Environ. Sci. Technol. 1988, 22, 1186-1 190
Yamaguchi, K.; Calderwood, T. S.; Sawyer, D. T. Znorg. Chem. 1986,25, 1289-1290. Nicholson, R. S.; Shain, I. Anal. Chem. 1964,36,706-723. Mullin, M. D.; Pochini, C. M.; McCrindle, S.; Romkes, M.; Safe, S. H.; Safe, L. M. Environ. Sci. Technol. 1984, 18, 468-476. Ballschmiter, K.; Zell, M.; Fresenius' 2.Anal. Chem. 1980, 302, 20-31.
(24) Roberts, J. L., Jr.; Calderwood, T. S.; Sawyer, D. T. J. Am. Chem. SOC.1983, 105, 7691-7696. Received for review October 8, 1987. Accepted March 14,1988. This work was supported by the National Science Foundation under Grant CHE-8516247 and by the Welch Foundation under Grant A-1042.
Small Molecular Weight Organic Amino Nitrogen Compounds in Treated Municipal Waste Watert Frank E. Scully, Jr.," G. Dean Howell, Helen H. Penn, and Kathryn Marina
Department of Chemical Sciences, Old Dominion University, Norfolk, Virginia 23529-0126 J. Donald Johnson
Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina 27599-7400
rn Concentrations of organic nitrogen compounds (total Kjeldahl nitrogen, total free amino acids, individual amino acids, and volatile amines) in primary and secondary effluents from municipal waste water treatment plants were measured and compared to concentrations of these compounds in primary and secondary effluents as reported by others. Primary treated municipal waste water effluents contained relatively low concentrations of dissolved free amino acids compared with those found in raw sewage as reported by others. A selective purge-and-trap method for the concentration of volatile amines was adapted to the analysis of waste water. Volatile organic amines, not previously reported in waste waters, were found in concentrations comparable to those of some of the amino acids. Secondary treatment generally reduces the concentrations of these amines below detection limits.
Introduction Typical nonnitrified municipal waste water effluents contain appreciable concentrations of ammonia (10-40 mg/L) ( I ) . When the water is disinfected by addition of chlorine, treatment plant operators rely on the rapid reaction of the ammonia with hypochlorous acid to produce monochloramine (NHzC1in eq l),the predominant disinfecting species in the effluent (1). In addition to amNHs HOC1 -* "$21 + HzO (1)
+
monia, waste waters contain a considerable number of organic amino nitrogen compounds which also react with chlorine but form nonbactericidal products (2-7). The organic N-chloramine products are particularly insidious because they respond to conventional chemical analysis for residual chlorine disinfectant concentration in the same way inorganic chloramines respond (8-10).Consequently, there has been concern that these methods (11)may be overestimating the disinfectant levels of treatment plant effluents (2-4). The compounds which compose the amino nitrogen fraction of waste water organics are poorly characterized. Jolley et al. (12)identified two nonvolatile amines, ten amino acids, and six purine or pyrimidine bases in HPLC concentrates of waste water effluents. Van Langenhove et al. (13) identified only two nitrogen-containingvolatiles, 'This paper was presented at the 193rd National Meeting of the American Chemical Society, Denver, CO, April 5-10, 1987. 1188 Environ. Sci. Technol., Vol. 22, No. 10, 1988
pyridine and trimethylamine, in an industrial waste water but suggested their presence was incidental. Several studies have identified most of the essential amino acids and quantitated them in raw sewage and in chlorinated raw sewage samples (14,15). As part of a program to evaluate the significance of amino nitrogen compounds in treated municipal waste waters, we have measured the concentrations of amino acids in unchlorinated primary and secondary effluents and identified and quantitated five volatile amines which appear to be natural components of municipal waste waters.
Materials and Methods General. All amines used as standards were of the highest purity available from Aldrich Chemical Co. and were used as received. Acid-washed Chromosorb W (60/80 mesh) was produced by Manville Products Corp. (Denver, CO) and obtained from Varian Associates. The copper chloride was reagent-grade obtained from Baker Chemical Co. Heptafluorobutyric anhydride (HFBA) was obtained from Pierce Chemical Co. (Rockford, IL). Chlorine-demand-free water (CDF water) (9) was used to prepare all solutions and to wash the XAD-2 and Dowex columns. Prepurified XAD-2 was obtained from Applied Science Laboratories (State College, PA) and further purified by Soxhlet extraction for 24 h each with methanol and methylene chloride. Before any waste water or standard solution was passed through the resin, it was washed successively with 300 mL of high-purity 2-propanol, 300 mL of high-purity methanol, with 300 mL of CDF water, and 300 mL of CDF water (adjusted to pH 2 with HC1. Dowex AG 50W-X8 cation-exchangeresin (100-200 mesh) in the hydrogen form was obtained from Bio-Rad Laboratories. Before any waste water or standard solution was passed through the resin, it was washed successively with 300 mL of 5050 methanol and 1 N HC1, 300 mL of 1 N HC1, 300 mL of CDF water, and 300 mL of CDF water adjusted to pH 2 with HC1. Equipment. A Hewlett-Packard Model 5890A gas chromatograph was operated in a purged splitless mode. A 0.32 mm i.d. X 30 m SPB-5 fused silica capillary GC column with a 0.25 pm film thickness (Supelco, Inc, Bellefonte, PA) was used. After a splitless injection for 0.50 min, purge was applied. After an initial temperature hold of 2 min at 50 "C, the column was temperature programmed at 10 "C/min to a final temperature of 150 OC and maintained at this temperature for 5 min. Chromatograms
0013-936X/88/0922-1186$01.50/0
0 1988 American Chemical Society
were recorded on a Hewlett-Packard Model 3393A recorder-integrator. A Hewlett-Packard Model 5985B GC/MS and RTE VI data system was used with 70 eV for electron impact ionization. Similar chromatographic conditions were used except the initial 50 "C was held 5 min and the temperature programmed at 5 "C/min to 150 "C and then at 20 OC/min to 250 "C. The mass spectrometer was scanned from 40 to 450 amu twice per second. The equipment used for high-performance liquid chromatography (HPLC) has been described previously (9). Description of Waste Water Samples. The waste waters used in this study were obtained from three different treatment plants. Plant 1is an advanced primary treatment plant which adds alum and polymer as coagulating agents. After flocculation and sedimentation the effluent is chlorinated before being discharged into an estuary. Plant 2 is a typical secondary treatment plant. Primary treatment consists of settling without chemical addition. The primary effluent is mixed with waste-activated sludge in an aeration tank in which dissolved oxygen is maintained above 2 mg/L. After final clarification (sedimentation) the water is disinfected with chlorine before it is discharged into an estuary. Both plants treat typical municipal effluent. Primary effluents from both plants typically contain ammonia concentrations ranging from 18 to 23 mg/L NH3-N and soluble total Kjeldahl nitrogen (TKN) concentrations of 1-5 mg/L as N. Plant 3 handles significant quantities of brewery waste with municipal waste. Primary effluent is subjected to an intermediary process using an oxidation tower followed by clarification and subsequent conventional activated sludge secondary aeration treatment. Final effluent is also chlorinated. Secondary effluent from this plant typically contains less than 1 mg/L NH3-N and soluble total Kjeldahl nitrogen (TKN) concentrations of 2-4 mg/L as N. During chlorine minimization studies at plant 3 it was determined that despite the presence of sufficient total residual chlorine (1.5-2.5 mg/L, 30 min contact time) adequate disinfection was not reliably obtained. Handling of Waste Water Samples. All waste waters examined in this study were collected prior to any chlorination step. Primary or secondary waste water effluents were obtained in 4-L glass bottles and sealed with PTFE-lined caps. Samples were acidified with high-purity concentrated sulfuric acid (10 mL/4-L sample), filtered through a Whatman Type F glass fiber filter, and stored at 4 "C until they were analyzed. Most were analyzed within 24 h. The sample from plant 1collected on 7/14/86 had been frozen immediately after filtration in a -80 "C freezer and analyzed 3 months later. Analyses. Acidified and filtered samples were analyzed in duplicate for soluble total Kjeldahl nitrogen (TKN) and ammonia within 14 days of sampling by U S . Environmental Protection Agency (EPA) Method 351.2 (16). A Scientific Instrument Corp. Model CFA-200 analyzer with a Model AD-20 block digestor was used. Volatile amines were analyzed by gas chromatography as their HFBA derivatives with electron capture detection (17) and by GC/MS comparison with standards. Before they were analyzed for total free amino acids, samples were taken to dryness by lyophilization and reconstituted with CDF water to give an overall 8-fold concentration. They were analyzed by precolumn derivatization and HPLC according to the method of Jones et al. (18). A one-point calibration with a standard sample of amino acids was used to approximate concentrations. Concentration of Amines from Waste Water Effluent. Waste water (2.0 L), which had been acidified and
filtered, was passed through a 2.5 cm X 4.0 cm column of XAD-2 resin to remove acids and neutrals before it was passed through a 2.5 cm x 12 cm column of Dowex AG 50W-X8 cation-exchange resin (100-200 mesh) in the proton form, The Dowex column was washed with 10 mL of high-purity water (adjusted to pH 2 with HCl). The column was eluted with 370 mL of 1 N KOH in water containing 10% methanol, The first 70 mL (column void volume) was discarded. The amines were recovered in the next 300 mL. The eluate was divided into separate 150-mL aliquots. Each was placed separately in a purge-and-trap apparatus and purged with high-purity helium. The purge-and-trap technique used here was an adaptation of the method of Chriswell(19). The apparatus was constructed from a 350-mL Pyrex gas washing bottle with side inlet (Corning 31750) and 24/40 gas outlet tube, In order to minimize condensation of water vapor in the trapping cartridges, the gas outlet tube was fitted with a 30-cm length of 5 mm i.d. glass tubing with a bend to direct the effluent in a vertical direction. The tube was wrapped in heating tape and heated to 55 "C. The end of this glass tube was fitted with a short length of polypropylene tubing, which enabled easy attachment and removal of trap cartridges. The trap cartridges were prepared from 5.75 in. long disposable pipets packed (5 mm X 4 cm) with copper chloride coated on acid-washed Chromosorb W (60/80 mesh) according to Chriswell (19). The cartridges and glassware used in the purge and trapping of amines were silanized by treatment with a 5% solution of chlorotrimethylsilane in toluene, rinsed with methanol, and ovendried. When a sample was to be purged, the washing bottle was immersed in a 65 OC water bath and the inlet connected by PTFE Swagelok fittings to a helium cylinder through a flow meter. With a minimum flow of helium purging the wash bottle, a 150-mL aliquot of column effluent from the Dowex column described above was placed in the bottle along with 1g of KOH and 50 g of KC1. A trap cartridge was fitted to the wash bottle and the helium flow increased to 380 mL/min. The amines and ammonia were trapped as copper complexes in the trap cartridges. When a cartridge became saturated, as indicated by a color change in the cartridge packing from green to blue, it was replaced by a fresh cartridge. Each 150-mL aliquot of Dowex column eluate was purged for a total of 2 h. When a waste water containing approximately 20 mg/L ammonia was analyzed, three cartridges were generally required for each 150-mL Dowex column eluate. Each copper chloride cartridge was placed in a GC oven at 100 "C and purged with high-purity N2 at a flow rate of 50 mL/min for 4 min to remove methanol. The cartridges were combined, and the volatile amines were recovered in the following fashion. The first cartridge was washed with 4.0 mL of 1 N KOH. The eluate from this cartridge was passed through a second, which was subsequently washed with a fresh 1.0mL of 1N KOH. The two washes were combined and passed through a third cartridge which was also rinsed by a fresh 1.0 mL of 1N KOH. This process was repeated with the remaining cartridges. The eluate from each cartridge was collected in a vial immersed in ice to minimize volatilization of amines. All washes were combined, saturated with KC1, and extracted with 1.00 mL of benzene. The benzene layer was recovered and dried over anhydrous sodium sulfate, and 0.50 mL was removed for derivatization with heptafluorobutyric anhydride (HFBA) (17). Environ. Sci. Technol., Vol. 22, No. IO, 1988
1187
Table I
step
sample manipulation
acidification and filtration of sample 2 XAD-2 resin for precleanup (removal of acids and neutrals) 3 Dowex cation exchange resin to preconcentrate amines 4 elution of Dowex with 1 N KOH containing 10% methanol 5 purge-and-trap of amines on copper chloride cartridges 6 purge methanol from trap cartridges with dry N2 and heat 7 recovery of amines from trap cartridge with 1 N KOH 8 extraction of amines into benzene 9 derivatization of amines with HFBA 10 analysis of HFBA-amines by GC/ECD -
Table 11. M i n i m u m Detection Limits and Overall Recoveries of Volatile Amines
amine
min detection limit: ppb
isoamylamine isobutylamine 2-methylbutylamine piperidine pyrrolidine
1.7 1.6
1
CDF water, acidified to pH 2 with HCl, was used as a system blank and was treated in a manner identical with that of waste water to ensure that no extraneous amines contaminated the system. A standard curve for each amine was prepared with 2-L solutions in CDF water containing 20 mg/L ammonia and amines with concentrations ranging from 8 to 40 pg/L in each of five amines: isobutylamine, 2-methylbutylamine, isoamylamine,pyrrolidine, and piperidine. These standard solutions were treated in a manner identical with waste water and analyzed by gas chromatography as described below. A total of seven separate solutions and seven blanks were used to obtain calibration points. Each point was determined from the average of the areas of gas chromatographic peaks obtained for three injections corrected for any contaminants in the blank. Corresponding solutions of the same amines were prepared in benzene in order to measure percent recoveries of each.
Results and Discussion Concentration and Analysis of Volatile Amines. The method used to concentrate and analyze volatile amines is summarized in Table I. Because the concentration of ammonia is 500-1000 times greater than that of any of the volatile amines identified in this study and because waste waters contain considerable quantities of organic compounds which might interfere with the analysis of the amines, extensive precleanup and concentration of the samples were performed. Beginning with 2 L of waste water the amines were concentrated 2000-fold before they were analyzed. To optimize the recoveries for each step, model solutions of three of the amines identified in wastewater, isoamylamine, piperidine, and pyrrolidine were used. The conditions described in each step represent a compromise between different conditions which maximized recovery of some amines while decreasing recovery of others. Parameters which were examined include the length of the XAD-2 column, elution of the Dowex column with basic solutions containing different organic cosolvents, volumes of Dowex column eluates subjected to purge and trap, and solutions and additives used to maximize recovery of the amines from the trap cartridges. The greatest losses of amines in the concentration steps occurred because of inefficient recovery of the amines from the XAD-2 and Dowex columns and because of losses in the purgeand-trap step. The sample preparation steps which followed purge and trapping appeared to account for only minor losses of the amines. Calibration curves were generated for each of the five amines identified in waste water. The correlation coefficients for isobutylamine and 2-methylbutylamine were better than 0.999; the correlation coefficients for piperidine and isoamylamine were better than 0.99; and the correlation coefficient for pyrrolidine was 0.96. The minimum 1188
Environ. Sci. Technol., Vol. 22, No. 10, 1988
recovery 53 f l % b 40 f I % b 51 f 6%' 36 2%' 8 2%c
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Flgure 1. Gas chromatogram of HFBA-derivatized purge-and-trap concentrate of volatile amines isolated from the primary effluent of plant 2. Amine derivatives (retention times): isobutylamine (5.4 min), 2-methylbutylamine (6.9 min), isoamylamlne (7.1 min), pyrrolidine (7.9 rnin), and piperidine (8.5 min). The isobutylamine peak overlaps with the diethylamine impurity in the triethylamine used in derivatization.
detection limits and overall percent recovery of each amine are given in Table 11. Analysis of Amino Acids. Amino acids were analyzed directly after lyophilization of the filtered waste water. Concentrations of amino acids in some of the samples were also analyzed after cleanup by passing samples through XAD-2 and Dowex cation-exchange resin, recovery of the amino acids by base washing the Dowex, and derivatization with o-phthalaldehyde. Results with and without cleanup in this manner did not differ. Occasionally samples contained interfering substances which precluded quantitation of some of the minor amino acids. Total free amino acid concentrations (TFAA)were determined by summing the concentrations of all the amino acids except proline, which could not be determined by the method used. Identification and Quantification of Amines. Figure 1is the gas chromatogram/electron capture detection of the HFBA-derivatized concentrate of the amines from a primary waste water effluent. Five volatile amines were identified in effluents: isobutylamine, 2-methylbutylamine, isoamylamine, pyrrolidine, and piperidine. The presence of all five amines was confirmed by comparison of the mass spectra of each HFBA-derivatizedamine with the GC/MS of a standard mixture of 32 ppb of each amine concentrated from aqueous solution and derivatized with HFBA. The total ion current reconstructed GC shown in Figure 2 demonstrates that these amines are the principal compounds present in the isolates. One other major component with a retention time of 12.8 min remains unidentified. Components which eluted earlier than 5 rnin in each chromatogram were similarly difficult to identify. That these particular amines were found is not surprising. Six of them can originate from biochemical decarboxylation of essential amino acids catalyzed by the
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