In the Laboratory
Gas Chromatography Analyses for Trihalomethanes: An Experiment Illustrating Important Sources of Disinfection By-Products in Water Treatment
W
Terese M. Olson* Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, MI 48109-2125;
[email protected] Alicia C. Gonzalez Metropolitan Water District of Southern California, La Verne, CA 91750 Victor R. Vasquez California Regional Water Quality Control Board, San Diego Region, San Diego, CA 92124
Background and Rationale Chlorination processes are an important and effective disinfection strategy in drinking water treatment. It is now well known, however, that side reactions of chlorine species with natural organic matter—or humic material—produce chemical disinfection by-products (DBPs) that can have toxic health effects (1–3). One important class of DBPs is trihalomethanes (THMs), which include compounds such as CHCl3, CHBrCl2, CHBr2Cl, and CHBr3. This experiment 1. Illustrates the types of precursor moieties in natural organic matter that react with chlorine to form THMs and other DBPs using a model substrate, resorcinol (1,3-dihydroxybenzene); 2. Explains how brominated THMs are formed during chlorination processes; 3. Demonstrates solvent extraction techniques and the measurement of extraction efficiencies for analytes in aqueous solution; and 4. Provides an environmentally relevant example of gas chromatography analysis for compound mixtures.
Other GC experiments have been developed that illustrate THM analysis (4 ) and THM formation using humic acid itself as the precursor (5). The experiment described herein represents an alternative approach that emphasizes the structural features of natural organic matter that are thought to give rise to THMs. By considering the extensively studied halogenation mechanism of a model precursor compound such as resorcinol, it is possible to better understand some of the facets of humic acid reactions with chlorine. Instruction on the essential features of the resorcinol chlorination mechanism is first provided with emphasis on why the formation of THMs leads to other organohalogen products as well. Students can compare their THM yields with those reported in the literature and then draw conclusions about the effect of bromide. It is also possible to discuss the practical objectives of pre-chlorination treatment strategies to minimize DBP formation with greater insight when students have explored the structural features of humic acids that give rise to DBPs.
The experiment is designed for an undergraduate junioror senior-level environmental chemistry course, but could easily be incorporated into graduate courses as well. No prior gas chromatography experience is assumed. Disinfection By-Product Formation Pathways The formation pathways of chlorination by-products have been extensively investigated since humic matter was first identified as an important precursor for trihalomethanes in water-disinfection processes (6 ). Humic substances are ubiquitous and complex macromolecular organic structures that form during the diagenesis of plant and animal matter. A review of the literature indicates that a variety of humic functional groups, including hydroxybenzenes (7 ), aliphatic carboxylic acids (8), and hydroxybenzoic acids (9), react with HOCl to form chloroform. Hydroxybenzenes with metasubstituted OH groups are particularly reactive and consequently the halogenation of these moieties has been the subject of much research. Most of these mechanistic studies have focused on model compounds, such as resorcinol (10–12): OH
OH
resorcinol,,
Reactions of HOCl with resorcinol are thought to proceed by the sequential electrophilic substitution of chlorine on the aromatic ring (12) followed by the hydrolysis of the pentachlorinated resorcinol (13), ring-opening to form keto carboxylic acids, decarboxylation, chlorination of the enol form of the resulting ketone, and base-catalyzed hydrolysis of the trichloromethyl ketone. The last two steps of the sequence are referred to as the “haloform” reaction in many organic chemistry texts (14 ). One example of the overall reaction scheme is shown in Scheme I, but the mechanism is complex and multiple chloroform formation pathways are thought to occur. Results of 13C labeling studies, however, have demonstrated that all of the haloform carbon originates from the activated C2 carbon of the 1,3-dihydroxybenzene (10).
JChemEd.chem.wisc.edu • Vol. 78 No. 9 September 2001 • Journal of Chemical Education
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In the Laboratory
orcinol (1-methyl 3,5-dihydroxybenzene), the chloroform yields at 10 min (11.6:1 Cl2/orcinol) were 95% of the ultimate yields (17).
O Cl + 5 H2O O
Experimental Overview
Cl O Cl *
Cl
H2O
* CHCl 2COCCl
CHCCl2CO2H
O Cl * CHCl 2COCCl
* CHCl 2COCCl * Cl3CCOCCl
− CO2
CHCCl2CO2H
HOCl
CHCHCl2
CHCHCl2
OH
−
* CHCl 2COCCl * Cl3CCOCCl
CHCHCl2 CHCHCl2
* CHCl 3 + Cl2HCHC
−
CClCO2
Scheme I An example of a reaction pathway for the production of chloroform by the chlorination of resorcinol.
Bromide concentrations in drinking water supplies are generally less than 3 mg/L; nevertheless, even small amounts of bromide in chlorinated drinking water supplies can lead to the formation of bromine-substituted haloform compounds (15). In the presence of HOCl, bromide is first oxidized to bromine, which is less extensively hydrolyzed than Cl2: Br᎑ + HOCl → HOBr + Cl᎑ HOBr + Br᎑ + H+
Br2 + H2O
(1)
Sample Generation The sample “unknown” in this experiment consists of a 2 × 10 ᎑6 M resorcinol solution that has been spiked with bromide and chlorinated at a 50:20:1 ratio of NaOCl:KBr:
(2)
Molecular bromine is an even stronger electrophile than HOCl and it reacts rapidly with many of the same humic functional groups that react with HOCl to yield bromoform and other brominated by-products. Br2 reacts initially with many phenols (as phenate), for example, at diffusion-controlled rates (16 ). The rapid kinetics of these reactions accounts for the relative importance of bromine-substituted haloform compounds in chlorinated drinking water. The experiment described in this paper also illustrates how competitive reactions between HOCl and Br2 with phenols lead to mixtures of chlorine- and bromine-substituted haloform compounds. THM yields obtained by chlorinating or brominating resorcinol have been reported for several reaction conditions and reaction times (see Table 1). In the case of chloroform production, research has demonstrated that ultimate THM yields increase sharply for reactant ratios ([HOCl]/[resorcinol]) up to 6 to 8, and then more slowly at higher ratios (10). With at least a tenfold excess of HOCl, ultimate chloroform yields are greater than 86% at neutral pH. Bromoform yields under the same conditions are less than chloroform yields but greater than 50%. The greater selectivity of bromine implies that the presence of bromide should cause the overall THM yield to decrease, although more halogen may ultimately be present as other organohalogen compounds. The rate of haloform production for meta-substituted dihydroxybenzene compounds is relatively rapid at neutral pH. Kinetic studies of resorcinol chlorination, for example, show that CHCl3 yields at 5 min are about 70% of the ultimate yields with a reactant ratio of Cl2/resorcinol of 12:1 (7). For 1232
Materials and Equipment All reagents required for this experiment are readily available commercially. Standard mixtures of the four trihalomethane compounds for GC calibration are also commercially available. The GC system is equipped with an electron capture detector and a fused-silica capillary column (HP-1) that contains a methyl silicone stationary phase. Good resolution of the four analyte and internal standard peaks is obtained over a run time of less than 5 min.
Table 1. Reported Haloform Yields from the Reaction of Halogen (X2) and Resorcinol at pH 7 Reaction time/h
[X2]:[Resorcinol]
T/°C
CHCl3
12:1
15
2
85
7
CHCl3
12:1
15
0.083
60
7
0.67
THM
Yield (%)
Ref
CHCl3
11.6:1
25
87.7
17
CHCl3
10:1
10
24
86
10
CHBr3
10:1
10
24
51
10
350
300
250
200
internal standard
Cl Cl
Pre-Lab Discussion and Safety Protocols Laboratory sessions begin with a brief overview of the objectives of the exercise, the rationale for the choice of resorcinol as a model substrate, and important features of the reaction mechanism. Principles of GC analysis and solvent extraction are reviewed briefly and a quiz based on the laboratory instructions and readings is usually administered. Safety protocols, hazards, and MSDS sheets are also reviewed.
bromoform
OH
chlorodibromomethane
*
Cl
5 HOCl
dichlorobromomethane
*
Cl
chloroform
Cl
Signal / mV
OH
150
100 0
1
2
3
4
5
Retention Time / min Figure 1. Gas chromatogram of THM compounds and 1,2,3-trichloropropane (internal standard) in a calibration standard containing 0.5 µg/mL of each THM in pentane; injection volume was 2 µL.
Journal of Chemical Education • Vol. 78 No. 9 September 2001 • JChemEd.chem.wisc.edu
In the Laboratory
resorcinol. The sample is immediately mixed and allowed to react in the dark for 10 min. Thiosulfate is added to quench the reaction. A 10-min reaction period was selected to minimize the total experiment time and volatilization losses.
Sample Extraction and GC Analysis Each group prepares a five-point calibration curve, determines the extraction efficiencies of the four THM compounds, CHCl3, CHCl2Br, CHClBr2, and CHBr3, and analyzes the unknown reaction mixture. An internal standard calibration approach is used in which four standard THM mixtures and a blank are spiked with 1,2,3-trichloropropane. Students are asked to determine the response factors and retention time windows (±3 s) of each compound. A typical calibration standard chromatogram is shown in Figure 1. Pentane is used to extract the THM compounds from aqueous samples after salt is added to promote the recovery. Extraction efficiencies of the THM compounds are determined by extracting an aqueous solution to which a known amount of a THM standard mixture and salt has been added. The pentane extracts are also spiked with the internal standard. The identification of trihalomethanes in the unknown sample is based on the retention time windows. Yields of the THM compounds are quantified using the student calibration curves and estimates of the extraction efficiencies. Hazards Students should be advised of the volatility and potential carcinogenicity of trihalomethanes and solvents used in the experiment. All sample preparations and extractions should be performed in fume hoods and the students should wear nitrile gloves and safety glasses. Since the GC analyses involve the use of electron capture detection (ECD), the students should be also be apprised before the experiments of the radioactivity exposure hazards associated with tampering with this sealed source detector. The ECD should comply with all safety inspection regulations and its users should be properly authorized. Results and Discussion After calibration and analysis of the samples, individual students are asked to present their results in the form of a report and to answer several post-laboratory questions. Average extraction efficiencies and THM yields (relative to the initial resorcinol concentration) are given in Table 2. The studentdetermined average total molar yield (i.e., moles of all THM compounds per mole of resorcinol) for a 10-min reaction Table 2. THM Extraction Efficiencies and Yields in Reaction Mixture Sample THM CHCl3
Extraction Efficiency a
Yieldb
0.64 (0.09)
0.17 (0.07)
CHCl2Br
0.71 (0.08)
0.23 (0.07)
CHClBr2
0.75 (0.03)
0.15 (0.05)
CHBr3
0.81 (0.02)
0.02 (0.01)
aValues bYields
in parentheses are standard deviations for three determinations. are molar fractions based on the initial resorcinol concentration.
period was 57 (±10)%. Based on the published yields of chloroform and bromoform in other resorcinol chlorination and bromination studies (see Table 1), the students’ average total THM yield is consistent with what might be expected for a reaction mixture. The students’ average yield, for example, is between the chloroform and bromoform yields reported by Boyce and Hornig (10). The most critical skills that students must master to obtain reproducible results include minimizing volatilization, transferring exact solvent volumes, and developing reproducible injection techniques. Suggestions to improve student performance in these areas are provided in the supplemental material.W With groups of about three students, each group having access to a GC, the laboratory exercise can be completed in approximately four hours. Alternatively, if only one GC station is available to the class, the laboratory can be conducted as a demonstration in which students prepare the standards and sample extracts and the laboratory instructor demonstrates the chromatography analysis. Acknowledgments We thank the National Science Foundation (DUE9452477) and its ILI program for funding the purchase of the gas chromatography instrumentation described in this paper. We are also grateful for support from NSF (BES9257896 award to TMO) and a Fluor Foundation Graduate Teaching Fellowship (award to VRV) that enabled the necessary curriculum development. The helpful comments of the reviewers are also acknowledged. W
Supplemental Material
Equipment, reagent, and stock solution lists, details of the experimental procedure, and suggestions for trouble shooting, possible variations of the experiment, and sample postlaboratory questions are available in this issue of JCE Online. Literature Cited 1. Cantor, K. P. Cancer Causes Control 1997, 8, 292. 2. Waller, K.; Swan, S. H.; DeLorenze, G.; Hopkins, B. Epidemiology 1998, 9, 134. 3. King, W. D.; Marrett, L. D.; Woolcott, C. G. Cancer Epidemiol. Biomarkers Prevention 2000, 9, 813. 4. Graham, R. C.; Robertson, J. K. J. Chem. Educ. 1988, 65, 735. 5. Brush, R. C.; Rice, G. W. J. Chem. Educ. 1994, 71, 293. 6. Rook, J. J. Water Treat. Exam. 1974, 23, 234. 7. Rook, J. J. Environ. Sci. Technol. 1977, 11, 478. 8. Rockwell, A. L.; Larson, R. A. In Water Chlorination: Environmental Impact and Health Effects; Proceedings of the Second Conference on the Environmental Impact of Water Chlorination, Gatlinburg, TN, Oct 31–Nov 4, 1977; Jolley, R. L.; Gorchev, H.; Heyward Hamilton, D. Jr., Eds.; Ann Arbor Science Publishers: Ann Arbor, MI, 1978; Vol. 2, pp 67–74. 9. Larson, R. A.; Rockwell, A. L. Environ. Sci. Technol. 1979, 13, 325. 10. Boyce, S. D.; Hornig, J. F. Environ. Sci. Technol. 1983, 17, 202. 11. Heasley, V. L.; Burns, M. D.; Kemalyan, N. A.; McKee, T. C.; Schroeter, H.; Teegarden, B. R.; Whitney, S. E.; Wershaw, R. L. Environ. Toxicol. Chem. 1989, 8, 1159.
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In the Laboratory 12. Rebenne, L. M.; Gonzalez, A. C.; Olson, T. M. Environ. Sci. Technol. 1996, 30, 2235. 13. Gonzalez, A. C.; Olson, T. M.; Rebenne, L. M. In Water Disinfection and Natural Organic Matter; Minear, R. A.; Amy, G. L., Eds.; ACS Symposium Series 649; American Chemical Society: Washington, DC, 1996; pp 48–62. 14. Solomon, T. W. G. Organic Chemistry, 6th ed.; Wiley: New York, 1996; pp 759–761.
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15. Richardson, S. D.; Thruston, A. D. Jr.; Caughran, T. V.; Chen, P. H.; Collette, T. W.; Floyd, T. L.; Schenck, K. M.; Lykins, B. W. Jr.; Sun, G.-R.; Majetich, G. Environ. Sci. Technol. 1999, 33, 3378. 16. Tee, O. S,; Paventi, M.; Bennett, J. M. J. Am. Chem. Soc. 1989, 111, 2233. 17. Norwood, D. L.; Johnson, J. D.; Christman, R. F.; Hass, J. R.; Bobenrieth, M. J. Environ. Sci. Technol. 1980, 14, 187.
Journal of Chemical Education • Vol. 78 No. 9 September 2001 • JChemEd.chem.wisc.edu
