150
HART READING, mm GLC. MAL, MAL-3
100
50
C I
I
30
40
ELUATE VOLUME,
503
cm
Figure 6. Separation of oligosaccharides of maltose and isomaltose series in 60% (w/w) ethanol at 75 OC Resin bed: 4 X 1300 mm Dowex 1-X8,Sodz-,8-13 pm. Linear flow rate: 1.7cm min-'. Glucose, maltose, maltotriose, isomaltose, and isomaltotriose, 10 mg of each
ture (75 "C or higher) although the equilibrium conditions are less favorable. The lower part of Figure 5 refers to the critical concentration of ethanol at 75 "C for the isomaltose series. No separation was obtained a t this temperature whereas, as expected, both the distribution coefficients and the separation factors increased by lowering the temperature. The results show that the critical concentration depends on the temperature. As expected, the critical concentration was lowered when the temperature was decreased. This can at least partly be ascribed to an increased mole fraction of water inside the resin a t low temperature. The dependence of the critical ethanol concentration on the type of glycosidic linkages (and also on the type of
sugar moieties) can be taken advantage of in separations of complex mixtures containing oligomers belonging to different oligomeric series. Figure 6 illustrates the separation of a mixture containing the first four members of the maltose series from isomaltose and isomaltotriose on a resin in the sulfate form. The substances were eluted a t the critical ethanol concentration for the maltose series (60% ethanol) and, as expected, the oligomers belonging to this series were obtained together with glucose in a single peak well separated from the individual oligomers belonging to tlie isomaltose series. In a subsequent run at higher ethanol concentration (70%), glucose and the oligosaccharides of the maltose series can be easily separated. From a practical point of view, it is important that when working a t 75 O C , the peak elution volumes were reproducible within f 1 % for a period of a t least 30 days and that the results were the same in runs with single species and with mixtures. The areas of the peaks were proportional to the amounts applied and, when the areas were compared to those in calibration runs made on the subsequent day with approximately the same amounts as those present in the sample, the precision was f 1 . 5 % or better.
LITERATURE CITED (1) R. L. Whistler and C. C. Tu, J. Am. Chem. SOC.,74,3609 (1952). (2)C. T. Bishop, Can. J. Chem., 33,1073 (1955). (3)M. John, G.Trenel, and H. Dellweg, J. Chromatogr.,42,476 (1969). (4)K. Chitumbo and W. Brown, J. Polym. Sci., 36,279 (1971). (5)W. Brown and 0. Andersson, J. Chromatogr.. 67, 163 (1972). (6)J. Havlicek and 0. Samuelson, Carbohyd. Res., 22,307 (1972). (7) E. Martinson and 0. Samuelson, J. Chromatogr.,50, 429 (1970). (8)J. Havlicek and 0. Samuelson, Chromatographia, 7, 361 (1974). (9)P. Jonsson and 0. Samuelson, Sci. Tools, 13, 17 (1966). (IO) B. Arwidi and 0. Samuelson, Sven. Papperstidn.. 68, 330 (1965).
(11) 0.Samuelson, "Ion Exchange Separations in Analytical Chemistry," Almqvist and Wiksell, Stockholm: Wiley, New York, 1963. (12)W. Brown and K. Chitumbo, Chem. Scr., 2, 88 (1972). (13) M. Mattisson and 0. Samuelson, Acta Chem. Scand., 12, 1386 (1958). (14)H. Ruckert and 0. Samuelson, Acta Chem. Scand., 11,315(1957). (15)0.Samuelson, in "ion Exchange 11," J. Marinsky, Ed., Dekker, New York. 1969. (16)0.Ramnas and 0. Samuelson, Acta Chem. Scand., 28,955 (1974).
RECEIVEDfor review March 25, 1975. Accepted May 12, 1975. Work supported by the Swedish Board for Technical Development.
Determination of Dimethylnitrosamine and Nitrosoproline by Differential Pulse Polarography Shaw Kong Chang and George W. Harrington Department of Chemistry, Temple University, Philadelphia, Pa. 79 722
Nitrosamines have been shown to be among the most potent chemical carcinogens ( I , 2) and, since nitrites are widely used as food preservatives, nitrosamines may be readily derivable from a variety of edible proteins during processing, cooking, or the digestive process. In view of their well established toxic effects, it is important that sensitive and accurate analytical procedures be developed for the determination of nitrosamines in a variety of media. The analytical chemistry of nitrosamines has received considerable attention. The subject has been discussed, in general, in a recent report by Wasserman ( 3 ) .Many analytical methods have been applied such as gas chromatography (31, mass spectrometry (31, spectrophotometry ( 3 ) , thin layer chromatography ( 3 ) and polarography (3-12).
While each of these methods can be highly sensitive, and reliable, they each'have certain unique disadvantages. Gas chromatography, for example, is restricted to volatile nitrosamines. Mass spectrometry and spectrophotometry, while highly sensitive, can be very difficult to interpret in the case of mixtures. Ordinary dc polarography which has been studied extensively in the case of nitrosamines has a limited sensitivity and rather poor resolution. The disadvantages associated with the polarographic technique can be minimized or eliminated by the use of differential pulse polarography. This technique has the further advantage of being capable of examining both volatile and nonvolatile nitrosamines. Differential pulse polarography has been thoroughly studied and is well established as a highly sensi-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 1 1 , SEPTEMBER 1975
1857
PH
Figure 2. Peak current vs. pH for 28.0 ppm DMN in buffered 0.1N KCI. -0.8
-0.9
voir
-1.0 VI
-1.1
-12
-1.3
SCE
Figure 1. Differential pulse polarograms of DMN in 0.1N KCI at pH 1.7 PIUS KN02.
Scan rate, 5 mV/sec. Pulse, 50 mV p-p. Ratios, DMN:NO?-. Curve(1) 1.0: 0.0, Curve(2) 1.0:0.10, Curve(3) 1.0:0.2,Curve(4) 1.0:0.50, Curve(5) l.O:l,O, Curve(6)1.0:2.0
tive analytical tool for the determination of a variety of substances (13, 14). It was the purpose of the present study to demonstrate the usefulness of differential pulse polarography for the determination of two structurally different nitrosamines in various media including those of biological or physiological importance. DMN and nitrosoproline were chosen because of the importance of the former as a carcinogen ( I ) and the presence of the nonvolatile nitrosoproline in processed meats ( 2 ) . The media chosen were water, blood serum, and methylene chloride. The last is important since it is the solvent used for the last step in the extraction of nitrosamines from processed foods ( 2 5 ) .
EXPERIMENTAL Chemicals. ACS reagent grade potassium chloride was recrystallized three times from triple-distilled water and vacuum dried in a desiccator. KC1 solutions prepared in triple-distilled water were further purified by extended electrolysis at -1.2 V (vs. SCE) using a large stirred mercury pool electrode. The nitrogen used was high purity dry nitrogen which was further purified by bubbling the gas through an oxygen scrubbing system consisting of zinc amalgam and chromous solution. The gas was saturated with water vapor by passing it through a solution of supporting electrolyte prior to use in the cell. The distilled water used throughout the investigation was triple-distilled. DMN was obtained from the Aldrich Chemical Company and used without further purification. Its purity was checked using a flame ionization detector gas chromatograph which showed it to be greater than 99.9% dimethylnitrosamine. Samples of nitrosoproline (>99.9%) were supplied by Eastern Regional Research Center, U S . Department of Agriculture. Apparatus. Differential pulse polarographs were determined using a Princeton Applied Research ModeJ 171 Polarographic Analyzer equipped with the PAR Model 172 Mercury Drop Timer. The mercury flow rate was 1.34 mg/sec in 0.1N KCl without applied potential. The column height was 75.6 cm. The pulse width of the Model 171 is 56 milliseconds. The delay time between pulses is established by the Mercury Drop Timer. Except as noted, a onesecond drop was used throughout and the scan rate was 5 mV/sec. Pulse heights of 50 and 100 mV were used in the course of the study. The electrochemical cell was a PAR Model 9343. The reference electrode was a saturated calomel electrode and the counter 1858
ANALYTICAL CHEMISTRY, VOL. 47,
Pulse, 50 mV p-p. ( 0 )first peak, (m) second peak electrode was platinum. For the studies involving serum extract, the counter was isolated from the main cell compartment by a medium porosity glass frit. All pH measurements were made using a Leeds and Northup extended scale Model X pH meter. Solutions. The usual analytical procedures were employed in the preparation of all solutions. The pH was adjusted using sulfuric acid or potassium hydroxide, as required, except in the case of serum samples where, as noted below, HC104 was used. Standard solutions of 5000-1000 ppm DMN, or nitrosoproline, were prepared in 0.1N KCl. Further dilution to the desired concentration was made using 0.1N KC1. Solutions were degassed for approximately ten minutes except as noted below in special experiments involving nitrite removal. The solutions were maintained in an atmosphere of Nz during each run. All polarographs were deter' C. mined a t 22 f 1 Serum Samples. Rat serum, supplied by the Fels Research Institute, Temple University, was diluted 1:5 with 0.1N KCl acidified to pH 0.5 with HClOI. This solution was digested for 10 minutes on a steam bath and then centrifuged. The supernatant was decanted into the electrochemical cell. Methylene Chloride Samples. Standard solutions of 259 ppm DMN were prepared in methylene chloride. More dilute solutions were prepared from this stock by serial dilution with methylene chloride. Twenty-five milliliters of the methylene chloride solution were mixed with 100 ml of 0.1N KC1 acidified to pH 1.7. The mixtures were stirred overnight (12-14 hr) a t 34-35', This treatment completely removed the CH2C12. The resulting aqueous solution was then run as noted above.
RESULTS As previously noted, the classical dc polarography of nitrosamines has been extensively studied. It has been noted (5, 8-11), for example, that the half-wave potential of the first wave of DMN in a KC1 supporting electrolyte is highly pH dependent. This was verified via differential pulse polarography. In buffered solutions (12 ), the peak potentials of the differential pulse polarograms exhibited the same linear dependence on pH over the range 1-5 as that shown by the half-wave potentials. In spite of its irreversibility (26), the wave in acidic medium, was relatively sharp and yielded a well defined differential pulse polarogram as shown by Curve 1 in Figure 1. The peak current in KC1 supporting electrolyte was also very pH dependent as shown in Figure 2. The pH chosen for the present study was 1.7, since this value yielded maximum current. At this pH, our data gave a diffusion coefficient of 9.0 X lod6 cm2/sec. which agrees quite well with previously reported values (4, 11). This same pH was used for nitrosoproline although its current vs. pH dependency was not studied in
NO. 11, SEPTEMBER 1975
~~
DMN concn in CHC12, P P ~
259 64.8 16.2 4.05
~~~
Recovery, %
97.8 101.4
+
100.0
+
94.6
DIMN:NOZ‘~
0.5
* 0.7 +
~~~~
Table 11. Percent E r r o r in the Determination of DMN in the Presence of Varying Amounts of Nitrite Ion
Table I. D M N Recovery from Methylene Chloride
DC polarography
...
...
1:0.60
0.4 1.4 2.8 4.2 5.6 7.8 2 0 .o
1:l.O
4 1 .O
.o
56.6
2.6 6.4 16.6 25.4 34.5 43.9 98.3 127.0 162 .O
l:o
0.5
1:0.05
0.7
1:O.lO 1:0.20
this investigation. This is reasonable since nitrosamines usually yield maximum currents a t low pH (10). As noted below, nitrosoproline yielded approximately the same results as DMN at this pH. At p H 1.7, the peak potential for nitrosoproline was -0.77 V. At pH 1.7, DMN and nitrosoproline both yielded well defined peaks in separate solutions as well as in mixtures. The curve for the mixture was simply the sum of the individual curves. Working curves of peak current vs. concentration were established for DMN in aqueous 0.1N KC1 and in rat serum extract. Nitrosoproline was studied in 0.1N KC1 only. The curve for DMN was linear over the range 100 ppm to 12 ppb. The standard deviation from linearity was 0.9% for fourteen different concentrations. Each concentration was run a t least four times. The precision from 100 ppm to 100 ppb was, in all cases, less than 1%.In four equal increments from 100 ppb to 12 ppb, the standard deviation of the precision increased from 1 to 10%. The lower limit corresponds to a concentration of 1.7 X lO-’M. Nitrosoproline yielded exactly the same results in terms of linearity and precision from 150 ppm to 10 ppb. The lower limit in the case of nitrosoproline corresponds to a concentration of 7.0 X 10-8M. Lower concentrations were not studied since the precision was obviously deteriorating at lower levels. Signal averaging or other computerized techniques would undoubtedly improve this situation. The lower limits are set by instrumental considerations in the case of nitrosoproline and the presence of trace amounts of oxygen in the case of DMN. The signal-noise ratio of the Model 171 is unfavorable at current levels below those correspclnding to
