Environ. Sci. Technol. 1997, 31, 1946-1952
Highly Selective Determination of Macromolecular Chlorolignosulfonic Acids in River and Drinking Water Using Curie-Point Pyrolysis-Gas Chromatography-Tandem Mass Spectrometry ALBERT-JAN BULTERMAN, WILLEM M. G. M. VAN LOON,* RUDI T. GHIJSEN, AND UDO A. TH. BRINKMAN Free University, Department of General and Analytical Chemistry, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands INA M. HUITEMA AND BOB DE GROOT Water Transport Company Rijn-Kennemerland (N.V. WRK), Laboratory for Special Research, P.O. Box 10, 3430 AA Nieuwegein, The Netherlands
A highly selective pyrolysis-gas chromatography-tandem mass spectrometry (Py-GC-MS-MS) procedure for the quantitative determination of high molecular weight (MW >1000) chlorolignosulfonic acids in river and drinking water is presented. The use of MS-MS considerably enhanced the selectivity toward 2-methoxy-5-chlorophenol (5-chloroguaiacol; a highly characteristic pyrolysis product of chlorolignosulfonic acids) as compared to SIM conditions. The dissociation of the parent ion at m/z 143 (C6H4ClO2+) into the daughter ion at m/z 115 (C5H4ClO+, loss of CO) was monitored due to its high selectivity in the complex pyrolysates. Standard addition quantification appeared to be mandatory due to a strong matrix dependency of the pyrolysis efficiency of 5-chloroguaiacol from the chlorolignosulfonic acids. A repeatability of 5% RSD (n ) 5) was obtained for a prepurified drinking water sample. The detection limit of the procedure is 0.5 µg/L (S/N ) 3) of chlorolignosulfonic acids in drinking water and has been decreased by a factor of 60 as compared to the former Py-GC-MS-SIM procedure. The CLSA concentrations in the river Rhine have decreased strongly during the past 5 years, leading to CLSA concentrations of ca. 26 µg/L at present. It was found that the waterworks WRK [which produces prepurified drinking water using coagulation with Fe(III)Cl3 and rapid sand filtration] showed an average water purification efficiency of CLSA of 51%; Sx ) 27%. The waterworks GWA (which produces finished drinking water using among others ozonization combined with biological active carbon filtration) showed a purification efficiency of 92-93% for CLSA, which resulted in low (0.6-0.8 ug/L) concentrations of CLSA in drinking water of the city of Amsterdam. The analytical strategy for the selective and quantitative determination of CLSA can possibly be applied to other synthetic polymer classes in water.
* Corresponding author fax: 31-20-444-7543.
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Introduction Chlorolignosulfonic acids (in this work abbreviated as CLSA) are produced as waste materials by sulfite pulp mills (1). CLSA are produced from lignins during sulfonation processes of wood chips and subsequent bleaching processes of the cellulose pulp produced. It has been reported that more than 70% of the effluents of these pulp mills consists of macromolecular CLSA (MW >1000) (1-3). Because these macromolecular CLSA cannot pass through the cell membranes of living organisms, they were at first considered to be biologically inactive (1). Recent research indicated however that the macromolecular fraction of kraft pulp mills, presumably composed of chlorothiolignins, was toxic to several marine species in their early life stages (4, 5). Therefore, macromolecular CLSA may possibly cause toxic effects on the freshwater aquatic ecosystem, but little information has been reported on this matter. Being anthropogenic pollutants, CLSA in principle may be considered undesirable in drinking water. A decade ago, the Water Transport Company RijnKennemerland (N.V.WRK) initiated a research program to develop analytical procedures for CLSA in pulp mill effluents, surface water, and drinking water (6-9). The program was entirely focused on CLSA because only sulfite pulp mills are present in the river Rhine basin. The program yielded a selective and quantitative Py-GC-MS-SIM method for macromolecular CLSA in river and drinking water (9). The Py-GC-MS-SIM analysis focused on the 5-chloroguaiacol substructure, which is in practice a specific pyrolysis product of CLSA. Chemical background signals, caused by pyrolysis products from aquatic humic substances, were very intense even under SIM conditions. The detection limit of the CLSA in drinking water was determined mainly by these interfering background signals. As the actual levels of CLSA measured in drinking water were close to or below the detection limit (9), the need arose for a more selective and sensitive method. The method can be improved at three stages, viz., the isolation, separation, and detection stages. The selectivity of the isolation procedure could be improved, or additional cleanup steps could be used. Size exclusion chromatography (SEC) was used in an attempt to separate the CLSA from aquatic humic substances (7), but this approach was not successful due to a strong overlap of their molecular weight distributions. Cherr et al. reported the successful separation of lignin-derived macromolecules into discrete bands by means of electrophoresis (5). Their procedure could possibly be applicable to separate CLSA and humic materials. Improvement of the GC analysis probably is difficult because of the high intensity of the chemical background signals. Although GC-GC techniques could provide a solution, the easiest and most promising alternative is the use of tandem mass spectrometry (MS-MS) to increase the selectivity of the method. Today, MS-MS is recognized as a powerful tool to filter out chemical background signals and to enhance the selectivity of MS; the loss in sensitivity is in general compensated by the gain in selectivity (10-12). As regards bio- and geopolymers, Py-MS-MS has been used to analyze lignin and cellulose (13), barley tissue (14), carbohydrates (15), miscellaneous biomaterials (16), kerogens (17), coal (18), and tar sand (19). In the case of complex samples, with this technique a large variety of pyrolysis products are introduced into the ion source of a mass spectrometer simultaneously. Consequently, many produced fragment (parent) ions will often contribute to a single m/z value. Several structurally different daughter ions produced after MS-MS of such a group of parent ions may still contribute to the same m/z
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1997 American Chemical Society
value, especially if compounds at trace concentrations are monitored. Therefore, with low-resolution MS-MS such a method probably is not sufficiently selective when trace analysis in complex samples has to be performed (20). Considering cost-effectivity, we prefer to perform Py-GCMS-MS instead of Py-high resolution-MS-MS. As regarding bio -and geopolymers, to our knowledge, Py-GC-MSMS has only been used to give structural evidence of pyrolysis products produced from hair (21). Therefore, this probably is the first report on aquatic trace analysis using Py-GCMS-MS. In this paper, we report the development of a Py-GCMS-MS procedure for the determination of CLSA in river water and drinking water. The four goals of this study were (a) to increase the selectivity of the currently available PyGC-MS-SIM method and to decrease the detection limit of the analysis of CLSA in complex aquatic humic matrices using MS-MS, (b) to investigate the standard addition quantification method in more detail, (c) to evaluate water purification efficiencies of CLSA in two waterworks, and (d) to propose a generally applicable analytical strategy for the quantitative determination of polymers in water.
Experimental Section Chemicals, Materials, and Instrumentation. Ultrapure water was obtained from a Millipore-Q system (Millipore, Bedford, MA). 5-Chloroguaiacol was purchased from Helix Biotechnology Corp. (Richmond, Canada). Amberlite XAD-8 resin (analytical grade) was purchased from Alltech (Deerfield, IL, mesh size 20-60) and additionally cleaned for 24 h by Soxhlet extraction with methanol. Other materials used are described elsewhere (8). Freeze-drying was performed with an Edwards micro modulyo freeze dryer (Sussex, U.K.). Curie-point pyrolysis was performed using a FOM 3-LX pyrolysis injector (FOMAMOLF, Amsterdam, The Netherlands). Ferromagnetic pyrolysis wires (Philips, Eindhoven, The Netherlands) with a Curie-point temperature of 510 °C were used. The pyrolysis energy (60 W) was supplied by a Horizon (Heathfield, Sussex, U.K.) Curie-point pyrolyzer. The pyrolysis head was surrounded by a home-made (Free University) external heating block and was mounted into an injection port of a Model GC 8065 gas chromatograph (Fisons, Weesp, The Netherlands). The GC system for Py-GC-MS-MS analysis contained a 0.5 m × 0.32 mm i.d. precolumn and a 30 m × 0.25 mm i.d. DB-5MS column (film thickness, 0.25 µm; J&W, Folsom, CA). A splitless injector was used for the optimization of collisioninduced dissociation (CID) conditions. The GC system was coupled to a Model Quattro triple-stage quadrupole mass spectrometer (Fisons). MS-MS was performed by CID with argon as the collision gas. Sampling. Sulfite pulp mill effluent samples were collected on March 22 and 23, 1994, from PWA Mannheim (Mannheim, Germany), Holtzmann Papier (Karlsruhe, Germany), Stracel (Strasbourg, France), Baienfurt der Feldmu¨hle (Baienfurt, Germany), and Cellulose Attisholz AG (Attisholz, Switzerland). A volume-averaged reference pulp mill effluent sample was composed using the individual effluent samples (9), and the DOC (160 mg/L) and AOX (3.0 mg/L) content were determined. Prior to the isolation, the reference sample was diluted to a DOC content of 4.7 mg/L and to an AOX content of 90 µg/L. The yield of CLSA from this reference sample was 12.0 mg. The following water samples were taken for CLSA analysis: Rhine River water at the inlet of the WRK (Nieuwegein, The Netherlands, indicated as Rhine water), prepurified drinking water at the outlet of the WRK (indicated as WRK water), dune-filtered water at the inlet of the municipal drinking water company of Amsterdam (GWA, Amsterdam, The Netherlands, indicated as Dune water), and drinking water at the outlet of the GWA after treatment with ozone
and biological active carbon filtration (indicated as GWA water). Isolation and Preparation of Samples for Py-GC-MS(MS) Analysis. The isolation procedure has been reported before (8) but has been improved in this study. Briefly, a water sample of 5 L was microfiltrated (0.45 µm) and acidified with 5 M HNO3 to pH 1. The sample was pumped through an XAD-8 column (flow, 3 mL/min), and the adsorbed material was desorbed with 25 mL of 0.1 M NaOH/methanol (50/50 v/v; flow, 1 mL/min). The eluate was subjected to cation exchange (1 mL/min), and 15 mL of water/methanol (50/50 v/v) was used to flush the cation-exchange column. Next, the sample was diluted to 100 mL with water and ultrafiltrated until the retentate volume was 10 mL. The ultrafiltration step was repeated once to remove methanol as much as possible. The retentate was then put into a 10-mL vial, frozen with liquid nitrogen, and subsequently freeze-dried. The yield of isolated material was weighed by transferring it with a small amount of methanol into a preweighed 1-mL vial. The methanol was then evaporated under nitrogen, the vial was weighed again, and the yield was calculated. Next, a solution of 8.0 mg/mL of the isolated material was prepared in water/ methanol (50/50 v/v), which was stored at -20 °C. Py-GC-MS(-MS) Analysis. A 5-µL droplet of the 8 µg/ µL sample solution was placed on the tip of a pyrolysis wire, rotated slowly, and dried under reduced pressure (0.2 atm). The pyrolysis wire was inserted into a low-dead-volume (80 µL) glass liner. The distance between the tip of the wire and the bottom of the glass liner was kept at 17 mm for maximum pyrolysis efficiency. Pyrolysis injector temperatures used were: injection port, 240 °C; external heating block, 150 °C; liner, 200 °C. The sample was conditioned in the liner for 2.5 min before pyrolysis, and the pyrolysis time was 4 s. The GC oven temperature was held at 35 °C for 2 min and was then raised to 300 °C at 20 °C/min. The GC-MS interface was held at 270 °C, and the ion source was held at 225 °C. The EI electron energy used was 70 eV, and the emission current used was 150-200 µA. SIM conditions used were as follows: selected ions, m/z 158, 160, 143, and 145, two ions monitored per run; monitoring time per mass, 0.08 s; interscan delay, 0.02 s; cycle time, 0.2 s. MS-MS conditions used were as follows: parent ions, m/z 158, 160, 143, and 145; daughter ions, m/z 143, 145, 115, and 117, two reactions monitored per run; span, 0.80 u; monitoring time per mass, 0.08 s; interscan delay, 0.02 s; cycle time, 0.2 s. A chlorine isotope ratio 95% confidence interval for m/z 117/115 of 32 + 12% was calculated (27 samples) and used as an identification criterion. CID Optimization. The pressure in the collision cell was optimized using the gas perfluorotributylamine. The dissociation of parent ion m/z 131 to its daughter ion m/z 69 was monitored. At the optimum collision energy for this dissociation, the pressure in the collision cell was optimized by monitoring the abundance of the daughter ion. The optimized gas pressure appeared to be reproducibly 5.0 × 10-5 mbar as measured in the analyzer. The collision energy of the CID of fragment ions of 5-chloroguaiacol was optimized using splitless injections of 2.2 ng of 5-CG and using a CID gas pressure in the analyzer of 5.0 × 10-5 mbar. Injection conditions used were as follows: injector temperature, 280 °C; injection volume, 1 µL; splitless time, 60 s. Quantification of the CLSA. A pyrolysis calibration curve (8-40 µg) was determined for macromolecular material isolated from the river Rhine. To correct for the pyrolysis matrix effect described above, a standard addition method was used (9). A sample is first analyzed (n ) 2) without the addition of the reference and, next, with a fixed amount of added reference material (n ) 2). A standard addition calibration curve was measured using 40 µg of macromolecular material isolated from prepurified drinking water and using standard additions of 0-16 µg. The concentration of
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CLSA in a sample can be calculated using eq I, which is a simplified form of the previously reported quantification equations (9).
[CLSA]sam )
DOCref QTsam Qpyref Ssam × py × T × (I) FDOC S Q sam Q ref sam+ref - Ssam |
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1
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2
3
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4
In this equation, [CLSA]sam is the concentration of CLSA in the sample, DOCref is the DOC of the reference, FDOC is the DOC fraction factor (0 < FDOC < 1) for the conversion of the absolute weight to the weight of the DOC, QTsam is the total amount of macromolecular material isolated from the sample, QTref is the total amount of macromolecular material isolated from the reference effluent sample, Qpysam is the pyrolyzed amount of the sample, Qpyref is the pyrolyzed amount of reference material, Ssam is the signal of the sample, and Ssam+ref is the signal of the sample with a standard addition. FDOC was calculated previously with isolated reference material (FDOC ) 0.53) (8), and it is assumed that FDOC is equal for the isolated material and the total organic compound fraction in the sample. The isolated volumes for the reference and the sample must always be equal. The AOX can be calculated using eq II.
AOXsam ) AOXref ×
QTsam Q
py sam
×
Qpyref Q
T ref
×
Ssam (II) Ssam×ref - Ssam
In this equation, AOXsam is the AOX of the sample and AOXref is the AOX of the reference. It is assumed that the AOX content of the isolated material and of the total organic compound fraction in the sample are equal. The pyrolysis efficiency is defined in this study as the percentage of a specific macromolecular substructure, which is transformed during pyrolysis into the gas phase and which becomes amenable to GC. The standard addition (SA) pyrolysis efficiency is defined as the increase of the 5-CG signal per microgram of reference CLSA added (4 µg of reference material added) to the sample that is determined (see eq III, abbreviations are explained above). Note that this SA pyrolysis efficiency depends on the sample matrix and on the instrumental sensitivity.
SA pyrolysis efficiency ) [Ssam+ref - Ssam]/Qpyref (III) The relative SA pyrolysis efficiency (0-100%) is defined in this study as the SA pyrolysis efficiency of a sample divided by the SA pyrolysis efficiency of another sample from a sample series determined on the same day, the latter with the highest SA pyrolysis efficiency. Evaluation of Water Purification of CLSA. The removal of CLSA in two waterworks was investigated: (a) the WRK, a waterworks that produces prepurified drinking water from river Rhine water, using coagulation of suspended matter with Fe(III)Cl3 and rapid sand filtration; (b) the GWA, the waterworks of the city of Amsterdam that produces finished drinking water using WRK water, which is additionally purified using subsequently dune filtration, aeration, rapid sand filtration, ozonization, hardness reduction, biological active carbon filtration, and slow sand filtration. It has been demonstrated by the GWA that by far the largest DOC removal in the purification system occurs in the ozonization and biological active carbon filtration steps. Therefore, the removal of CLSA by these two purification steps was studied in particular.
Results and Discussion Setting up the Py-GC-MS-MS Method. (a) Optimization of CID. The first step in finding suitable MS-MS pathways
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FIGURE 1. EI mass spectrum of 5-chloroguaiacol (5-CG).
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 7, 1997
FIGURE 2. Relationship between the CID daughter ion intensity and the collision energy (eV) as monitored for the dissociations m/z 158 f m/z 143, m/z 143 f m/z 115, and m/z 158 f m/z 115, respectively. is to select suitable parent ions. In the EI mass spectrum of 5-chloroguaiacol (see Figure 1) three prominent ions and their chlorine isotopes were observed, viz. at m/z 115, m/z 117, m/z 143, m/z 145, m/z 158, and m/z 160. These ions all appear to be suitable parent ions because of their high relative abundance and their relatively high m/z values. However, in the mass spectrum (Figure 1) no abundant fragments below m/z 115 could be observed. Therefore, it is unlikely that the parent ions at m/z 115 and m/z 117 will produce abundant daughter ions, and therefore they were not investigated further. Only the fragmentation pathways of the parent ions at m/z 158 and m/z 143 were further investigated at this stage because the reactivity of their 37Cl isotope ions (m/z 145 and m/z 160) is identical. The dissociation of the ion at m/z 158 to m/z 143 occurs via loss of a methyl radical (CH3•). The dissociation of the ion at m/z 143 to m/z 115 occurs via loss of CO. Although a daughter ion at m/z 115 was observed for the parent ion at m/z 158, a direct dissociation is not very likely. Presumably, two dissociations occur in succession, i.e., loss of CH3• followed by loss of CO. Optimization of the collision energy (see Figure 2) showed that the dissociations of the ion at m/z 158 to m/z 143 and of the ion at m/z 143 to m/z 115 both have an optimum at ca. 11 eV. The dissociation of the ion at m/z 158 to m/z 115 however has an optimum around 100 eV (not reached in Figure 2), which also indicates that this pathway occurs via a double dissociation. At its optimal collision energy, the latter dissociation was much less sensitive than the former two, and therefore it was not studied further. The dissociation from the parent ion at m/z 143 to m/z 115 is 2.1 times more
FIGURE 4. Mass chromatograms of the pyrolysate of GWA water sampled in July 1995. (A) m/z 143/145 f m/z 115/117, the high frequency noise is removed by smoothing; (B) m/z 158/160.
FIGURE 3. Mass chromatograms of the pyrolysate of WRK water sampled in July 1995; (A) m/z 158/160 f m/z 143/145; (B) m/z 143/145 f m/z 115/117; (C) m/z 158/160; (D) m/z 143/145.
sensitive as compared to the dissociation at m/z 158 to m/z 143 (see Figure 2). (b) Comparison of CID Pathways Using a Drinking Water Sample. In order to compare the selectivity of both dissociations, they were studied for macromolecular material isolated from WRK water (Figure 3), which represents a complex aquatic humic matrix. The difference in selectivity between the selected ions and dissociations can be observed clearly. In Figure 3A (lower trace), there is still an interfering peak to the left of the 5-chloroguaiacol peak (5-CG), which is absent in Figure 3B (both traces). The 37Cl chromatogram of Figure 3A (upper trace), in which the interfering peak cannot be seen, demonstrates that this peak does not originate from a chloroguaiacol isomer. There is good agreement in the overall profiles of the chromatograms of Figure 3, panels A and C, which indicates the low selectivity of the loss of CH3•. This agreement cannot be seen if one compares Figure 3, panels B and D, which indicates the high selectivity of the loss of CO. Clearly, the dissociation of the parent ion at m/z 143 to the daughter ion at m/z 115 is the most selective. For this reason, it was selected to be implemented in the PyGC-MS-MS method. Comparison of the Py-GC-MS-MS and the Py-GCMS-SIM Method. For a valid comparison, both methods should be applied to a set of river and drinking water samples. The results for WRK water have already been discussed above (Figure 3). The other samples studied were river Rhine water and GWA water (Figure 4). With river Rhine water (figure not shown), both methods appear to be suitable. With Py-GCMS-SIM, a shoulder occurs on the left side of the 5-CG peak (figure not shown). In going from WRK water to GWA water (Figure 4B), not only does this shoulder becomes larger but also on the right side of the 5-CG peak a shoulder appeared.
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TABLE 1. Py-GC-MS-MS Results for River and Drinking Water date
19-7-95
16-8-95 8-11-95 3-1-96 24-4-96 14-8-96 4-12-96 6-9-95
10-6-96
sample Suwannee HA Suwannee FA Rhinee WRKf GWAg Rhine WRK Rhine WRK Rhine WRK Rhine WRK Rhine WRK Rhine WRK Duneh 1 Dune 2 GWA 1 GWA 2 Dune GWA pure CLSA
yield of HMWa material (mg/5 L)
relative pyrolysis efficiencyb (%)
2.2 0.9 0.3 5.7 1.6 2.3 1.5 2.0 1.6 3.0 2.3 3.5 3.0 3.2 2.9 2.9 2.3 0.6 0.6 2.1 0.4
100d 82 51 100 64 100 45 100 76 100 67 100 55 100 100 88 100 50 34 100 90 30
concn of CLSAc (µg/L)
RSDd (%)
contribution of CLSA to AOXc (µg/L)
