Characterization of High Molecular Weight Disinfection Byproducts

The SEC−UV chromatograms and SEC−ESI-MS spectra show that coagulation could ... Environmental Science & Technology 2015 49 (24), 14239-14248...
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Environ. Sci. Technol. 2005, 39, 963-972

Characterization of High Molecular Weight Disinfection Byproducts from Chlorination of Humic Substances with/without Coagulation Pretreatment Using UF-SEC-ESI-MS/MS X I A N G R U Z H A N G , * ,† R O G E R A . M I N E A R , * ,† A N D SYLVIA E. BARRETT‡ Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, and Metropolitan Water District of Southern California, La Verne, California 91750

Chlorinated disinfection byproducts (DBPs) generated from the reaction of the disinfectant chlorine with naturally occurring humic substances in raw water have been intensively studied over the past three decades, yet only a fraction of the total organic halogen (TOX) formed during chlorination has been chemically identified or even well characterized. The majority of the unknown portion of the TOX is likely attributable to high molecular weight (MW) DBPs (above 500), which may have potential adverse health effects. In this work, typically dosed chlorinated Suwannee River fulvic acid (SRFA) samples with and without coagulation pretreatment were separated and fractionated by using ultrafiltration (UF) and size exclusion chromatography (SEC) techniques. The SEC fractions corresponding to the high MW region were concentrated with nitrogen sparging and characterized by negative ion electrospray ionization mass spectrometry (ESI-MS) and ESI-MS/MS. The results demonstrate that the ESI-MS/MS precursor ion scan is an effective tool for the selective detection of the electrospray ionizable chlorine-containing compounds in a complex mixture. Many high MW chlorine-containing DBPs were tentatively found in the UF-SEC fractions of the chlorinated SRFA samples with/without coagulation pretreatment. The SEC-UV chromatograms and SEC-ESIMS spectra show that coagulation could significantly reduce the formation of high MW chlorinated DBPs.

Introduction Chlorinated disinfection byproducts (DBPs) as a result of the reaction of the disinfectant chlorine with naturally occurring humic substances in raw water have been intensively studied over the past 30 years, yet only a fraction of the total organic halogen (TOX) formed during chlorination has been chemically identified or even well characterized (1-6). Richardson compiled a review on drinking water DBPs, * Corresponding author phone: (928) 284-4009; fax: (928) 2844083; e-mail: [email protected] (R.A.M.); phone: (574) 631-0907; fax: (574) 631-9236; e-mail: [email protected] (X.Z.). † University of Illinois at Urbana-Champaign. ‡ Metropolitan Water District of Southern California. 10.1021/es0490727 CCC: $30.25 Published on Web 01/15/2005

 2005 American Chemical Society

in which she listed 259 halogenated DBPs identified in chlorinated drinking water samples or chlorination of humic substances (7), almost all of which were low molecular weight (MW) DBPs and were identified by gas chromatography/ mass spectrometry (GC/MS) or derivatization-GC/MS (8, 9). But GC/MS generally is not amenable to the identification of high MW DBPs (MW > 500), which may constitute ∼50% of TOX (10, 11) and may account for 41-64% of mutagenicity in chlorinated drinking water using the Salmonella histidine reverse mutation assay (10). Thus, liquid chromatography (LC)/MS seems to be a logical choice for identifying high MW chlorinated DBPs. Due to the high chemical background inherent in LC/MS, a successful study involving the identification of unknown nonhalogenated DBPs in the ozonated water by LC/MS has also employed derivatization. The MWs of those highly polar carbonyl compounds identified in that study are less than 160 (12). The chlorine-containing DBPs (not including bromine-containing species) that have been well identified to date generally have MWs of less than 220, e.g., 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (217.4) and 5,5,5-trichloro-4-oxopentanoic acid (219.5) (7). It is most likely that a chlorine-containing DBP with MW above 220 contains multiple hydrophilic functional groups such as -COOH, -OH, dO, and -NH2, which makes the full derivatization of each functional group very difficult. Recently, Zhang et al. (13) reported several chlorine-containing DBPs with molecular ions m/z 313, 355, and 357 in a highly dosed (100 mg/L as C and 50 mg/L as Cl2) chlorinated Suwannee River fulvic acid (SRFA) sample using an electrospray ionization tandem mass spectrometry (ESI-MS/MS) method. To our knowledge, there has been no direct mass spectrometry evidence in the literature for the presence of high MW chlorine-containing DBPs (with molecular ions m/z > 500). Using LC/MS in search of high MW chlorine-containing DBPs in chlorinated drinking water is challenging. The difficulty lies in the following issues: (1) Assuming a size exclusion chromatography (SEC) column is used for the LC fractionation, the chlorine-containing compounds may be eluted with retention times from 30 to 1623 min (14). If one fraction is collected per minute, there are ∼1600 fractions per sample. How are those fractions with high MW chlorinated DBPs to be located? (2) Inorganic ions in water samples may cause high chemical background in the LC/MS chromatogram (8), and may suppress the electrospray ionization of chlorinated DBPs. In a chlorinated drinking water sample, there are always some inorganic ions such as sodium, chloride, sulfate, and, possibly, phosphate and arsenate due to the use of coagulating, chlorinating, buffering, and quenching agents. How are those inorganic ions eliminated prior to the mass spectrometry analysis? (3) A chlorinated humic substance sample or sample fraction could produce an ESI-MS spectrum with hundreds to thousands of ion peaks distributed on almost every mass unit. How are the chlorinecontaining ions differentiated from the chlorine-free ions by mass spectrometry? A solution to the first issue can be found in Zhang and Minear’s work (14), in which they introduced radioactive 36Cl into an SRFA sample to label the chlorine-containing DBPs. By combining the fractionation techniques of ultrafiltration (UF) and SEC with the detection of 36Cl, UV, and dissolved organic carbon (DOC), the high MW chlorinated DBP region in the SEC-36Cl profiles of the chlorinated sample with and without UF was defined. The SEC-UV and SECDOC profiles were found to be approximate alternatives to the SEC-36Cl profiles for the high MW region. The second VOL. 39, NO. 4, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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issue can be resolved concomitantly with the first issue owing to the use of UF. It has been demonstrated that for the UF cell with a 500 nominal MW cutoff membrane, the commonly known low MW DBPs (e.g., haloacetic acids and trihalomethanes) and some inorganic ions (e.g., sodium and chloride) could be effectively flushed out, and most of the phosphate ions could also be flushed out (14, 15). The third issue is essentially to find the fingerprint chlorine of a chlorine-containing compound with appropriate ESIMS or ESI-MS/MS. ESI is a soft ionization technique used in conjunction with mass spectrometry that usually generates a molecular ion or pseudomolecular ions even for thermally unstable and polar molecules (16). Because chlorine has two natural isotopes, 35Cl and 37Cl, with an abundance ratio of ∼3:1, compounds containing different numbers of Cl give different chlorine patterns by ESI-MS. Abundance of isotopic peaks can be expressed by the binomial expansion (17)

(a + b)n ) an + nan-1b + n(n - 1)an-2b2/2! + ... + bn where n is the number of chlorine atoms in a compound and a and b are the natural abundances of the two stable isotopes. For instance, a compound containing two Cl atoms shows a chlorine pattern by ESI-MS of A:A + 2:A + 4 = 9:6:1, in which A, A + 2, and A + 4 contain two 35Cl atoms, one 35Cl atom and one 37Cl atom, and two 37Cl atoms, respectively; a compound containing three Cl atoms shows a chlorine pattern by ESI-MS of A:A + 2:A + 4:A + 6 = 3:3:1:0.1, in which A, A + 2, A + 4, and A + 6 contain three 35Cl atoms, two 35Cl atoms and one 37Cl atom, one 35Cl atom and two 37Cl atoms, and three 37Cl atoms. But if the concentration of this compound were too low, or the chlorine pattern of this compound were partly overlapped with those of other compounds in a mixture, none of the ions (A, A + 2, A + 4, A + 6) could be definitely considered as a chlorine-containing ion. Thus, appropriate ESI-MS/MS is needed to aid in this determination. Initially, a Finnigan TSQ 7000 ESI-quadrupole ion trap mass spectrometer at Kyoto University, Japan, was tested. It was found that a chlorine-containing compound could produce chloride ions, 35Cl- and 37Cl-, by ESI-MS/MS product ion scan after the instrument was optimized in carrier fluid flow rate, collision gas pressure, and collision energy (13). A Micromass AutoSpec time-of-flight mass spectrometer can detect cloride ions, 35Cl- and 37Cl-, from standards using ESI-MS/MS product ion scan, but could not find any unknown chlorine-containing DBPs from different batches of chlorinated SRFA samples or their SEC fractions by ESIMS/MS product ion scan (18). Later, some standard chlorinecontaining compounds were tested with a Micromass triplequadrupole mass spectrometer at the Mass Spectrometry Center at the University of Illinois; chloride ions were readily observed with this instrument by ESI-MS/MS product ion scan. However, when an SEC fraction of an ultrafiltered chlorinated SRFA sample was analyzed to determine which ions in the ESI-MS spectrum with ∼1000 major ions contain chlorine by ESI-MS/MS product ion scan, it was realized that such an analysis by testing one ion per injection is very time-consuming and labor intensive, and requires a prohibitive cost and a large volume of sample. From a practical point of view, it is nearly impossible to determine whether each ion in the SEC fraction contains chlorine or not by ESI-MS/MS product ion scan. Fortunately, the triple-quadrupole mass spectrometer at the Mass Spectrometry Center was found to have a useful MS/MS mode, precursor ion scan, which can be used to look for the precursor of a particular fragment. Assuming that the fragment were selected as the chloride ion m/z 35 or 37, it would be possible to find all the chlorine-containing DBPs in a mixture by ESI-MS/MS precursor ion scan. 964

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To characterize high MW DBPs, one cannot neglect the practical aspects of the treatment processes, in which coagulation is required in most cases to remove natural organic matter (NOM). According to the disinfectant (D)/ DBP rule, if the total organic matter (TOC) is above 2.0 mg/L, it needs to be removed by 15-50% depending on the raw water TOC level and alkalinity (19). Ebie et al. (20) found that the coagulation/sand filtration pretreatment removed about 58% of mainly larger NOM molecules, leading to a shift of the distribution from an MW range of 7500-1150 to one of 3500-1150. Vilge-Ritter et al. (21) reported that coagulation primarily eliminates the NOM molecules with an apparent MW larger than 1500, especially larger than 5000. Both studies show that although coagulation eliminates extremely large NOM molecules, there are still some relatively large molecules left with an MW range of 500-3500. The remaining NOM fractions after coagulation may still react with the disinfectant chlorine to form high MW chlorinated DBPs. For the limited papers in the literature that reported the study of the formation and characterization of high MW chlorinated DBPs (10, 11, 14), coagulation seemed to be overlooked. In this work, typical dose chlorinated SRFA samples with and without coagulation pretreatment were prepared, and were separated/fractionated with the techniques of UF and SEC. The SEC fractions then were concentrated by N2 gas sparging and analyzed by negative ion ESI-MS and ESI-MS/ MS. The specific objectives of this work were (1) to examine the feasibility of exploiting ESI-MS/MS precursor ion scan in the selective detection of chlorine-containing compounds in a mixture and to find the direct mass spectrometry evidence for the presence of high MW chlorine-containing DBPs in chlorinated humic substance samples and, meanwhile, (2) to characterize high MW chlorinated DBPs and to examine the effect of coagulation on the formation of high MW chlorinated DBPs by comparing SEC-UV chromatograms and SEC-ESI-MS spectra.

Experimental Methods Jar Test for Coagulation. SRFA from the International Humic Substances Society was added to Milli-Q water to simulate water of 3.0 mg/L DOC, which was buffered to pH 7.5 with phosphate. Milli-Q water was supplied by a Millipore MR3 purifier system. Alum (aluminum sulfate, Al2(SO4)3‚14H2O) was used as coagulant. The coagulant doses ranged from 10 to 70 mg/L. The simulated water was clarified at room temperature using a jar test setup: The coagulation occurred under rapid mix conditions (3 min at 100 rpm), during which the coagulant was added to 1000 mL of the water. Flocculation occurred during the slow mix period (30 min at 35 rpm). The sample jar was then removed from the stirrer, and the flocs were allowed to settle for 30 min. Finally, the supernatant was collected, and was filtered with a Millipore 0.45 µm membrane. The concentrations of SRFA in the simulated water and the clarified water were determined by UV absorbance at 254 nm (Beckman spectrophotometer) and DOC (Shimadzu 500 carbon analyzer). For the simulated water (3.0 mg/L SRFA as C), both UV and TOC measurements show that the optimum alum dose was around 40 mg/L, which would be used in the preparation of the sample with coagulation pretreatment. Preparation of Chlorinated SRFA Samples. SRFA from the International Humic Substances Society was employed as a representative of humic substances. Ultra-high-purity (UHP) chlorine gas (99.97%) was purchased from Scott Specialty Gases. HOCl stock solution was prepared by the absorption of the UHP chlorine gas with Milli-Q water, and was standardized by the iodometric method (22). For the chlorinated SRFA sample without coagulation, the initial concentration of SRFA was 3.0 mg/L as C (the average DOC

level in raw waters), pH was controlled at 7.5 with 7.0 mL of 0.1 M phosphate buffer, and the initial concentration of HOCl was 1.5 mg/L as Cl2. For the chlorinated SRFA sample with coagulation, the initial concentration of SRFA was 3.0 mg/L as C, and pH was controlled at 7.5 with 7.0 mL of 0.1 M phosphate buffer. Coagulation was performed with the same procedure as used in the jar test with the optimal alum dose (40 mg/L). The concentration of DOC after coagulation was measured, and HOCl was added accordingly at a Cl2:C weight ratio of 1:2. Because phosphate buffer had been used in the sample preparation in ref 14, it was also used here to prepare samples with composition similar to that in ref 14 so that the SEC elution behavior variation could be minimized and the defined high MW chlorinated DBP region by the radioisotope 36 Cl would remain valid. Although the presence of phosphate in a sample may suppress the electrospray ionization of organics with ESI-MS or ESI-MS/MS, most of the phosphate would be expected to be flushed out of the UF cell with sufficient Milli-Q water (15), and the residual phosphate would be expected to be eluted into several SEC fractions due to the size exclusion effect. For both samples, Br- was not added for the sake of simplifying the reaction mixture, and the reaction was quenched with 0.25 mL of 0.1 M NaAsO2 after a contact time of 24 h. Each sample had a volume of 3300 mL. Removal of Low MW DBPs and Inorganic Ions. UF was first conducted in a stepwise procedure with a 400 mL Millipore stirred cell. The membrane was made of cellulose acetate with a nominal MW cutoff of 500. The UHP nitrogen gas applied to the 400 mL cell was kept at 45-50 psi. The UF was performed in a stepwise manner in which initially 350 mL of the chlorinated SRFA sample was added to the cell. Each time when half the volume of the sample in the cell passed through the membrane, the initial volume was restored with the chlorinated SRFA sample, or with Milli-Q water only after all the 3300 mL sample was fed to the cell. A total volume of about 4000 mL of Milli-Q water was used for flushing in the stepwise procedure. The solution in the cell was mixed continuously with a magnetic stir bar throughout the process. Then, the retentate (nominal MW > 500) in the 400 mL cell was transferred to the 10 mL cell of a continuous UF device (14). Another 350 mL of Milli-Q water was used for continuous flushing. The UF was stopped when 4 mL of retentate remained. All the UF processes were performed in a cold room at 4 °C. The ultrafiltered chlorinated SRFA samples without and with coagulation pretreatment are coded as S(I) and S(II), respectively. Fractionation of High MW DBPs with SEC. The same HPLC system, same SEC column, same eluent, same flow rate, and same injection loop as in ref 14 were used. Briefly, SEC was conducted with a Beckman Gold HPLC system, consisting of two model 110B solvent delivery pumps, a model 166 programmable UV detector, and a data-collecting computer. The column used was a 25 × 200 mm BIAX (Chrom, Germany) with a column packing of Toyopearl HW 50S resin (Japan). The eluent was 0.03 M NH4HCO3 with a flow rate of 0.80 mL/min. NH4HCO3 was used as the SEC eluent because it is “volatile” and well suited for electrospray ionization. The UV wavelength was set at 254 nm. In ref 14, by combining the fractionation techniques of UF and SEC with the detection of 36Cl, UV, and DOC, the high MW region in the SEC-36Cl profiles of the chlorinated sample with and without UF was defined within retention times of 40-65 min. For the same system and column under the same conditions, the SEC elution behavior for the samples should not change. The injection loop was 500 µL. One fraction of 0.80 mL was collected per minute into a 7 mL glass vial with a fraction collector. The vials with collected fractions within retention times of 40-65 min were sealed with polypropylene screw caps and Teflon-lined septa, and kept in a refrigerator at 4

°C. The fraction collected within a retention time of 40-41 min is coded as #41; the fraction of 41-42 min is coded as #42, and so forth. The sample ran about 6 h (to ensure that all the materials from the previous injection were flushed out), then all the vials in the refrigerator were put back into the fraction collector, another 500 µL of the sample was injected, and fractions were collected in the same pattern. The fraction with the same retention time was collected into the same vial. The above process was repeated until fractions from five runs were collected. All the fractions, each with a final volume of 4 mL, were stored at 4 °C. For the SEC column with the eluent of 0.03 M NH4HCO3 at a flow rate of 0.80 mL/min, a semilog-linear calibration curve was defined by polystyrenesulfonates (14):

log Msec ) -0.0424(RTsec) + 5.4838

(1)

By using eq 1, the MW of an analyte, Msec, corresponding to the SEC retention time, RTsec, was determined. Removal of NH4HCO3 and Concentration of SEC Fractions. A high concentration of NH4HCO3 (used as the eluent) in each SEC fraction could complicate MS spectra due to the formation of adducts, and lower the electrospray ionization efficiency for organics. Most of the NH4HCO3 in the sample fractions was demonstrated to be effectively removed by nitrogen sparging. The carbon content of 0.03 M NH4HCO3 was 360 mg/L as C. A given volume of 0.03 M NH4HCO3 solution in Milli-Q water was sparged dry with UHP nitrogen gas, the same volume of Milli-Q water was added, and then the carbon content was measured to be 1.86-3.38 mg/L as C, which indicates that more than 99% of NH4HCO3 was removed. Another reason for using nitrogen sparging is for concentration. After a 4 mL fraction was sparged dry, 400 µL of Milli-Q water was added, then the fraction was transferred to a 1 mL graduated screw thread vial with open-top closure and a Teflon-faced silicone septum, and kept at 4 °C for further analysis with mass spectrometry. Characterization of Chlorinated DBPs in the Concentrated Fractions. The mass spectra were acquired on a Micromass Quattro I mass spectrometer (VG Quattro triple quadrupole, FISONS Instruments). The ESI source operates at atmospheric pressure. The solution of a sample is sprayed through a needle at a potential of 3.5 kV. ESI-MS operation parameters were set as follows: sampling loop, 10 µL; carrier flow, 1:1 water/acetonitrile; carrier flow flow rate, 15 µL/min; ESI, negative; source temperature, 65 °C; source cone, 35 V. Quadrupoles 1 and 3 (Q1 and Q3) were set to unit resolution in all experiments. The scan range for each fraction was m/z 10-4000. Under the selected instrument condition, the total ion current (TIC) and ESIMS spectrum of each fraction corresponding to the high MW region in the SEC-36Cl profile were obtained. The background noise was determined from a baseline spectrum over the same mass range. The average background intensity was determined and subtracted from the intensity data of the analyte spectrum. Each ESI-MS spectrum was then transferred to an Excel spreadsheet. Assuming that all the ions were singly charged molecular ions, the MS number-average MW (Mn) of each fraction based on ion intensity distributions was calculated according to accepted methods (23): N

Mn )

∑ i)1

N

niMi/

∑n

(2)

i

i)1

where ni is the number of molecules of molecular weight Mi. In the ESI-MS spectrum, ni and Mi correspond to the intensity and the mass-to-charge ratio of ion i, respectively. VOL. 39, NO. 4, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. SEC-UV chromatograms of chlorinated and nonchlorinated SRFA (3 mg/L as C) with/without coagulation pretreatment. Besides all the parameters for ESI-MS, additional parameters were set for ESI-MS/MS: collision gas (argon) pressure, 1.8 × 10-3 mbar; collision energy, 200 eV. Under such an instrument condition, ESI-MS/MS precursor ion scan and product ion scan were conducted for a standard sample and several fractions having relatively high ion intensity in the ESI-MS spectra. The standard sample consisted of 10 mg/L each of dichloroacetic acid (DCA), potassium hydrogen phthalate (PHP), 2,4,6-trichlorophenol (TCP), and chlorophenol red sodium salt (3′,3′′-dichlorophenolsulfonephthalein, CPR) in Milli-Q water. DCA, PHP, TCP, and acetonitrile were of analytical grade, CPR was of reagent grade, and all were purchased from Aldrich. CPR is not a DBP but was used to simulate chlorine-containing DBPs with relatively high MW.

Results and Discussion SEC-UV Chromatograms. Figure 1 shows the comparison of SEC-UV profiles of SRFA samples with and without coagulation. After coagulation, the DOC level in the SRFA sample decreased from 3.0 to 1.1 mg/L. For the SEC column under the specified conditions, the MW of each fraction (Msec) can be estimated according to eq 1. For the fractions with Msec of 5000, 3000, and 1000, the reductions of UV absorbance at 254 nm were 96%, 64%, and 22%, respectively. These data indicate that coagulation could significantly reduce SRFA from the simulated waters (about 60% as C). Coagulation removed the components of SRFA selectively; the higher the MW, the higher the removal, leading to a dramatic decrease of the average MW of the SRFA sample. After coagulation, the majority of the SRFA components with MW less than 3000 remained. The results are in good agreement with others’ findings (20, 21). Though coagulation eliminates extremely large humic substance molecules, there are still some relatively large molecules with MW in the range of 500-3500 left. These components remain after coagulation and can still react with the disinfectant chlorine to form high MW chlorinated DBPs. Figure 1 also shows the comparison of SEC-UV profiles of the SRFA sample before and after chlorination. For either the coagulated or the noncoagulated SRFA sample, chlorination lowered the UV absorbance almost evenly along the high MW region due to its destruction of some chromophores that absorb UV radiation, which means that chlorination did not appreciably change the MW distribution of the SRFA sample. Therefore, the effect of coagulation on the MW distribution of the SRFA sample was passed on to the chlorinated SRFA sample; i.e., coagulation not only significantly reduced the formation of high MW chlorinated DBPs, but also lowered the average MW of high MW chlorinated DBPs. ESI-MS Spectra. Fractions that represent the high MW region with retention times of 40-65 min, which has been defined by the SEC-36Cl profile under the specified conditions (14), were employed for the ESI-MS analysis. Figure 2 displays 966

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the ESI-MS spectra of some representative high MW SEC fractions from the ultrafiltered chlorinated SRFA samples with and without coagulation pretreatment. These spectra indicate that each SEC fraction of the ultrafiltered chlorinated SRFA samples was still a complex mixture containing hundreds of major components. For each fraction of the samples, the ESI-MS spectrum was displayed as a distribution of ions from m/z 10 to m/z 4000 or more. The intensive ions appeared below m/z 1500, especially less than m/z 1000, and the most intensive ions appeared in the range of m/z 100-500. After the most intensive ion region, there was a high-mass tail extending to around m/z 4000. Thin single peaks with relatively large m/z values are most likely attributed to electronic noise spikes or single ions. For each fraction of the sample, the complexity of the ESI-MS spectrum makes unlikely the direct identification of chlorine-containing ions according to the chlorine patterns seen by ESI-MS, and also makes nearly impossible the determination of whether each peak in the ESI-MS spectrum contains chlorine by using the ESI-MS/MS product ion scan (13). An evident phenomenon in Figure 2 is that for the representative high MW fractions, the ion intensities in the fraction with coagulation pretreatment [S(II)#m] were much weaker than those in the corresponding fraction without coagulation pretreatment [S(I)#m]. This can be quantitatively reflected by TIC, an indicator for the total charges of all the ions formed from ESI or for the total number of electrospray ionizable molecules provided that all the ions are singly charged. The relationship between TIC and SEC retention time of the ultrafiltered chlorinated SRFA samples with/ without coagulation pretreatment is illustrated in Figure 3. The trends of the curves of TIC versus SEC retention time generally were consistent with the SEC-UV profiles. Coagulation pretreatment significantly brought down the TICs of the high MW SEC fractions, with the reductions ranging from 36% to 90%. One of the important properties of high MW DBPs is the MW distribution, especially the average MW (Msec or Mn). To calculate Mn of high MW DBPs in the SEC fractions, the remarkable discrete ions observed in the ESI-MS spectra of SEC fractions #53-57 (as indicated in fraction S(I)#57 in Figure 2) need to be differentiated. These discrete ions corresponded to m/z 177, 195, 217, 293, 315, 391, 413, 489, etc. Fragmentation of ions m/z 195, 217, and 293 by MS/MS gave product ions with m/z 63, 79, 97, and 195, which indicates that these discrete ions came from the buffer phosphate and its polymers. This was expected because although more than 90% of the phosphate could be flushed out of the UF cell (15), the residual phosphate was eluted into several SEC fractions corresponding to the SEC retention time of phosphate (centered at 55 min). Assuming that the presence of phosphate does not affect the ESI-MS ion distribution of organics in an SEC fraction, the intensive discrete ions corresponding to phosphate and its polymers in fractions #53-57 can be excluded from the Excel spreadsheet transferred from the ESI-MS spectrum, and then Mn can be calculated according to eq 2. The values of Mn for the representative high MW SEC fractions are also shown in Figure 2. According to the separation mechanism of SEC, larger size or higher MW compounds are eluted earlier. This is manifested by the Mn values in Figure 2: For the ultrafiltered chlorinated SRFA sample without coagulation pretreatment, Mn decreased from 708 (fraction #41) to 426 (fraction #57). The average MW of each SEC fraction can also be estimated as Msec according to eq 1. The calculated values indicate that Msec decreased from 5560 (fraction #41) to 1170 (fraction #57). It is noteworthy that the values of Mn from the ESI-MS spectra were much lower than those of Msec from the SEC retention times. In the study of MW distribution of humic

FIGURE 2. ESI-MS spectra of some representative high MW SEC fractions from the chlorinated SRFA samples without coagulation [S(I)] and with coagulation [S(II)]. The x-axis and y-axis of each chart indicate the m/z ratio and ion intensity, respectively.

FIGURE 3. Relationship of ESI-MS total ion current and SEC retention time of the ultrafiltered chlorinated SRFA samples without coagulation [S(I)] and with coagulation [S(II)]. substances, the low MW bias by ESI-MS for the high MW region has been attributed to multiple charging (24, 25), selective ionization (26), or fragmentation (27). In consideration of the belief that coagulation generally removes large particles/high MW compounds (28) and of the fact that coagulation mainly removed species with m/z 100-500 in the ESI-MS spectra (Figure 2), the low MW bias by ESI-MS

for the high MW DBPs was most likely attributed to multiple charging or fragmentation. McIntyre et al. (27) studied humic substances by negative ESI-MS with a Micromass triplequadrupole mass spectrometer, and found that the source cone potential has a dramatic effect on the fragmentation of humic substances: the distribution of ions shifted to lower masses with an increase in the source cone potential, and the maxima occurred at ∼160 Da at a source cone potential of 100 V. They suggested that a source cone potential of 35 V be used to acquire the ESI-MS spectra of humic substances because no fragmentation seemed to occur at that potential. Since the source cone potential in this work was set at 35 V, the fragmentation could be ruled out to a great extent. Therefore, the low MW bias by ESI-MS for the high MW DBPs was most likely attributed to multiple charging. Kujawinski et al. (29) suggested that negative ion mode spectra were more likely to contain multiply charged ions. Brown et al. (30) pointed out that the formation of multiply charged negative ions in the ESI experiment is consistent with the polycarboxylic acid nature that is usually attributed to fulvic acid. While numerous workers have investigated humic substances by ESI-MS, the “mystery” for low MW bias by ESI-MS has not been resolved yet. Stenson et al. (26) reported that the vast majority of ions present in spectra of Suwannee VOL. 39, NO. 4, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. MS spectra of a standard sample of 10 mg/L each of DCA, PHP, TCP, and CPR: (a) ESI-MS scan, (b) ESI-MS/MS precursor ion scan of m/z 35, and (c) ESI-MS/MS precursor ion scan of m/z 37. River humic substances are singly charged in ESI Fourier transform ion cyclotron resonance mass spectrometry, but other workers (18, 24, 25, 29, 30) found that the peak(s) can be observed along the baseline approximately halfway between the M (12Cn) and M + 1 (13C12Cn-1) peaks, which is an indication of multiply charged ions. Surprisingly, for the representative fractions as shown in Figure 2, Mn of a fraction from the sample with coagulation [S(II)#m] was greater than that of the corresponding fraction from the sample without coagulation [S(I)#m]. This seems to be unexpected but readily understood because according to eq 2, Mn is determined by m/z ratios in the range of 104000 and the intensity of all the ions. Coagulation mainly reduced the intensities of ions with m/z 100-500 in the ESIMS spectra (Figure 2), i.e., in the lower end of the m/z scan range, thus resulting in higher Mn. This further reflects the low MW bias by ESI-MS. ESI-MS/MS Spectra. A standard sample of 10 mg/L each of DCA, PHP, TCP, and CPR was used to test the triple968

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quadrupole mass spectrometer. Figure 4a shows the ESI-MS spectrum of the standard sample and the molecular ion structures of the four species. There were four major groups of peaks seen by ESI-MS. The first group had peaks at m/z 127.2, 129.2, and 131.2, which correspond to the ion of [DCA - H]-. The second group had only one major peak at m/z 165.2, which is [PHP - K]- because this compound contains no chlorine. The third group had peaks at m/z 195.2, 197.2, 199.2, and 201.2, with an intensity ratio of approximately 3:3:1:0.1, which correspond to the ion of [TCP - H]-. The fourth group had peaks at m/z 421.2, 423.2, and 425.2, with an intensity ratio of approximately 9:6:1, which should be [CPR - Na]-. The ESI-MS/MS precursor ion scan was performed for the standard sample. Figure 4b shows the precursor ions of m/z 35 of the sample, which consisted of three groups of peaks. The first group had peaks at m/z 127.2 and 129.4, the second group had peaks at m/z 195.2 and 197.3, and the third group had peaks at m/z 421.2 and 423.2. The three

FIGURE 5. ESI-MS/MS product ion scans of a standard sample of 10 mg/L each of DCA, PHP, TCP, and CPR: (a) product ions of m/z 421.2, (b) product ions of m/z 423.2, and (C) product ions of m/z 425.2. groups correspond to [DCA - H]-, [TCP - H]-, and [CPR Na]-, respectively. It was expected that no peaks would be observed for [PHP - K]- because this precursor ion could not contribute to the formation of m/z 35, 35Cl-. The first strong peaks from each chlorine-containing compound were m/z 127.2, 195.2, and 421.2. Figure 4c shows the precursor ions of m/z 37 of the sample, which was also made up of three groups of peaks. The first group had a peak at m/z 129.2, the second group had peaks at m/z 197.3 and 199.0, and the third group had peaks at m/z 423.2 and 425.2. The three groups correspond to [DCA - H]-, [TCP - H]-, and [CPR - Na]-, respectively. As expected, there were no peaks present for [PHP - K]- because this precursor ion could not contribute to the formation of m/z 37, 37Cl-. The first strong peaks from each chlorine-containing compound were m/z 129.2, 197.3, and 423.2. These spectra indicate that (1) the ESI-MS/MS precursor ion scan can selectively detect those chlorine-containing compounds in a mixture, (2) between the first strong peaks detected by “precursor ions of m/z 35”

and those detected by “precursor ions of m/z 37”, there is a 2 unit difference, and (3) precursor ions of m/z 35 are more sensitive in finding the chlorine-containing compounds than precursor ions of m/z 37 (because the natural abundance ratio of 35Cl to 37Cl is about 3:1). It needs to be pointed that the ESI-MS/MS precursor ion scan can only detect those chlorine-containing compounds that are ionized by negative ion ESI. Thurman et al. (31) developed an ionizationcontinuum diagram to determine whether ESI works on a compound. Negative ion ESI works well on ionic species, and also works well on polar species where there is acidity in solution. Humic substances/chlorinated humic substances generally are rich in carboxylic and phenolic groups, which make the high MW chlorinated DBPs suited for analysis with negative ion ESI. With the aid of the precursor ion scan in finding the chlorine-containing ions, the ESI-MS/MS product ion scan was used to confirm whether those ions could produce product chloride ions. Figure 5a shows the spectrum of VOL. 39, NO. 4, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. ESI-MS/MS precursor and product ion scans applied to SEC fraction S(I)#49: (a) precursor ions of m/z 35 (scan range m/z 100-2000), (b) product ions of m/z 1110.9 (scan range m/z 30-42), (c) product ions of m/z 1351.6 (scan range m/z 30-42), (c) product ions of m/z 1469.5 (scan range m/z 30-42), and (e) product ions of m/z 1769.8 (scan range m/z 30-42). product ions of m/z 421.2 of the standard sample. It contained a product ion at m/z 35.0 corresponding to 35Cl-. Figure 5b shows the spectrum of product ions of m/z 423.2 of the sample. It contained product ions at m/z 35.0 and 37.3, with an intensity ratio of approximately 1:1, which correspond to 35Cl- and 37Cl-, respectively. Figure 5c shows the spectrum of product ions of m/z 425.2 of the sample. It contained a product ion at m/z 36.9 corresponding to 37Cl-. Therefore, the product ion scan can be used as an effective tool in confirming the chlorine-containing compounds. For the ultrafiltered chlorinated SRFA samples, several SEC fractions with relatively higher values of TIC were selected, and were analyzed with the ESI-MS/MS precursor ion scan by setting m/z at 35 and 37, respectively. It had been expected that, by comparing the spectra of precursor ions of m/z 35 and 37, the chlorine-containing DBPs in each fraction could be determined because there is a 2 unit difference between the first strong peaks detected by precursor ions of m/z 35 and those detected by precursor ions of m/z 37. But the chlorine-containing ions detected by precursor ions of m/z 37 generally were of very low abundance and in most cases could not be discerned. Therefore, an alternative method was used, analyzing the same fraction twice with precursor ions of m/z 35. Then, the ions with relatively strong intensities that were reproducible in the spectra of precursor ions of m/z 35 were selected, and were further analyzed with the ESI-MS/MS product ion scan. Note 970

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that a very narrow scan range (m/z 30-42) was set for the product ion scan to improve the sensitivity in finding the product chloride ion (13). To gain additional information that could potentially be used to propose structures for the high MW TOX, full-scan product ion scans (not just around m/z 35) of the ions that showed the presence of chlorine were performed, but the spectra obtained were too complex to draw any conclusions. Figure 6a shows the spectrum of SEC fraction S(I)#49 by the ESI-MS/MS precursor ion scan. By using precursor ions of m/z 35, a series of precursor ions with relatively strong intensities were selected through duplicate injections including m/z 305.1, 394.0, 1110.9, 1228.0, 1351.6, 1469.5, 1769.8, and 1770.8. Then these ions were examined by the ESI-MS/MS product ion scan. Ions m/z 305.1, 1110.9 (Figure 6b), 1351.6 (Figure 6c), 1469.5 (Figure 6d), and 1769.8 (Figure 6e) were found to produce a prominent 35Cl- product ion peak; i.e., the ratio of 35Cl- ion intensity to background ion intensity is greater than 2.0. Ion m/z 394.0 produced a weak 35Cl- product ion peak; i.e., the ratio of 35Cl- ion intensity to background ion intensity lies within 1.5-2.0. The 35Clproduct ion peak produced from ion m/z 1228.0 or 1770.8 could not be discerned from the background peaks; i.e., the ratio of 35Cl- ion intensity to background ion intensity is below 1.5. It is not surprising that the 35Cl- product ion peak from a high MW chlorinated DBP generally was much weaker than those from commonly known low MW chlorinated DBPs.

FIGURE 7. ESI-MS/MS precursor and product ion scans applied to SEC fraction S(I)#53: (a) precursor ions of m/z 35 (scanned from m/z 100 to m/z 2000 and expanded from m/z 400 to m/z 505) and (b) product ions of m/z 476.1 (scanned from m/z 30 to m/z 42). This confirms the previous findings (14): The Cl:C atomic ratios of the high MW DBPs (roughly a constant, 0.025) are much lower than those of the commonly known chlorinated DBPs. The slight shift of the SEC-36Cl profile from the SECDOC profile indicates that the chlorine incorporation into some humic substances is not a completely even process; i.e., some DBPs may not contain any Cl atom, and some DBPs may have a higher Cl content than the average. It is probable that precursor ions of m/z 35 mainly detected those high MW chlorinated DBPs that have a higher Cl content than the average, and have higher concentrations. By applying the ESI-MS/MS precursor ion scan and product ion scan to SEC fraction S(I)#51, it was found that ions m/z 279.1 and 559.2 produced a prominent 35Cl- product ion peak, and ion m/z 1119.4 produced a weak 35Cl- product ion peak. It is of interest that these three ions could come from the same compound (X) with an MW of 1120.4 because ions m/z 279.1, 559.2, and 1119.4 can be ascribed to its ions with four charges [X - 4H]4-, two charges [X - 2H]2-, and one charge [X - H]-, respectively. Similarly, by applying the ESI-MS/MS precursor and product ion scans to SEC fraction S(I)#53, ions m/z 476.1 (Figure 7), 1219.5, and 1870.3 were found to produce a prominent 35Cl- product ion peak and ions m/z 237.1 and 1936.6 produced a weak 35Cl- product ion peak. By applying the ESI-MS/MS precursor and product

ion scans to SEC fraction S(I)#55, ions m/z 292.2 and 489.5 were found to produce a prominent 35Cl- product ion peak. Sample S(II) was a representative sample starting with a typical dose of NOM with coagulation. For this sample, fraction #51 was first analyzed with the ESI-MS/ MS precursor ion scan. On the basis of the spectrum of precursor ions of m/z 35, some precursor ions were selected and analyzed with the ESI-MS/MS product ion scan. Ions m/z 217.0, 606.6, 805.8, and 1141.5 were found to produce a prominent 35Clproduct ion peak. Ions m/z 296.9, 578.7, and 666.9 were found to produce a prominent 35Cl- product ion peak and a 37Clproduct ion peak. Ions m/z 283.4, 1275.2, and 1391.7 produced a weak 35Cl- product ion peak. Although it is still difficult to obtain structural information of high MW DBPs owing to the unlikely possibility that any particular peak in the ESI-MS spectrum by the triplequadrupole mass spectrometer is due solely to a single structural or compositional isomer, this work is the first step in resolving the unknown high MW portion of TOX formed from chlorination, which had eluded researchers for several decades. The mass spectrometry results demonstrate that the high MW chlorine-containing DBPs could be formed during chlorination. The results also indicate that the ESIMS/MS precursor ion scan is a powerful tool in selectively detecting the chlorine-containing compounds in a complex VOL. 39, NO. 4, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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sample, and the ESI-MS/MS product ion scan can be used to confirm whether those compounds can produce chloride ions. Precursor ion scans of PO3- have been employed for the selective detection of proteins with improved signal-tonoise ratio (32). There are obvious extensions of the technique to the selective detection of some chlorine-containing toxic compounds such as chlorinated pesticides in the environment.

Acknowledgments We acknowledge funding for the research from the United States Environmental Protection Agency (Grant No. R82683401). The Quattro mass spectrometer was purchased by the Department of Chemistry, University of Illinois at UrbanaChampaign, in part with a grant from the Division of Research Resources, National Institutes of Health (RR 07141). We thank Vernon Snoeyink of the University of Illinois at UrbanaChampaign for suggesting that the effects of coagulation on the formation of high MW DBPs be investigated, and Cordelia Hwang, Yingbo Guo, and Stuart Krasner of the Metropolitan Water District of Southern California for useful discussions.

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Received for review June 20, 2004. Revised manuscript received November 13, 2004. Accepted November 24, 2004. ES0490727