) M. F. Argus and C. Hoch-Ligeti, J . Natl. Cancer Inst., 27, 695 (1961). (14) H. Druckery, R. Preussman, D. Schmahl, and M. Miller, Naturwissenscbaften, 48, 134 (1961). (15) E. Boyland, R. L. Carter, J. W. Gorrod, and F. J. C. Roe, Eur. J . Cancer, 4, 233 (1968). (16) H. Lund, Acta Chem. Scand., 11, 990 (1957). (17) L. Holleck and R . Schindler, Z . Nectrochem., 62, 942 (1958). (18) "Official Methods of Analysis", 12th ed., Association of Official Analytical Chemistry Washington, D.C., 1975. (19) R . A. Nicholas, and J. B. Fox, Jr., J . Assoc. Off. Anal. Chem., 56, 922 (1973). (20) S . S.Mirvish, J. Sams, T. Y. Fan, and S. R. Tannenbaum, J . Natl. Cancer Inst., 51, 1833 (1973). (21) S. Mirvish, Toxicol. Appl. Pharmacol., 31, 325 (1975). (22) S. Mirvish, J . Natl. Cancer Inst., 44, 633 (1970). (23) J. H. Ridd, Quart. Rev., Chem. Soc., 15, 418 (1961). (24) E. Kalatzis and J. H. Ridd. J . Chem. SOC.6 ,529 (1966). (25) T. Y . Fan and S. R. Tannenbaum, J . Agric. FocdChem., 21, 237 (1973). (26) C. A. Bunton, D. R. Llewellyn, and G. Stedman, J . Chem Soc., 568 (1959). (27) R. K. Skogerboe and C. L. Grant, Spectrosc. Lett., 3, 215 (1970). (28) J. 0 . Ingle, Jr., J . Chem. Educ., 51, 101 (1974). (29) S. R. Tannenbaum, A. J. Sinskey, M. Weisman, and W. Bishop, J . Natl. Cancer Inst.. 53, 79 (1974). (30) P. N. Magee, R. Montesano, and R. Pruessmann in "Chemical Carcinogens," ACS Monograph, No. 173, C. E. Searle, Ed., 1976, Chapter 11.
Table VII. Concentration (ppm) of NANO, in Frankfurter: Comparison of 1 : l O vs. 1:lo0 Dilution Concentration ( p p m ) o f NANO, in frankfurter 1:lOO ( n = 4 ) 1 : l O (n = 4) Sample D 23.44 i 0.10 23.56 i 0.28 Sample E 25.44 * 0.09 25.49 i 0.24 ACKNOWLEDGMENT The authors thank Walter Fiddler of The Eastern Regional Research Laboratories of the U S . Department of Agriculture for his generous contribution of materials for Griess reagent. LITERATURE CITED P. N. Magee and J. M. Barnes, Brit. J . Cancer, 10, 11 (1956). W. Lijinsky and S. S. Epstein, Nature (London), 225, 21 (1970) I. A. Wolff and A. E. Wasserman, Science, 177, 15 (1972). E. Szekely, Talanra 15, 795 (1968). E. Sawicki, T. W. Stanley, I. F-faff, and A. Damico, Tabnta, 10, 641 (1963). Princeton Applied Research Application Brief N-1, Princeton, N.J., 1974. S. W. Boese, V. S. Archer, and J. W. O'Laughlin. Anal. Chem.. 49. 479 (1977). S. Yanaqida, D. J. Barsotti, G. W. Harrinqton, and D. Swern. Tetrahedon Lett., 28, 2671 (1973). S. K. Chang and G. W. Harrington, Anal. Chem., 47, 1857 (1975). S. E. Abanoli, J. A. Popp, S. K . Chang, G. W. Harrington, P. D. Lotlikar, D. Hadjiolov, M. Levitt, S. Rajalakshmi, and D. S.R. Sarma, J . k t / . Cancer Inst.. 58. 263 (1977). S.K. Chang, G. W . Harrington, H. S. Veale, and D. Swern, J . Org. Chem., 41, 3752 (1976).
RECEIVED for review March 31, 1977. Resubmitted July 18, 1977. Accepted September 12, 1977. Presented a t the 11th MARM, April 1977, Newark, Del. T h e investigation was supported by P H S Research Grant CA-18618 from the National Cancer Institute.
Fourier Transform Infrared Analysis below the One-Nanogram Level R. Cournoyer," J. C. Shearer, and D. H. Anderson Industrial Laboratory, Eastman Kodak Company, Rochester, New York
The interfacing of microscopy with Fourier Transform infrared (FTIR) spectroscopy Is a useful combination allowing samples of less than 1 ng to be identified. The sample, usually supported by a thin sodium chloride plate, is centered in a aperture 50 to 200 pm in diameter. The sample mount is oriented in an 8X beam condenser In an FTIR spectrometer where multiscan signal averaging techniques produce a spectrun wlth the desired signal-to-noise ratio. One or two hours of total analysis time Is generally required. Polymers and other solids as well as oils and various liquids have been Identified. The small amounts of material require that the entire sample preparation be done under a microscope.
T h e characterization of samples too small to be visible to the naked eye is restricted to the domain of the microscope. In many cases characterizations can be made accurately and quickly with t h e microscope alone, but some microsamples d o not yield to such analysis or do so only with great difficulty. These samples require the interfacing of modern instrumental techniques with classical microscopic analysis. The interfacing of microscopy with Fourier Transform infrared (FTIR) spectroscopy is a useful combination allowing preparations of less than 1 ng of sample to be identified. The sample, usually supported by a thin sodium chloride plate, is centered
14650
in an aperture 50 to 200 ,urn in diameter. The sample mount is oriented in an 8X beam condenser in an FTIR spectrometer where multiscan signal averaging techniques produce a spectrum with the desired signal-to-noise ratio. One or two hours of total analysis time is generally required. Polymers and other solids as well as oils and various liquids have been identified using this approach. EXPERIMENTAL Instrumentation. A stereo and compound microscope as well as microtomes, hot stages, or any other apparatus appropriate for the particular samples at hand are required. A fine pointed probe (dissecting needle), forceps, and other paraphernalia suitable for micromanipulation are also necessary. A Digilab FTS-14 Fourier Transform spectrometer equipped with the standard nichrome wire source and TGS detector, and a Perkin-Elmer 8X reflecting beam condenser were used to produce the spectra. Sodium Chloride Plates. Thin sodium chloride plates (200-500 pm thick) are prepared by cleaving rock salt used in standard infrared work. The salt crystals are first cut into rectangles (approximately x cm) with a clean, single edged stainless steel blade. This rectangle is transferred with forceps to a clean microscope slide and placed under a stereo microscope. The rectangle is stood on edge and cleaved into two plates of equal thickness with a clean, unused single edged blade. The process is repeated until plates of the desired thickness are obtained. Plates without both faces freshly cleaved are discarded to avoid possible contamination. A plate is selected that is flat and has flawless domains, and is trimmed to 1-2 X 3-4 mm. Some infrared ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
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