Environ. Sci. Technol. 1993, 27, 720-724
NChloroaldimines. 4. Identification in a Chlorinated Municipal Wastewater by Gas Chromatography/Mass Spectrometry? Barbara Conyers, Ellzabeth Walker, and Frank E. S~ully,Jr.’ Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, Virginia 23529-0 126
G. Dean Marbury Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina 27599-7400
N-Chloroaldimines are formed in aqueous solution by chlorination of nonpolar a-amino acids with 2 2 equiv of aqueous chlorine at pH 7. Four N-chloroaldimines were identified in chlorinated municipal wastewater with varying degrees of confidence. The compounds were isolated from wastewater by ether extraction and concentrated by Kuderna-Danish evaporation before GC/MS analysis.The presence of N-chloroisobutyraldimine(I),a product of the chlorination of valine, was confirmed in two municipal wastewaters (primary effluents) by comparison of its retention time and E1 and CI mass spectra with those of standards. The identity of N-chloro-3-methylbutyraldimine (11,from leucine), N-chloro-2-methylbutyraldimine (111, from isoleucine), and N-chlorophenylacetaldimine (IV, derived from phenylalanine) in the wastewaters was confident based on a comparison of retention times and CI mass spectra. However, the chromatographic peaks were too small to obtain E1 spectra clear enough to make a confirmed assignment. The identity of IV was tentative since its presence was indicated by its retention time and CI spectrum in the multiple-ion detection mode and was only suggested on a second instrument in the E1 mode.
Introduction
Although disinfection of waters and wastewaters has been practiced for over 70 years, there still remains a poor understanding of the reactions of chlorine with organic compounds in these waters. As a composite of a group of compounds, chloramines are considered to be among the most toxic components of chlorinated sewage to some species of fish, although sensitivity can vary with life stage and body weight ( I , 2). Chloramines also maintain their toxicity longer and generate stronger fish avoidance reactions than free residual chlorine (HOC1+ C10-) (3-7). However,there is confusion associated with these studies, since the term “chloramines” is used to refer to all compounds that respond to conventional analyses as “combined residual chlorine”. Thus, studies which have utilized model solutions of inorganic chloramines (mixtures of NH&l and NHC12) have been used to imply that all compounds which respond to residual chlorine analysis as combined residual chlorine are toxic (3,8-14). The organic chloramines present in chlorinated wastewaters are poorly characterized. However, because of their biogenic nature, N-chlorinated organic amino nitrogen compounds are likely to have a more insidious effect on aquatic life than their inorganic counterparts. N-Chlorinated organic nitrogen compounds can also interfere in the establishment of reliable disinfecting + Part 3: Conyers, B.;Scully, F. E.,Jr. Enuiron. Sci. Technol. 1993,27, 261-266. 720
Environ. Sci. Technol., Vol. 27, No. 4, 1993
conditions in wastewaters. They are not effective bactericides and yet give a positive response to conventional methods of determination of disinfectant concentration (15-23). Recently we characterized a new class of byproducts of the chlorination of amino acids in model solutions and in wastewaters (24-26). N-Chloroaldimines are formed when amino acids react with 2 equiv of aqueous chlorine to form N,N-dichloramino acids. N,N-Dichloramino acids are unstable and decompose by competing pathways (26) to form nitriles and N-chloroaldimines (eq 1). N-Chloro-
-
2HOC1
H,N+CHRCOO-
-+
C1,NCHRCOORC(=NCl)H
RC=N (1)
aldimines respond to residual chlorine analysis in the same way monochloramine does and yet they are stable in the presence of free residual chlorine. They are likely to account for at least part of the unusually stable combined residual chlorine present in wastewaters containing free residual chlorine. Our early experience with N-chloroaldimines (24) suggested that they were only stable in dilute aqueous solution and would not withstand the rigors of conventional methods of concentration and gas chromatographic or GC/ MS analysis. This belief limited our approach to the identification of these compounds in wastewaters to the use of radiotracers. Our most recent experience with N-chloroaldimines has shown us that these compounds can be chromatographed and identified by GC/MS (25, 26). As an extension of this work we have characterized four N-chloroaldimines by electron impact (EI) and chemical ionization (CI) mass spectroscopy and performed exact mass measurements on them. Using information from these studies, we then analyzed concentrated ether extracts of chlorinated wastewaters and identified four N-chloroaldimines in these extracts. Experimental Section
General Information. All chemicals were reagent grade or better. Amino acids were obtained from Sigma Chemical Co. Reagents and methods used for the analysis of residual chlorine by the DPD ferrous titrimetric method are described elsewhere (method 408 D) (27). All aqueous reagents, prepared and standardized as previously described, were made using chlorine-demandfree (CDF) water (28, 29). Instrumentation. All GUMS analyses were carried out on 32 mm i.d. X 30 m DB-5 fused-silica capillary GC columns with a 0.25-pm film thickness (J&W Scientific). GC/MS 1, operated in the CI mode, has been described previously (25). In the full-scanmode spectra were scanned from m/z 56 to 200 each second (15 microscans). The 0013-936X/93/0927-0720$04.00/0
0 1993 American Chemical Society
instrument was also operated in the multiple-ion-detection (MID) mode to detect N-chlorophenylacetaldimine in wastewaters using m/z 91,118,154 (MH+),and 156 (MH + 2)+. GC/MS 2 was a Hewlett-Packard Model 5890 gas chromatograph interfaced to a VG70-250SEQ mass spectrometer (resolution 1000or 10 000; 10%valley definition). In the full-scan mode (resolution lOOO), the magnet was scanned from 550 to 45 amu at 1 s/decade. The spectrometer was operated in the electron impact (EI) and isobutane chemical ionization (CI) modes. Elemental compositions of amino acid chlorination products were derived from accurate mass measurements. For accurate mass measurements, a low-resolutiontechnique employing perfluorokerosene as an internal reference compound was used, enabling operation at full sensitivity. Selected ion monitoring of the (M - 1) and [(M - 1) + 21 ions was performed for each N-chloroaldimine. The chromatographic parameters were the same for both systems. The two methods used to isolate the chlorination products were headspace analysis and ether extraction. Compounds isolated by headspace analysis were chromatographed using a splitless injection and a temperature program of 40 "C for 0.5 min followed by a temperature rise of 10 "Cirnin to 220 "C. An injector temperature of 220 "C was used on GC/MS 1while an injector temperature of 250 "C was used on GUMS 2. Chloroaldimines isolated by ether extraction were chromatographed using a split/splitless injection and a temperature program of 40 "C for 3 min and a rise of 5 "C/min to 170 "C. This enabled separation of the N-chloroaldimines formed from leucine and isoleucine. Because of the thermal lability of N-chlorophenylacetaldimine, it was chromatographed with an initial temperature of 60 "C followed immediately by a 10 "C/ rnin rise to 170 "C. Preparation and GUMS Analysis of Model NChloroaldimines. The mass spectrometric analyses of the chlorination products of the amino acids valine, isoleucine, and leucine were performed using headspace analysis. Model solutions of isoleucine,leucine, and valine, M, were prepared in 0.025 M monobasic sodium 1.43 X phosphate (pH 7.0withNaOH). Aliquots were chlorinated to a Cl/N molar ratio of 2.0 before analysis. The amino acid solutions were stirred constantly while being chlorinated, sealed in serum vials, and incubated for 30 rnin in the dark. The sealed vials were then heated for 15 rnin in an 80 "C water bath, and 25-50 pL of headspace was analyzed by GUMS. The N-chloroaldimine product of the chlorination of phenylalanine was analyzed by ether extraction. The amino acid solution (15 mL, 1.43X M) was chlorinated to a Cl/N molar ratio of 2:l and incubated for 30 min in the dark. The mixture was extracted with 3 mL of freshly distilled ethyl ether before GC/MS analysis. Description and Handling of Wastewater. The wastewaters used in this study were samples of primary effluentobtained on November 18and December 10,1991, from a typical secondary treatment plant described in previous work as plant 2 (30). Handling of samples has been described previously (30,311. Total Kjeldahl nitrogen (TKN) and ammonia concentrations were determined by the Hampton Roads Sanitation District Commission (HRSD) laboratory according to published standard procedures (27).
100
20
1
0 0:OO
1:00
300 4:OO Retention Time (min)
200
5:OO
6:OO
Figure 1. Total ion chromatogram of the volatile chlorination products of an isoleucine model solution (1.43 X M in 0.025 M sodium phosphate, pH 7.0) chlorinated to a CVN molar ratio of 2.0, showing 2-methylbutyronltrile (retention time, 2.4 min) and Kchloro-2-methylbutyraldimine (retention time, 3.9 min).
GC/MS Analysis of N-Chloroaldimines in Chlorinated Wastewater. To 1 L of wastewater was added 3.45 g of monobasic sodium phosphate to produce a 0.025 M solution, and the pH was adjusted to 7 with sodium hydroxide. While being stirred, the solution was chlorinated to 210 mg/L and incubated in the dark at ambient temperature for 30 min. Potassium chloride (60 g) was added to the wastewater before it was extracted with freshly distilled ether (3 X 65 mL). The ether extracts were combined and concentrated to 10 mL by KudernaDanish evaporation at 45-50 "C. The residual was concentrated further to a final volume of 1 mL under a stream of dry nitrogen gas and stored over a small amount of anhydrous sodium sulfate. The concentrates were stored in a freezer (-4 "C) until 1-2-pL aliquots were analyzed by GC/MS. Results and Discussion
Electron Impact Mass Spectra. When separate solutions of the amino acids valine, leucine, isoleucine, and phenylalanine were chlorinated at pH 7 with 2 equiv of aqueous chlorine, GC analysis of each solution revealed the formation of two volatile products (Figure 1)associated with the chlorination of that amino acid. The mass spectrum of the peak which eluted earlier in each chromatogram matched that of an authentic sample of the nitrile that was expected by the breakdown of each NJV-dichlorarnino acid (see eq 1). The spectrum of the later peak was due to the corresponding N-chloroaldimine. The major ions in the electron impact (EI) mass spectrum for each chloroaldimine are listed in Table I. A chlorine isotope cluster was observed in the spectrum of N-chlorophenylacetaldimine(IV), but was not found in the other E1 spectra (Figure 2). The E1 mass spectrum of N-chloroisobutyraldimine (I) instead contained a chlorine isotope cluster corresponding to (M - H).+ Aliphatic nitriles, compounds closely related to N chloroaldimines, also exhibit negligible parent ions, and the (M - 1)+ion is generally more abundant than the (M)+ ion (32). The mass spectra of N-chloro-3-methylbutyraldimine (11)andN-chloro-2-methylbutyraldimine (111)(MW = 119) each contained an unusual cluster of ions around the mass corresponding to the molecular weight (Table 11). Exact mass measurements on the cluster of ions derived from I11 Environ. Sci. Technol., Vol. 27,
No. 4, 1993 721
Table I. Major Ions in the 70-eV Electron Impact Mass Spectra of N-Chloroaldimines Formed on Chlorination of Amino Acids and Wastewater
m/z (re1 intens)
compd (precursor) (CH&CHCH=NCl (I) (valine) (CH3)&HCH2CH=NC1 (11) (leucine) CH3CH&H(CH3)CH=NCl(III) (isoleucine) CsH5CH2CH=NC1 (IV) (phenylalanine)
N-chloroisobutyraldimine(I) (wastewater)
106 (6), 105 (l),104 (15), 91 (6), 89 (16), 77 ( E ) , 74 (6), 70 (9),69 (loo), 68 (Id), 67 (21), 59 (lo), 55 (IO), 54 (6), 53 (44), 51 (8) 122 (2), 120 (5), 118 (ll),106 (29), 105 (8), 104 (87), 79 (32), 77 (80), 70 (8), 69 (13),68 (lo), 64 (5), 62 (7), 57 (31), 56 (6),55 (7), 44 (6), 43 (loo), 41 (77), 40 (15), 39 (48), 38 (11),37 (5) 122 (5), 120 (16), 118 (12), 106 (4), 104 (12), 93 (40), 92 (lo), 91 (89), 90 (14), 84 (26), 82 (5), 79 (17), 78 (6), 77 (40), 70 (13), 69 (28), 68 (17), 67 (lo), 64 (1%62 (20), 57 (67A 56 (73L ~ (38), 41 (loo), 40 (15L 39 (53), 38 (15) 55 (76), 54 (48),53 (12), 52 (13), 51 ( 1 1 ~ 4 (3 1 3 42 155 (6), 153 (17), 120 (16), 119 (81), 118 (94), 117 (loo), 116 (88), 104 (13),102 6% 93 (1% 92 (74),91 (99), 90 (91),89 (87), 88 (18), 87 (IS), 86 (IO), 78 (18), 77 (45A 76 75 (19), 74 (22), 66 (13), 65 (78), 64 (38), 63 (75), 62 (39), 61 (15), 53 (7), 52 (26), 51 (TI),50 (50) 106 (4), 105 (l),104 (12), 91 (5), 89 (15), 76 (2), 70 ( l l ) , 69 (loo), 68 (IS), 67 (25), 63 (8), 55 (ll),54 (12), 53 (70), 52 (8),51 (20)
Table 11. Exact Mass Measurements of the Major Ions Associated with N-Chloroaldimines compd (precursor)
major ion
calcd mass of parent ion
exact mass found
I (valine) I1 (leucine) I11 (isoleucine) IV (phenylalanine)
(M - H)+ (M - H)+ (M - H)+ M+
104.0256 118.0424 118.0424 153.03453
104.0267 118.0445 118.0428 153.0341
100
80
100 80
,+
60
118,0019
1
20 0 116
60
70
80
90
100
110 120 130 140 150
d z
Flgure 2. Electron impact mass spectrum of I derived from the chlorination of valine at pH 7.0.
(resolution, 6454; Figure 3) indicated the presence of a mixture of two chlorine isotope clusters, one corresponding to (M - H)+ (m/z 118.0019 and 119,9990)and the other corresponding to (MH)+(m/z120.0177and 122.0147).The presence of both of these clusters appears to be the result of an ion-molecule reaction taking place in the mass spectrometer, like that which aliphatic nitriles undergo (32). Major ions present in the mass spectra of all the N-chloroaldimines can be explained by the fragmentation of 11: 5 7h
3
7
CH,
j
I
I
CH,-CH-CH~+C I 4 104/106
!
1
N-CI
1 1
118/120
t
-t-----" H
In addition, a McLafferty rearrangement results in the loss of propylene to produce the mlz 77 and 79 cluster. For compounds which cannot undergo a McLafferty rearrangement (I, IV), the base peak corresponds to the loss of HC1 to produce the nitrile cation radical. Chemical Ionization Mass Spectra. Major ions in the isobutane and methane CI mass spectra of each Environ. Scl. Technol.. Vol. 27, No. 4, 1993
122.0147 119.0062
121.0619
123.0769
I
117
118
119
120
121
122
123
m/z
Flgure3. Accuratemass measurementof major ions in the E1spectrum of 111 in the range mlz 117-123.
loo] 80 yH3 N-CI CH~-CH-C@ 'H
i2o.oin
40
g -
,
50
722
-E
I 1 0 0 200 300 4 0 0 500 6 0 0 700 800 900 10001100 Retention Time (min)
Flgure 4. Total ion (EI) chromatogram (obtalned on GC/MS 2) of a concentrate of an ether extract of a sample of wastewater collected on Nov 18, 1991, and chlorinated to 210 mglL aqueous chlorine (pH 7.0). Retentiontimes of Kchloroaldimlnes: I, large peak at 2.95 min; If, 6.8 min; 111, 6.9 min.
chloroaldimine are listed in Table 111. One of the major fragmentation processes evident in all CI spectra is loss of HC1 by a @-eliminationfrom the (MH)+ion to produce a protonated nitrile.
-
-HCI
RCH=HN+C~ RC=N+H Use of isobutane as the ionizing gas gave a higher abundance of the parent ions of IV than methane. Thermal Lability of N-Chloroaldimines. Analyses of N-chlorophenylacetaldimine were only successful on the magnetic sector instrument after on-column injection was employed. Subsequently, it was recognized that all of the chloroaldimines studied are sensitive to GC injection temperatures. A study of the chlorination products of isoleucine revealed that with an injection temperature of 250 "C no N-chloroaldimine was observed and that, as the injection temperature was lowered, a chromatographic peak for the compound appeared and grew in height until a maximum was reached at injection temperatures of 1200 "C. It also appeared that N-chlorophenylacetaldimine decomposed to some extent on the column, since the faster
Table 111. Major Ions in the 100-eV Chemical Ionization Mass Spectra of N-Chloroaldimines Formed on Chlorination of Amino Acids and Wastewater m/z (re1intens)a compd (precursor)
VG70-250SEQ spectrometerb
Finnigan Model 800 ion trap spectrometer'
108 (33),106 (loo), 72 (13),71(9),70 (29), 56 (9),55 (la), 52 (16) 108 (25), 106 (loo), 72 (49), 70 (90) 122 (30), 120 (loo), 119 (5),86 (20),85 (IO), 84 (26),69 (20), 122 (32), 120 (loo), 86 (46), 84 (63), 64 (121, 58 (41), 57 (94) 77 (33), 70 (13), 69 (15) 122 (31), 120 (loo), 91 (29), 86 (43),84 (60), 70 (13) 122 (37), 120 (loo), 86 (12),85 (a), 84 (21), 58 (28),57 (57) I11 (isoleucine) 156 (2), 154 (5), 119 (6), 118 (63),117 (Id), 92 (IS), 91 (100) IV (phenylalanine) 156 (37), 154 (94), 120 (63), 119 (34), 118 (loo), 117 (23), 93 (la), 92 (37),91 (68), 69 (13) 108 (34), 106 (loo), 77 (4),72 (lo), 71 (6), 70 (16), 56 (3),55 (2),52 (10) I (wastewater) NAd 122 (34), 120 (loo), 86 (7), 84 (32),69 (17),64 (11), 58 (23), 57 (56) I1 (wastewater) NAd 122 (33), 120 (loo), 86 (5),85 (7),84 (16),76 (11): 58 (26), 57 (65) I11 (wastewater) NAd 156 (l),154 (4), 118 (72), 91 (80) IVI (wastewater) NAd a Except for parent ions, only intensities of > l o % reported for standards. Ionizing gas was isobutane. c Ionizing gas was methane. d Concentrates of wastewater extracts were not analyzed by CI MS on this instrument. e Ions with abundance of > l o % not found in standards. f N-Chlorophenylacetaldimine was detected only in the MID scan mode monitoring only the four ions listed. I (valine) I1 (leucine)
Table IV. Chemical Characteristics of Primary Wastewater Effluent from Plant 2, Norfolk, VA selected amino acids and other chem params valine phenylalanine 1eu cine isoleucine chloramine maximum breakpoint total Kjeldahl nitrogen (as N), 19.8 mg/L ammonia nitrogen (as N)
wastewater concn 1" 26 1.3pM 0.39 pM 0.29 pM 0.38pM 120 mg/L 200 mg/L 24.05 mg/L 18.5 mg/L
0.36pM 0.50 pM 0.21 pM NAc 120 mg/L 200 mg/L 24.38 mg/L 18.5 mg/L
Nov 18,1991. Dec 10,1991. Because ofinterference, thisamino acid could not be analyzed in this sample. a
l o80 o]
20 - 51
-
104
I,,,
.
,
.
,I
I.
,.
mlz
Figure 6. E1 mass spectrum of I in wastewater as determined on GC/MS 2.
200 3:22
I""~"'~I"~'""'I"''""'I""""'I 300 400 500 600 501 6:41 8:21 1001
700 11.41
Scan Number (time In mln)
Figure 5. Total ion (Cl)chromatogram (obtained on GC/MS I) of a concentrate of an ether extract of a sample of wastewater collected on Nov 18, 1991, and chlorinated to 210 mg/L aqueous chlorine (pH 7.0). Retention tlmes of Kchloroaldimines: I, 5.1 min; 11, 8.0 min; Ill, 7.8 mln.
the compound was eluted from the column, the greater the chromatographic peak area. N-Chloroaldimines in Wastewaters, Characteristics of the two wastewaters used in this study are given in Table IV. Each was chlorinated to 205-210 mg/L Clz to produce a free residual chlorine level of
