The Analytical Approach Edited by Claude A. Lucchesi
Identification of Organic Compounds in an Industrial Wastewater Ronald A. Hites School of Public and Environmental Affairs and Department of Chemistry 400 E. Seventh St. Indiana University Bloomington, Ind. 47405
Viorica Lopez-Avila Midwest Research Institute 425 Volker Blvd. Kansas City, Mo. 64110
For several years, our laboratory has been engaged in the identification of organic compounds in industrial wastewaters and in rivers (1-6). T h e purpose of these studies has been t o identify the exact molecular struc tures of the compounds present so t h a t the origin, environmental fate, and toxicity of a wide variety of com pound types could be assessed. Our approach has been based on survey analyses ("What is present?") rather t h a n specific compound analyses ("How much of compound χ is present?"). Because the compounds are found at very low concentrations (ppb) and in quite Complex mixtures, we have used gas chromatographic mass spectrometry (GC/MS) as our primary analytical tool. Using mod ern, commercially available instru mentation, G C / M S data can usually be obtained without excessive effort; the interpretation of these data, how ever, is not always so straightforward. In most cases, we have had to collate many different types of information in order to solve these interpretive prob lems. This paper presents such a prob lem and its solution. One industrial wastewater which we have analyzed quite extensively is t h a t of a specialty chemicals plant (3). This plant operates in a batch production mode, generally following a weekly schedule. A wide range of compounds including pharmaceuticals, herbicides, antioxidants, thermal stabilizers, ul traviolet light absorbers, optical brighteners, and surfactants is pro
duced. Water is used in synthesis pro cesses, in the recovery of solvents, in steam jets, and in vacuum p u m p seals. T h e wastewater is neutralized in ei ther of two 1-million-gal equalization tanks, passed through a trickling filter for biological degradation, and clari fied in a 150 000-gal tank with a resi dence time of 3 h. T h e water spills over from the clarifier at a rate aver aging 1.3 Χ 10 6 gal/day and enters a river through an underground pipe about 100 yards away. Only about one-fourth of the total biochemical oxygen demand (BOD), which aver ages 12 000 lb/day, is removed by the waste t r e a t m e n t system. In the course of the complete organ ic analysis of this wastewater, several unknown but related mass spectra were encountered. Figure 1 gives these six mass spectra. Spectra A and Β were obtained by GC/MS, while spec t r a C - F were obtained by collecting fractions after separation by high pressure liquid chromatography. Each fraction was collected in a 5 ml pearshaped flask and was evaporated t o dryness on a rotary evaporator. T h e sample was redissolved in 5-10 μΐ.. of dichloromethane and transferred to a capillary tube (1.5-1.8 X 50 mm) which was introduced into the mass spectrometer via the direct probe. This technique required 10-15 min per fraction; even when the fraction contained more than one compound, clean mass spectra for each compound could usually be obtained because of differential volatility of the various components. T h e use of H P L C as a supplement to G C / M S is an important feature of this study. Among other benefits, it indicates t h a t the compounds are in digenous to the sample and are not formed by pyrolysis during the G C / M S analysis itself. Clearly spectra A - F are related to one another. T h e y all show isotopic clusters which indicate the presence of two or more chlorine atoms; several
1452 A • ANALYTICAL CHEMISTRY, VOL. 51, NO. 14, DECEMBER 1979
have intense peaks a t m/e 218 and 252; and spectra A, E, and F indicate molecular weights differing by 126 a m u (288, 414, and 540). None of these spectra were in an index of known mass spectra (7), nor were they re trieved by a computerized search of mass spectra of known compounds (8). Spectrum A indicates a molecular weight (MW) of 288 and has an isoto pic cluster corresponding to CI3. (For reference, the expected distributions of the Cli to CI5 isotopic clusters are given in Figure 2). We consulted a list of commercially manufactured organic compounds which was ordered by exact molecular weight; the only com pound listed with MW = 288 and with three chlorines was 5-chloro-2-(2,4dichlorophenoxy)-phenol. (This list is available in microfiche form on re quest to the author.)
ci—\S— ο—χ/-C1 OH
(A)
CI
Furthermore, this compound is manu factured by the company whose waste water we were studying. Spectrum A agrees with this structure; it shows ions due to the loss of one CI (m/e 253) a n d two CI (m/e 218). T h e authentic compound A was obtained; its mass spectrum and GC and H P L C reten tion times agreed with the unknown. We can now consider structure A proven and use this structure as a springboard from which the structures of compounds B - F can be reached. At this point it is helpful to our sub sequent discussion if we outline the synthetic scheme which we presume is used in the commercial production of compound A (see Figure 3). T h e first step is the reaction of 2,5-dichloronitrobenzene with 2,4-dichlorophenol; the resulting trichloro-nitro-diphenylether is then reduced with Raney nickel to the amino compound. Final ly, the diazonium salt is formed and 0003-2700/79/A351-1452S01.00/0 © 1979 American Chemical Society
r
A
M—2CI
288 290
Γ Ή Γ |HI mpi uiyii ιιφι « y i iilft «qui aip my illui|uu ιιιρι mpi ^M'hyu wyw w| 180 200 220 240 260 280 300 320 M + Β M—2CI 252 289 217 M—Cl 287
ill
219
,.291
ι
L
ρ I»I[IM aii|H» tu|i»i m^iiu'lBJlIti iui|im wiim iuyllHla[iiii mi|tw wf m J|ftl m p mqai ni|
180
200
220
240
260
280
300
320
c M + 252
254
M—CO~Cl 189
M-CI
,|191 217i 2 1 9 ,256 |Bi main ιιινίιι in iHjir itajun ιΙΙΙΙικι «up ni>|iiu iii^aPituu «φ· IIH|«U imp IAJHII in 180 200 220 240 260 280 300
M---CO—Cl
|274
2071,209
i
im aiiim lapw u p » iibim uiiiw BIWI min» mi|m BIBUII ini^rluim « y » »HHI wpn n i
180
200
240
260
280
300
320
218
E
r
220
416 M + 414
252 |220
254
272
j418
W|liPlyu ayi'iiyj'illliii m j i l r i i p mynfhipw ίΐι[*ι niiput IIMJIIH UIUIIII wyn iw|iin niyiii awim iui|iiii muiin imjim IMII» IIII|IIIH||IW i^ 200 220 240 260 280 300 320 340 360 380 400 420 218 252 220
200
220
2 5 4
I
l
M +
542
5 4 0
I544 (546
|i» ι«|ΐΙΙΒφΜΐ ηψί mi|iui'iillllu ιΐ[ΐιιι wipi iiii|nn iinjnii mi|»u IIIIJIW impw i y u I»[HII ai|»n alljllii iiii|iiiHili[iii ιιιρι nii|H IEI|M w|im M|in mijim mi|Mi ιιιιρ i«|ia ιηιυ *ψ· «ι MrWH 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540
Figure 1. Mass spectra of unknown compounds A-F which were isolated from the wastewater of a chemical plant Conditions: electron impact, 70ev, HP 5982A mass spectrometer
M
M
+2
+4
+2
M
+2
M
+4
+2
+4
+6
M
+2
+4
+8
Figure 2. Characteristic multiplets of peaks caused by the presence of one to five chlorine atoms in a molecular or fragment ion; they are all spaced two mass units apart
hydrolyzed to give compound A. None of these reactions is 100% complete; thus, many of the precursors can be present in small amounts at any step of the process. Let us now move on to spectrum B; it indicates a molecular weight of 287 and the presence of three chlorine atoms. T h e spectrum is very similar to spectrum A except it is shifted down by one mass unit. Replacing the OH (17 amu) in compound A by NH2 (16 amu) could account for this shift. This compound is commercially available and is, in fact, the synthetic precursor of compound A (see Figure 3).
Cl
(B)
In spectra A and B, the group of peaks due to the loss of CI and HC1 is complex (see m/e 251 to 256). In spec t r u m B, for example, m/e 252, 254, and 256 are due to the loss of CI from the intact molecule. Since there are still two chlorine atoms in this ion, the expected isotopic abundance is 252: 100%, 254: 65%, and 256: 11%. T h e in tense ions at m/e 251, 253, and 255 in spectrum Β are due to the loss of HC1 from the molecule and probably have the structure .0
CI
CI
T h e corresponding ions in spectrum A are at 252, 254, and 256 and probably have the structure
Τ (T
ing hydrolysis of the diazonium salt intermediate
Cl
Spectrum C shows a similar ion cluster at 252, 254, and 256 which is also the molecular weight. By analogy with the above ion structure, we formed the hypothesis t h a t compound C is a dichlorodibenzodioxin which was formed as a by-product during the production of compound A. This hy pothesis is supported by the ion a t m/e 189 which is due to the loss of CI and CO from the molecule. This is a characteristic fragmentation for chlorodibenzodioxins (9). Spectrum C, of course, cannot tell us where on the ring the chlorines are positioned. However, since we know the substitu ent positions of the precursor, we can predict t h a t compound C is the 2,7dichloro isomer
- a£&Xa
(D)
Assuming t h a t this is the correct reac tion, we can position the chlorines as shown. Spectra A, E, and F indicate molec ular weights differing by 126 amu and show the presence of 3,4, and 5 chlo rine atoms, respectively. T h e 126 amu difference is most likely
—Ο
V CI
OH CI -HCl
XX°0XX
(C)
CI ° CI Spectrum D shows a molecular ion at m/e 270 which contains three chlo rine atoms and an ion at 207 due to the loss of CO and CI. An elemental composition of C12H5CI3O is suggested by these data and by analogy with compound A. It takes very little imagi nation to put together a trichlorodibenzofuran structure from this infor mation. Such a compound could be formed by the loss of nitrogen fol lowed by intramolecular coupling dur
1454 A • ANALYTICAL CHEMISTRY, VOL. 5 1 , NO. 14, DECEMBER 1979
We can attach this moiety to com p o u n d A in at least two ways (see Fig ure 4). These particular positional iso mers were selected because either could be formed by the reaction of compound A with 2,4-dichlorophenol which is probably present as residual starting material (see Figure 3). Compounds Εχ and E2 can be dis tinguished from each other based on t h e electron impact induced cleavages indicated in Figure 4. We see t h a t both compounds would give fragment ions at m/e 253 and 269 (with 2 chlo rines) due to the loss of the dichloro substituted ring; however, only com p o u n d E2 could give ions a t 287 or 271
(with three chlorines). Spectrum Ε (see Figure 1) shows a small ion clus ter at 272 with a Cl 3 isotopic pattern. This could correspond to the fragment expected at m/e 271 with an addition al proton added by a rearrangement of the phenolic hydrogen. In addition, the methane chemical ionization mass spectrum of compound Ε (see Figure 5) shows an ion cluster a t m/e 287 with 3 chlorines; this corresponds to the two-ring, two-oxygen fragment shown in Figure 4. This chemical ionization spectrum definitively indicates t h a t the hydroxy group is on the terminal ring rather t h a n the middle ring; thus, we believe structure E2 is the correct assignment for compound E.
~^-c
+
NO2
c-
HO-
-CI
) CI
—(
NO:
'
sli
c-
Q-
•
-c,
-0-
NH2
CI
1
(1) NaNOa, H + (2) H2O, Δ
Spectrum F indicates a molecular weight of 540 and has an isotopic pat tern indicative of five chlorine atoms. Since its molecular weight is 126 amu greater t h a n compound E, we hypoth esize t h a t it was formed by the reac tion of 2,4-dichlorophenol with com p o u n d E; this is analogous to the for mation of compound Ε itself. T h e po sitions of the substituents can be de termined from the assumed precursors and from the mass spectrum in a man ner similar to compound E. Note that t h e fragment ions a t m/e 272, 274, and 276 in spectrum Ε are shifted by 126 a m u in spectrum F and now appear at m/e 398, 400, and 402 and t h a t these ions have a four chlorine isotopic pat tern. These facts indicate t h a t the hy droxy group in compound F is also in the terminal ring. Therefore, com pound F has the structure:
In order to verify these structures, which all seem to result from the ther molysis of compound A, 1 g of this compound was heated at 250 °C for 60 min in a 100 m L round-bottom flask fitted with a reflux condenser and a thermometer. T h e products were ex tracted with dichloromethane and an alyzed by the H P L C / M S technique outlined above. T h e chromatogram and peak identifications are shown in Figure 6. Clearly, several reactions have occurred. Degradation of com pound A to dichlorophenol (MW = 162, presumably the 2,4-isomer) has taken place; this is apparently fol lowed by a reaction of compound A with dichlorophenol to produce the
0,-
-0OH
-0, CI
Figure 3. Synthetic scheme which presumably was used for the industrial production of 5-chloro-2-(2,4-dichlorophenoxy)phenol [compound A ] Reference: E. Model and J. Bindler, Swiss Patents 428,758 to 428,760 (see Chem. Abst. 68, 12690-2)
Figure 4. Two possible structures for compound Ε showing masses of expected fragment ions ANALYTICAL CHEMISTRY, VOL. 5 1 , NO. 14, DECEMBER 1979 • 1455 A
Figure 5. Methane chemical ionization mass spectrum of compound
three ring compound E. This com pound in turn reacts with dichlorophenol to yield compound F. Further reactions of this type give even larger compounds of this nature; note the compound of MW = 666 (540 + 126) in Figure 6 which has five rings. Simi lar compounds containing six to nine rings are probably formed as well (see arrows in Figure 6) b u t good mass spectra could not be obtained of these peaks. T h e peaks numbered 2 to 9 in Figure 6 are compounds of the form
OHL where η = 2,3, ...9. Compound C (the dichlorodioxin) is also formed by the thermal cyclization of compound A (see Figure 6, MW = 252). Compound Β (the amino species) and compound D (the trichlorodibenzofuran) are not found in the products of this thermolysis. This is expected since compound Β is actually the syn thetic precursor of A, and D is formed from its diazonium salt. This set of identifications demon strates a number of things: • Several by-products are formed during the commercial production of compound A and are discharged with the process wastewater. • T h e interpretation of groups of mass spectra of related compounds is far more productive t h a n treating each as an isolated case. For example, it would have been very difficult to in terpret spectrum F without first hav ing deduced structures A and E. • T h e use of production informa tion (compounds in commercial pro duction and their industrial synthe ses) is essential for the efficient inter pretation of mass spectra of com pounds isolated from the environ ment. • Considerable information about an unknown chemical production pro cess can be obtained by a careful anal ysis of the wastewater of t h a t process.
Figure 6. High pressure liquid chromatogram of the reaction products formed by heating 5-chloro-2-(2,4-dichloro-phenoxy)-phenol [compound A] at 250° for 60 min HPLC conditions: μ Bondapak C l e , 20-100% CH3CN in H2Ot 2 ml/min, 254 nm. The peaks which were identified by mass spectrometry as compounds A, C, E, and F are so indicated. The compound with MW = 162 is 2,4-dichlorophenol
Acknowledgment T h e cooperation of the company of ficials and plant personnel is appre ciated. This work has been supported by the Chemical T h r e a t s to M a n and the Environment Program of the Na tional Science Foundation (Grant No. ENV-75-13069) and by the U.S. Envi ronmental Protection Agency (Grant No. R 806350). References (1) G. A. Jungclaus, L. M. Games, and R. A. Hites, "Identification of organic com pounds in tire manufacturing plant wastewaters," Anal. Chem., 48,1894-96 (1976). (2) L. M. Games and R. A. Hites, "Compo sition, treatment efficiency, and environ mental significance of dye manufactur ing plant effluents," ibid., 49,1433-40 (1977). (3) G. A. Jungclaus, V. Lopez-Avila and R.
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1979
A. Hites, "Organic compounds in an in dustrial wastewater: a case study of their environmental impact," Environ. Sci. Technol., 12,88-96(1978). (4) L. S. Sheldon and R. A. Hites "Organic compounds in the Delaware River," ibid., 12,1188-94 (1978). (5) L. S. Sheldon and R. A. Hites "Sources and movement of organic compounds in the Delaware River," ibid., 13, 574-79 (1979). (6) L. S. Sheldon and R. A. Hites, "Envi ronmental occurrence and mass spectral identification of ethylene glycol deriva tives," Sci. Total Environ., 11,279-86 (1979). (7) Eight Peak Index of Mass Spectra, Mass Spectrometry Data Centre, Read ing, United Kingdom (1974). (8) S. R. Heller, G. W. A. Milne and R. J. Feldmann, "A computer-based chemical information system," Science, 195, 253-59 (1977). (9) N. P. Buu-Hoi,G. Saint-Ruf and M. Mangane, "The fragmentation of dibenzo-p-dioxin and its derivatives under electron impact," J. Heterocycl. Chem., 9,691-93 (1972).
