(5) A. Katchalsky and P. F. Curran, "Nonequilibrium Thermodynamics in Biophysics," Harvard University Press, Boston. MA, 1965. (6) L. C. Craig and A. 0. Pulley, Biochemistry, I, 89 (1962). (7)B. K. Fritzinger, S.K. Brauman, and D. J. Lyman, J. Biomed. Mater. Res., 5, 3 (1971).
RECEIVEDfor review September 3, 1974. Accepted Febru-
ary 3, 1975, Significant portions of this paper were presented a t the Symposium on Membranes in Separation Processes, Case Western Reserve University, Cleveland, OH, May 8-10, 1973. This work was supported under National Science Foundation Grant No. GH-38996X.
Description of the Thermal Energy Analyzer (TEA) for Trace Determination of Volatile and Nonvolatile N-Nitroso Compounds David H. Fine,' Firooz Rufeh, David Lieb, and David P. Rounbehler Thermo Electron Corporation, 85 first Avenue, Waltham, MA 02 154
Many N-nitroso compounds are known to be carcinogenic ( 1 , 2 ) , and there is much concern about their possible widespread occurrence in the environment (3).Analytical methods, sensitive a t the pg/kg level in the original material, are available for the more volatile N-nitrosamines (4, 5 ) . The procedures involve concentration and cleanup by distillation and/or extraction, followed by separation by gasliquid chromatography (GLC) and detection by either the Coulson electrolytic conductivity or the alkali flame ionization detectors (6).Because they are nonspecific to N-nitroso compounds, the GLC detectors are useful for screening purposes only; confirmation by GLC-mass spectrometry is still mandatory (7, 8). Judging from the wide variety of complex secondary amines occurring in nature, a large group of naturally-occurring high molecular weight N-nitroso compounds is to be expected. As a result of their high molecular weight, they would be nonvolatile, and not amenable to GLC analysis or to cleanup by steam or vacuum distillation. For this reason, adequate methods are not yet available for the analysis of nonvolatile N-nitroso compounds. A new detection technique, called the Thermal Energy Analyzer (TEA), which is both highly sensitive and highly selective to all N-nitroso compounds, has been reported recently (9, 10). We report here on a practical TEA system which may also be used as a detector for gas-liquid chromatography (11). The scope and limitations of the technique are described in terms of sensitivity to different Nnitroso compounds, and quantitative limitations are placed on selectivity and performance. The detector as described here is an analytical tool capable of selectively detecting sub-pg/kg amounts of N-nitroso compounds in complex biological materials and foodstuffs. The fundamental principles and the theoretical concepts underlying the technique are described elsewhere (12).
EXPERIMENTAL Description of the TEA. A dilute solution containing the N nitroso compound is injected directly into a catalytic pyrolyzer. T h e pyrolyzer inlet is constructed in a manner similar to a gas chromatograph injection port, with provision for preheated carrier gas, a septum, and electrical heaters for maintaining the temperature in excess of 275 "C. A schematic of the TEA is shown in Figure 1. At the same time as vaporizing the solvent, the N-nitroso compound is decomposed into a nitrosyl radical and an organic radical. The organic fragment either decomposes further (13, 1 4 ) or rearranges to give a stable product, which together with the solvent vapor and the nitrosyl radical are swept through a capillary Author to whom correspondence should be addressed. 1188
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ANALYTICAL CHEMISTRY, VOL. 47, NO, 7, JUNE 1975
restriction into an evacuated reaction chamber, maintained a t reduced pressure by a 140 l./min rotary vacuum pump. Ozone, formed by high voltage electric discharge (7500 volts across a 1-mm glass dielectric) in oxygen, also enters the reaction chamber through a capillary restriction, where it reacts with the nitrosyl radical yielding electronically excited NO**. The excited NO** rapidly decays back to its ground state, with the emission of light in the near infrared region of the spectrum. T h e intensity of the emission is detected by means of a S-20 photomultiplier tube (RCA 3000 CN), in conjunction with a red optical filter (Corning CS2-60). T h e photomultiplier response is amplified electronically and displayed on a chart recorder. In order to minimize photomultiplier tube dark current, the tube is kept in a shieldfd housing maintained a t -20 "C. Chemicals. Standard solutions containing approximately 1 pg N-nitroso compound per ml solvent were made up gravimetrically. Depending on the solubility and stability of the particular N-nitroso compound, either ethanol or dichloromethane was used as the solvent. Compounds for interference testing were chosen for one of several reasons: because of their structure and the presence of either NO? or NO functional groups; because of their great abundance in many foodstuffs and biological materials; because they may be formed in the TEA by rearrangement and pyrolysis of parent species; or because of their frequent use as solvents either in gas chromatography or in high performance liquid chromatography. A stock solution containing 0.1 pg of N - nitrosodiphenylamine per ml of dichloromethane was prepared. Using the stock solution as the solvent, 1%solutions of the compounds to be tested were made u p gravimetrically. If the compound was not soluble in dichloromethane, another solvent was used, both for the standard solution and also for the test solution. All the solvents, the N-nitroso compounds, and the potential interferences were used as received, without further purification. Procedure. As in gas chromatography, 1- to 10-pl liquid samples were introduced, by means of a hypodermic needle, directly into the flash vaporization chamber of the TEA.
RESULTS The integrated TEA response to approximately 1 pg/ml solutions of twelve different N-nitroso compounds in ethanol is shown in Table I. Calibration curves for five N-nitroso compounds of different chemical structure (N-nitrosodimethylamine, N-nitroso-N-ethylaniline, 9-nitrosocarbazole, N-nitroso-Nmethyl urethane, and N-methyl-N-nitroso-N-nitroguanidine) are shown in Figure 2. Table I1 is a listing of those compounds which gave no detectable response on the TEA (a N-nitroso compound to interference ratio of greater than 10,000 to 1). No compounds have been found which give a negative response. Table I11 lists those compounds for which a positive response was observed. The magnitude of the response is re-
Table I. TEA Response Factors for Different N-Nitroso Compounds (5-11 injections) N -Nitroso compound
N-nitrosodimethy lamine N-nitrosodiethy lamine
N-Nitrosodipropylamine N-Nitrosodiphenylamine N-Nitroso-N-ethylaniline 9 -Nit rosocarbazo le N-Nitroso-N-methyl urethane N-Nitroso -N-phenylbenzylamine E thyl-N-nitrososarcosinate N-Methyl-N-nitroso-,V-nitroguanidine N-Nitrosopiperidine Dinitrosopiperazine
Concn,
Measured response,
Response per
Relative response,
Mol wt
udml
integrated units
nitrosyl group
nitrosyl mole basis
74 102 130 198 150 196 132 212 146 147 114 144
0.964 1.07 0.84 1.86 1.17 1.99 0.52 2.10 2.15 4.71 0.93 1.99
235 2 04 128 189 145 167 74 166 258 590 172 434
18.1 19.4 19.8 20.1 18.6 17.6 18.8 16.7 17.5 18.4 21.2 15.7
1.07 1.09 1.11 1.03 1.03 1.04 0.92 0.97 1.02 1.17 0.87
1 .oo
Figure 1. Schematic of the thermal energy analyzer
ported on a mole basis as a response ratio ( R R ) ,where:
(RR)= TEA r e m o n s e f r o m mole of comDound TEA r e s p o n s e f r o m mole of N-nitroso compound An RR of 1 would indicate that the TEA response is the same as that for a N-nitroso compound. A response of, say, 0.0004 would mean that the TEA response was 1 X lo-* times what a N-nitroso compound would have given a t the same mole concentra t'ion. DISCUSSION Response to N-Nitroso Compounds. The TEA response characteristics on a weight N-nitroso compound per unit volume of solvent basis vary markedly (see Table I). However, the relative mole response per nitrosyl radical (correcting the integrated value for molecular weight and the number of nitrosyl groups in the molecule) is seen to be essentially constant. The N-nitroso ureas are unstable in ethanol, and their response was instead evaluated using dichloromethane as the solvent. Again, a relative response factor close to unity was observed. The largest variation observed was &15%,which was obtained between dinitrosopiperazine and N-nitrosopiperidine. Although the differences between N-nitroso compounds are relatively small, a difference of f 1 5 % is probably too large to be explained by experimental error alone, The reason for some of the small difference may reside in competition for the nitrosyl radical nitrogen between different chemical paths, leading to Nz, N20, diazo, or other compounds (15, 16). Despite these small differences, the essential predictability of the TEA response to N-nitroso compounds having vastly differing chemical structures is established. Because of the wide variety of the functional groups R1 and Ra in the general formula:
Figure 2. Calibration for N-nitrosodimethylamine (0),N-nitroso-Nmethyl urethane (O), N-methyl-N-nitroso-N-nitroguanidine(A),N-nitroso-Nethylaniline (O),and 9-nitrosocarbazole (W)
which have been evaluated, and because all N - nitroso compounds which have been tested give a positive response on the TEA regardless of the nature of R1 and R2, it may reasonably be assbmed that the TEA will respond to all N-nitroso compounds. For the N-nitrosamines, calibration has already been shown to be linear over six orders of magnitude (IO). The linearity of the calibration for five different N-nitroso compounds in the concentration range from 10 ng/ml to 100 pglml is shown in Figure 2. I t may reasonably be assumed that the TEA response will likewise be linear for other Nnitroso compounds. Potential Interferences. The only compounds found so far which give a rapid TEA response, similar to that observed for a N-nitroso compound, are organic and inorganic nitrites and the unstable compound 2,2',4,4',6,6'-hexanitrodiphenylamine (see Table 111). Introduction of nitrates causes an erratic response, which changes the base line. The response from 10 p1 of an aqueous solution containing 1 mg sodium nitrate per ml can last from several hours to several days. I t is believed to be due to the relatively nonvolatile compound slowly decomposing inside the hot injection port. The small TEA response observed for other compounds listed in Table I11 may be due to the compound itself, or it may be due to impurities. Some of these possibilities are discussed below: ANALYTICAL CHEMISTRY, VOL. 47, NO. 7 , JUNE 1975
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Table 11. Representative List of Compounds Which Were Found T o Give No Interference on the T E A Acetic acid Ethyl carbamate Oxalic acid Acetone Ethylene glycol 12-Pentane Acetonitrile Fluorobenzene Phenyl hydrazine Alizarin red Gasoline d, Z-Phenylalanine Ammonia (gas) Glycerol p-Phenylazoaniline Benzene d-Glucose Phosphoric acid Benzylsalicylate Glutamic acid Propane @as) 2 -Butoxy ethanol n-Hexane Pyridine Carbon dioxide Hydrogen (gas) Quinine Carbon disulfide Hydroquione Sodium acetazolamide Carbon monoxide (gas) 8 -Hydroxyquioline Sulfadiazine Carbon tetrachloride Inosine Sulfanilic acid Chloral hydrate d, 1-iso-leucine Tetrahydrofuran Chlorobenzene Methane (gas) Theophylline 1-C hloropropane Methyl acetate Toluene 2 -C hloropropane N-Methyl bisacrylamide 2,4,6 - Trichlorophenol Cyclohexane 2 -Methyl butane 2,2,4 - Trimethylpentane d, 1-Tryptophane Cyclopentane Methyl formamide 1,2-Dichloroethane Methyl isobutyl ketone Urea 2,3 -Dichloropropane Methyl orange Uric acid Diethy lether Methyl red Urethane Dimethylamine (gas) Naphthalene Water p-Dioxane Nitrogen (gas) Xylene Diphenylamine Nitrobenzene Ethyl acetate o-Nitrotoluene ~~~
~~~
Table 111. Mole B a s i s Response Ratio for Those Compounds Which Give a Detectible Response on the T E A Compound
N-Nitroso compounds 2,2',4,4', 6,6'-hexanitro diphenylamine Isopentylnitrite d, Z-Cyclohexylaminenitrite Pentylnitrite Sodium nitrite (in H,O) Sodium nitrate (in H20) Nitric acid Dimethyl sulfoxide Hydrazine (95%) 5 -Nitrouracil p-Nitrosodiphenylamine 3 -Nitrophthalidimide Nitromethane Ammonium hydroxide Dimethylglyoxime Dimethy lamine hydrochloride Diphenyl carbazone Aniline 2 -Nitroso-1 -naphthol
Response ratio (RR)
1.o 1.4 1.o 1.o 1.o 1 .o
-1.0 (very slow response lasting from hours to days) -1.0 (very slow response lasting from hours to days) 0.03 0.03 0.017 0.0050 0.0030 0.0018 0.0016 0.0010 0.0009 0.0007 0.0003
0.0002
P-Nitrosodiphenylamine (Baker Chemical) was recrystallized from hot benzene and air-dried. The T E A response ratio after recrystallization was found t o decrease from 0.005 t o 0.002, indicating that the observed TEA response was due t o an impurity. A likely impurity would be diphenylnitrosamine, formed as a by-product during manufacture. Dimethylamine Hydrochloride (Baker Chemical) was recrystallized from hot chloroform. The T E A response ratio was observed t o decrease from 0.00092 t o 0.00035. Dimethylamine gas itself (Matheson Gas Products) does not 1190
ANALYTICAL CHEMISTRY, VOL. 47, NO. 7, JUNE 1975
give a response on the TEA, reinforcing the possibility of a small impurity being the cause of the interference. T h e method used to prepare the dimethylamine hydrochloride is unknown, but it is of interest t o note that a commonly used procedure used to be catalytic hydrogenation of N nitrosodimethylamine. Dimethyl-sulfoxide (Baker Chemical) is prepared by the air oxidation of nitrogen oxides. It is possible that traces of nitric oxide remain in the dimethyl sulfoxide. Dimethylglyoxime (Baker Chemical) has a TEA response equivalent to 0.1% mole impurity. Iso-amyl nitrite may be the impurity, for it is used as the starting material in the bulk synthesis of dimethylglyoxime. Selectivity testing for classes of compounds which might cause a n interference can never be complete, and whatever the number of compounds which have been tested, it can never be exhaustive enough. This is particularly true for the TEA detection system which relies for its selectivity not on a unique fundamental physical phenomenon, but rather on discrete chemical and physical processes occurring in a prescribed sequence. Selectivity. The first step in the TEA requires that the compound be flash pyrolyzed in the presence of a catalyst, the temperature being in the range 275-300 OC, with the release of a nitrosyl radical. Many compounds may pyrolyze in this temperature range, but only a compound which already possesses a labile nitrosyl group will release a nitrosyl radical. Other fragment species will also, naturally, be formed. The nitrosyl radical is then swept into a chamber where it reacts with ozone to produce light in the near infrared region of the spectrum. Other compounds such as ethylene and carbon monoxide also react with ozone t o produce a luminescence, but the wavelength of their luminescence is in the blue region of the spectrum. There is no fundamental reason why other compounds should not react with ozone to produce a n infrared luminescence, but to date none has been found. If another compound or compounds are found which react with ozone t o produce a luminescence in the infrared, their effect may be eliminated by interposing a cold trap between the catalytic pyrolyzer
and the ozone reaction chamber. At a temperature of -154 OC, the vapor pressure of the nitrosyl radical is greater than one atmosphere, whereas the vapor pressure of almost all organic compounds except for methane, ethane, acetylene, etc., is substantially less than one atmosphere. Thus, if a cold trap were used, the only possible source of interference would arise from compounds with labile nitrosyl groups. The fact that reasonable recoveries of N - nitrosamines a t the sub-100 pg/kg concentration level have been obtained from foodstuffs ( 1 0 ) gives further evidence for the apparent selectivity of the technique.
ACKNOWLEDGMENT We are indebted to F. Campagna of Thermo Electron Corporation for technical assistance. We thank E. K. Weisburger of the National Cancer Institute, Bethesda, MD, for the N-nitroso compounds used in this study.
LITERATURE CITED (1) P. Magee and J. Barnes, Adv. Cancer Res., 10, 164 (1967)
(2) H. Druckrey. R . Preussman, and S. Ivanokovic. Ann. N.Y. Acad. Sci., 163, 676 (1969). (3) W. Lijinsky and S.S.Epstein, Nature (London), 225, 21 (1970). (4) J. M. Essigmann and P. issenberg, J. Food Sci., 37, 684 (1972). (5) W. T. Iwaoka, M. S.Weisman. and S. R. Tannenbaum, Third Meeting on the Analysis and Formation of N-Nitroso Compounds, Lyon, France, Oct. 1973, to be published by IARC, Nov. 1974. (6) J. F. Palframan, J. McNab. and N. T. Crosby, J. Chromatogr., 76, 307 (1973). (7) G. M. Telling, T. A..Bryce, and J. Aithorpe, J. Agric. Food Chem., 19, 937 (1971). (6) T. A. Gough and K. S.Webb, J. Chromatogr., 79, 57 (1973). (9) D. H.Fine, F. Rufeh, and B. Gunther. Anal. Lett., 6, 731 (1973). (10) D. H. Fine, F. Rufeh, and D. Lieb, Nature (London), 247, 309 (1974). (1 1) D. H. Fine and D. P. Roubehler, J. Chromatogr., In press (1975). (12) D. H. Fine, D. Lieb, and F. Rufeh, J. Chromatogr., 107,35~~-fl9757.-~ (13) E. M. Burgess and J. M. Lavanish, Tetrahedron Lett., 20, 1221 (1964). (14) C. H. Bamford, J. Chem. Soc., 12 (1939), (15) E. H. White and R . J. Baumgarten, J. Org. Chem., 29, 2070 (1964); 20, 3636 (1964). (16) E. H. White and C. A. Aufdermarsh. J. Am. Chem. SOC., 83, 1174 (1961).
RECEIVEDfor review December 23, 1974. Accepted January 28, 1975. This work was performed pursuant to Contract NOlCP45623 with the National Cancer Institute, U S . Department of Health, Education, and Welfare.
Application of the Methylthymol Blue Sulfate Method to Water and Wastewater Analysis J. M. Adamski and S. P. Villard Ontario Ministry of the Environment, P.O. Box 213,Rexdale, Ontario, Canada
The metallochromic indicator Methylthymol Blue, as described by Korbl and Pribil (I, 2) received much attention when Lazrus, Hill, and Lodge ( 3 ) applied it to an automated spectrophotometric technique for the determination of sulfate ion in rainwater. The gravimetric method is presently the specified standard sulfate method for water and wastewater analysis ( 4 ) ; however, the tedious, time-consuming nature of this method presents a definite disadvantage to environmental laboratories which must handle a large number of samples. The faster Methylthymol Blue technique is more adaptable to routine laboratory procedures if it can be applied to these same types of samples. A comparative study was therefore conducted to determine which technique was analytically preferable when applied to a series of water and wastewater samples.
EXPERIMENTAL Apparatus. A modified version of the Methylthymol Blue technique as described by Lazrus et al. (3)was employed using a Technicon AutoAnalyzer I system with the appropriate accessories described in Figure l . T h e ion exchange column designated in this figure is constructed from a 23-cm glass tube with a n internal diameter of 2 mm. I t was packed with Amberlite IR-120 cationic exchange resin and plugged on both ends with glass wool. T h e premixer units, also shown in Figure 1, were constructed from short lengths of Solvaflex tubing inserted into each other to form a chain. All the appropriate apparatus for performing a gravimetric analysis is discussed in “Standard Methods” ( 4 ) . Reagents. T h e Methylthymol Blue colorimetric reagent was prepared by dissolving 0.1182 g of Methylthymol Blue [(3,3’bis(N,N- di(carboxymethyl)aminomethyl)thymolsulfone)phthalein pentasodium salt](Eastman Catalog No. 8068), in approximately 100 ml of distilled water. This solution was then mixed with 25 ml of barium chloride stock solution, 4 ml of 1.ON hydrochloric acid,
and 400 ml of 95% ethanol and diluted to 1 1. with distilled water. T h e barium chloride stock solution was prepared by dissolving 1.526 g of barium chloride dihydrate in distilled water and diluting t o 1 1. T h e dilution water used in the analysis contained small amounts of sulfate and a detergent concentrate, BRIJ-35 (Technicon Chemical Formula No. AR 110-62), in various proportions depending on the range of analysis. T h e dilution water required for analysis in the 0-50 mg/l. range was prepared by mixing 3 ml of a 1000 mg/l. sulfate standard and 3 drops of Brij-35 with distilled water and diluting to 2 1. T h e dilution water required for analysis in the 0-200 mg/l. range was prepared by mixing 0.75 ml of a 1000 mg/l. sulfate standard and 3 drops of Brij-35 with distilled water and diluting to 2 1. All the appropriate reagents for performing a gravimetric analysis are described in “Standard Methods” ( 4 ) . Procedure. T h e color reagent used in the Methylthymol Blue technique contained equimolar quantities of Methylthymol Blue and barium ion in an aqueous-ethanol solution adjusted to a p H between 2.5 and 3.0 with 1.ON hydrochloric acid. When a water sample containing sulfate reacts with this reagent, barium sulfate is produced. After allowing sufficient time for the production of barium sulfate, the pH is re-adjusted between 12.3 to 13.0 with 0.18N sodium hydroxide. A t this high pH, any barium ions remaining in solution will complex with available Methylthymol Blue leaving a n amount of uncomplexed Methylthymol Blue in solution which is equivalent to the quantity of sulfate removed as barium sulfate. T h e uncomplexed Methylthymol Blue is then determined colorimetrically. Figure 2 was derived to show the locations of maximum colorimetric sensitivity. These scans were performed on a Unicam 1800 double beam spectrophotometer using 1-cm cells and distilled water as a reference. A wavelength of 460 nm was chosen for all our experiments. Calibration of the AutoAnalyzer system was performed for three sulfate ranges: 0-15 mg/l. (no dilution), 0-50 mg/l. (3-fold dilution). and 0-200 mg/l. as sulfate (18-fold dilution) (See Figure I). Curvature in the calibration was corrected by the addition of sulfate t o t h e dilution water when operating in the 0-50 mgil. and the 0-200 mg/l. ranges. Curvature could not be corrected in the 0-15 ANALYTICAL CHEMISTRY, VOL. 47, NO. 7, JUNE 1975
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