Grantee Research Project Results
2001 Progress Report: Molecular Weight Separation and HPLC/MS/MS Characterization of Previously Unidentified Drinking Water Disinfection By-Products
EPA Grant Number: R826834Title: Molecular Weight Separation and HPLC/MS/MS Characterization of Previously Unidentified Drinking Water Disinfection By-Products
Investigators: Minear, Roger A. , Barrett, Sylvia
Institution: University of Illinois Urbana-Champaign , Metropolitan Water District of Southern California
EPA Project Officer: Hahn, Intaek
Project Period: November 1, 1998 through October 31, 2001
Project Period Covered by this Report: November 1, 2000 through October 31, 2001
Project Amount: $363,591
RFA: Drinking Water (1998) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The overall objective of this project is to develop new approaches for better characterization of disinfection by-products (DBP) molecular weight profiles by using tandem mass spectrometry (MS/MS) techniques. A prerequisite to making such procedures meaningful is the development of pre-separation procedures that will simplify the MS data. The specific objectives of this reporting period are to: (1) obtain size exclusion chromatography (SEC)-Cl profiles of chlorinated natural organic matter (NOM) samples, which are the basis for further fractionation, concentration, and MS/MS analysis; (2) separate/ fractionate chlorinated NOM and better define the chlorinated high molecular weight (MW) DBPs with ultrafiltration (UF) and SEC techniques with a combination detection of 36Cl, ultraviolet (UV), and dissolved organic carbon (DOC); (3) roughly estimate MW distribution and Cl/C atomic ratios of high MW chlorinated DBPs from chlorinated NOM; and (4) examine the effects of contact time, pH, and NOM on the formation of high MW DBPs. The past research period focused on searching for the chloride ion from MS/MS experiments in various samples, including chlorinated Suwannee River fulvic acid (SRFA) solutions, and fractionated chlorinated SRFA solutions obtained from UF followed by SEC. Additional investigations include: (1) the instrument's ability to perform electrospray high resolution analysis for accurate mass measurements; (2) ionization suppression effect in the presence of SRFA; and (3) preliminary solid phase extraction (SPE) of mixed standards.
Progress Summary:
Activities at MWDSC
Experimental Approach. At Metropolitan, chlorination of SRFA solutions was conducted at a starting concentration of SRFA of 1000 ppm in Milli-Q water, using sodium hypochlorite (NaOCl) at pH 7, and chlorine to total organic carbon (Cl2:TOC, w/w) ratios of 1:1 and 1:10. No quenching reagents or preservatives were added to the reaction mixtures afterwards. A 1 ml aliquot of acetonitrile (CH3CN) was added to 1 ml of each chlorinated SRFA solution prior to MS analysis. The chlorination of SRFA solutions used for the UF-SEC experiments were carried out at the University of Illinois-Urbana Champaign (UIUC).
Results and Discussion
Comparison of Electrospray (ESI) Positive Ion and Negative Ion Modes. Similar to the mass spectra of SRFA solutions obtained under ESI negative ion mode, mass spectra of SRFA solutions under the positive ion mode also displayed ion peaks at every mass. An alternating mass pattern was observed, with strong peaks at every other mass and weak peaks in between the strong peaks. The mass spectra of chlorinated SRFA solutions at Cl2:TOC ratios of 1:1 and 1:10 presented lower peak intensities compared to unreacted SRFA; however, the shifting of the ion distribution envelope of SRFA after chlorination toward lower masses observed under the ESI negative ion mode was not obvious under the ESI positive ion mode.
MS/MS Experiments of Chlorinated SRFA Solutions. Samples were introduced into the ESI source through loop injections with a 10 µl loop and a carrier fluid of 1:1 CH3CN:H2O. To ensure that precursor ions for the MS/MS experiments were selected automatically for loop injections, two reference compounds were used and compared for calibrating the first stage in the ESI negative ion mode: Ivory soap and Polyethylene Glycol (PEG) 600 diacid.
Due to the fact that the precursor ions of interest from chlorinated SRFA solutions generally are low in intensity, every effort has been made to achieve the optimized sensitivity for MS/MS experiments:
· To increase the number of scans with each sample injection, so as to increase the ion peak sensitivity, a flow rate of 20 µl/minute was used for the following MS/MS experiments.
· The instrument is capable of acquiring multiple precursor ions within one sample injection, but doing so decreases the acquisition time spent on each individual ion, which decreases the sensitivity of each ion. Therefore, usually only one ion was acquired during each sample injection.
· Two different ways to calibrate the ion masses at the first stage were investigated: using location data within the MS/MS experiment setup, versus setting up a separate surface insulation resistance (SIR) voltage reference file. The two calibration methods gave comparable results, and the one using location data within the MS/MS experiment setup was used throughout the experiments.
· It also was found that chlorination of freshly prepared SRFA solutions led to better defined mass spectra in terms of peak width and intensity than those from previously prepared SRFA solutions.
· To evaluate possible different intensities of the same ion generated by the two different sample injection methods, i.e., direct infusion versus loop injection, dichloroacetic acid (DCAA) was used as a model compound. It was found that direct infusion into the ESI source offered a five to six fold advantage in peak intensity compared to that from loop injection, possibly due to the loop injection's dilution effect.
· The different collision effects of two collision gases, xenon (Xe), and methane (CH4), were tested using 257 m/z from DCAA [35Cl2CHCOOH + 35Cl37ClCHCOOH -H]- as the precursor ion. CH4 gave stronger fragment ions of 35 and 37 than those from Xe.
MS and MS/MS Experiments of UF SEC Fractionated Chlorinated SRFA Solutions. Nine UF SEC fractionated chlorinated SRFA samples sent from UIUC were received at Metropolitan. The full scan mass spectra of the nine samples were obtained in both ESI positive and negative ion modes from 100 to 1,000 m/z. One common phenomenon was that for the same sample, the ion peak intensities from the ESI negative ion mode were stronger and better defined than those from the ESI positive ion mode. Two samples also were scanned up to 2,000 m/z in the ESI negative ion mode in search of high mass ions, but the ion peaks with mass higher than 800 m/z generally were weak in intensity and narrow in peak width, and no significant ions were observed above 800 m/z.
Using the experimental conditions optimized for the MS/MS experiments with chlorinated SRFA solutions, MS/MS experiments on two fractions with larger amounts of sample volume were conducted with CH4 as the collision gas, a maximum mass range of 1,000 m/z, a collision energy of 400 ev, and the time of flight (TOF) detector set at 2,100. No chloride ion was observed from these experiments. From another experiment, one fragment ion was observed at 26 m/z, suggesting a possible cyanide ion (CN-).
Accurate Mass Measurement. The instrument's ability to perform accurate mass measurements was demonstrated using two model compounds, DCAA and coumarin-3-carboxylic acid (CCA) at a mass resolution of 5,000. The ions of interest were the solvent adduct ion of DCAA, [DCAA+H2O+CO2-H]-, at 188.936 m/z, and the molecular ion of CCA, [CCA-H]-, at 189.018 m/z. The reference compound used was PEG 600 diacid. At a mass resolution of 5,000, the two peaks can be well separated. The ion corresponding to the DCAA adduct ion was 188.948 m/z, which was 12.5 mDa off the true value, and that corresponding to CCA was 189.021 m/z, which was 1.8 mDa off the true value. The DCAA adduct ion peak was much weaker than the CCA molecular ion, which contributed to the bigger margin of error.
Ionization Suppression Effect in the Presence of SRFA. Possible ionization suppression effects caused by the presence of a complex mixture such as SRFA solutions were evaluated by obtaining MS of DCAA in 1:1 CH3CN:H2O, at 250 ppm SRFA solution, and 500 ppm SRFA solution at two concentration levels, 1 ppm and 20 ppm. The qualitative results obtained from full scan spectra offered a rough estimation of the ionization suppression effect caused by the presence of SRFA in the solution; however, it is postulated that these effects strongly are compound and concentration dependent.
Preliminary Solid Phase Extraction (SPE) Experiments. A preliminary SPE experiment was carried out using a mixture of six chlorinated standards: 4-chlorobenzoic acid (CBA); chlorosuccinic acid (CSA); 3,5-dichloro-4-hydroxybenzoic acid (DCHBA); chloroacetic acid (CAA); DCAA; and dichlorophenol (DCP). The recoveries of standards from two separate runs were different and further experiments are needed.
Activities at UIUC
Experimental Approach. Carrier free stock HO36Cl solution was prepared via the reaction of Na36Cl with KMnO4, and H2SO4 with collection of the 36Cl2 gas in aqueous solution. Initial concentrations of NOM and HO36Cl/HOCl were 100 mg/L as C and 50 mg/L as Cl2, respectively. SRFA and Suwannee River humic acid (SRHA) were used as two representatives of NOM. Contact time (1, 24, and 120 hours) and pH (5.5, 7.5, and 9.5) were examined as variables. Removal of lower molecular weight material and inorganic 36Cl was by UF conducted in a continuous manner with a 500 Da membrane. Size exclusion chromatography was conducted using a 25 x 200 mm BIAX (Chrom, Germany) with a column packing of Toyopearl HW 50S resin (Japan). Polystyrene sulfonates (PSS) with MW of 18k, 8k, 4.6k, and 1.8k (Polysciences Inc., MA) were used as high MW markers.
Results and Discussion. The SEC-36Cl profile of the ultrafiltered chlorinated SRFA sample demonstrated that chlorine is incorporated into SRFA across the entire molecular weight range. Therefore, all sample fractions are appropriate for MS and MS/MS characterization study.
Elution Profiles
· Cl/C Atomic Ratios of High MW DBPs. By determining the carbon content of SEC fractions Cl/C ratios were determined. In the high molecular weight region the ratio was relatively constant and low, roughly 0.025.
· Effect of Contact Time. As contact time increased from 1 to 120 hours, the amount of high molecular weight DBPs decreased, which is very likely due to the formation of more low molecular weight DBPs, and the occurrence of more oxidation reactions.
· Effect of pH. As pH increased from 5.5 to 9.5, the formation of high molecular weight DBPs increased.
· Effect of NOM. Compared with SRFA, SRHA produced less total DBPs, but those DBPs had relatively higher molecular weight. This confirms that SRFA relatively has lower average molecular weight and is more reactive.
Future Activities:
Future efforts will include:
(1) Characterization of the chlorine-containing UF/SEC fractions by electrospray/MS/MS.
(2) The performance of the two mass spectrometers used so far (the AutoSpec at Metropolitan and the Quattro I at UIUC) will be compared, using the same samples, in terms of the sensitivity of chloride ion detection from the MS/MS experiments.
(3) Calculation of number average molecular weight and weight average molecular weight will be carried out on SRFA solutions, chlorinated SRFA solutions, and fractionated chlorinated SRFA solutions as a means for characterization. The results may give indications as to the effects of chlorination and fractionation by SEC.
(4) The Micromass AutoSpec MS at Metropolitan is capable of scanning in the ESI mode to 4,000 m/z. Future fractionated chlorinated SRFA samples will be scanned up to 4,000 m/z in search of possible high mass ions.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 25 publications | 10 publications in selected types | All 7 journal articles |
---|
Type | Citation | ||
---|---|---|---|
|
Ikeda K, Arimura R, Echigo S, Shimizu Y, Minear RA, Matsui S. The fractionation/concentration of aquatic humic substances by the sequential membrane system and their characterization with mass spectrometry. Water Science & Technology 2000;42(7-8):383-390. |
R826834 (2001) |
Exit |
|
Zhang X, Minear RA. Characterization of high molecular weight disinfection byproducts resulting from chlorination of aquatic humic substances. Environmental Science & Technology 2002;36(19):4033-4038. |
R826834 (2001) |
|
Supplemental Keywords:
drinking water, disinfection by-products, molecular weight, HPLC, SEC, MS/MS., RFA, Scientific Discipline, Water, Waste, Chemical Engineering, Physics, Environmental Chemistry, Chemistry, chemical mixtures, Analytical Chemistry, Drinking Water, alternative disinfection methods, complex mixtures, monitoring, public water systems, human health effects, molecular weight separation, exposure and effects, chemical byproducts, disinfection byproducts (DPBs), exposure, community water system, HPLC, toxicity, treatment, tandem mass spectrometry, water quality, DBP risk management, drinking water contaminants, drinking water treatment, other - risk managementProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.