Environ. Sci. Technoi. I W 3 , 27, 2387-2396
Quantitative Analysis of Sulfonic Acid Groups in Macromolecular Llgnosulfonic Acids and Aquatic Humic Substances by Temperature-Resolved Pyrolysis-Mass Spectrometry Wiliem M. G. M. van Loon,*pt Jaap J. Boon,? and Bob de Groots
Unit for Mass Spectrometry of Macromolecular Systems, FOM-Institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam, The Netherlands, and Laboratory for Special Research, Water Transport Company Rijn-Kennemerland, Groenendael 6, 3439 LV Nieuwegein, The Netherlands
A novel, selective and quantitative pyrolysis-mass spectrometric (Py-MS) procedure for sulfonic acid groups present in macromolecular lignosulfonicacids and aquatic humic substances is presented. The procedure is based on the specific Py-MS detection of sulfur dioxide, which is a characteristic pyrolysis product of sulfonic acid groups. Using this procedure, specific mass spectrometric evidence for the general occurrenceof significant amounts of sulfonic acid groups in macromolecular aquatic humic substances is presented for the first time. The presence of sulfonic acid groups in aquatic humic substances fundamentally limits their isolatability on hydrophobic resin types. Several aquatic humic substance samples appear to contain sulfone structural units. The Py-MS determinations are reproducible (RSD 1000) and oligomeric (MW 200-1000) chlorolignosulfonic acids, lignosulfonic acids, chlorothiolignins, and thiolignins (kraft lignins) are dissolved in the extraction liquors and subsequently recovered or discharged into surface waters ( 4 ) . Annually, 9.3 million t of lignosulfonic acids are burnt for energy generation or discharged (5). The interest in these large discharges is primarily fueled by the potential toxicity of chlorolignosulfonic acids and chlorothiolignins, because these macromolecules can partially be degraded by microorganisms into low molecular weight toxic chlorinated aromatic compounds (1, 6, 7). This study focuses on the Rhine River Basin, in which only sulfite pulp mills, discharging sulfonated lignins, are present. Chlorolignosulfonicacids and lignosulfonicacids, when dissolved in water at pH 7, are very hydrophilic macromolecules because of their high sulfonic acid contents. Consequently, these macromolecules are expected to be difficult to remove by water purification plants and, consequently, are thought to be present in drinking water produced from river water receiving pulp mill effluents (8). For these reasons, waterworks along the Rhine River t FOM-Institute for Atomic and Molecular Physics. !Water Transport Company Rijn-Kennemerland.
0013-936X/93/0927-2387804.00/0
0 1993 American Chemlcai Society
are highly interested in specificand quantitative analytical procedures for chlorolignosulfonic acids and lignosulfonic acids. We reported a specific and quantitative pyrolysisGC-MS (Py-GC-MS) procedure to detect trace concentrations of chlorolignosulfonic acids in river water and drinking water, using specific chloro-2-methoxyphenyl substructures (9). Since lignosulfonic acids are discharged combined with chlorolignosulfonic acids, the presence of the former compounds in river water and drinking water is also diagnostic for the presence of chlorolignosulfonic acids. In fact, lignosulfonic acids and chlorolignosulfonic acids share their sulfonated character. Therefore, we aimed at the development of a specific and quantitative analytical procedure for lignosulfonic acids to complement the procedure we already reported for chlorolignosulfonicacids (9). The analysis of lignosulfonic acids in pulp mill effluents has been reviewed recently ( 4 ) . The specific or selective detection of lignosulfonic acids in river water and drinking water presents a problem. No recent reviews have appeared on the analysis of lignosulfonic acids in these water types, and a short review is presented here. Several multiple wavelengthUV (10-12), fluorescence(1315),and polarographic (16)procedures have been proposed for the selective and quantitative analysis of lignosulfonic acids in river water (10-14, 16) and seawater (15). A fundamental disadvantage of these spectrometric and electrochemical procedures is that the insight into the substructures which are actually detected is limited, and consequently the selectivity of these procedures is often unclear or questionable. Therefore, a mass spectrometric identification and quantification procedure for lignosulfonic acids in river water and drinking water is highly desirable. Several analytical pyrolysis-mass spectrometric studies have appeared in which the formation of SO2 from sulfonic acid groups is demonstrated (3, 17, 18). Jakab et al. demonstrated the SO2 formation from sulfonic acid groups using thermogravimetry-mass spectrometry (TG-MS)(17). Van de Meent et ai. reported the quantitative analysis of lignosulfonic acids in biopolymer mixtures by Py-MS combined with discriminant analysis (18). Van Loon et al. (4) reported that Py-MS of lignosulfonic acids yields relatively large amounts of SOZ,which appeared to be a characteristic pyrolysis product of sulfonic acid groups. The pyrolysis reaction is shown in eq I; R represents a macromolecular residue:
-
PYr
+
-
+
R-SO,H R' 'SO,H SO2 'OH (1) It was proposed that the sulfonic acid group can be used to analyze lignosulfonic acids in river water and drinking water specifically and quantitatively (4). However, a prerequisite for a sufficiently specific determination of Environ. Sci. Technol., Vol. 27, No. 12, 1993 2387
anthropogenic lignosulfonic acids in river water is that natural background concentrations of sulfonic acid groups in aquatic humic substances are relatively low or zero. Until now, no evidence regarding the presence of sulfonic acid groups in aquatic humic substances has been reported. Therefore, we assumed the natural concentrations of sulfonic acids in dissolved organic carbon (DOC) to be negligible and aimed at the development of a specific and quantitative analytical procedure for sulfonic acid groups in macromolecules isolated from river water. Because the investigations presented here aim at the analysis of the sulfonic acid group, both chlorolignosulfonic acids and lignosulfonic acids are referred to as lignosulfonic acids hereafter. Functional group analysis of carboxylic acid, phenolic groups, and methoxylgroups in humic substances is a welldeveloped field and has been reviewed by Perdue (19). Sposito et al. provided indirect evidence for the presence of sulfonic acid groups in soil-sludge mixtures with high sulfur contents (4-12%) by a combination of IR spectrometry and titrimetry (20, 21). Their titrimetric data indicate a strongly acidic fraction (pK, < L5), and their infrared data indicate sulfone groups. However, they did not provide MS evidence for this functional group. These sulfonic acid groups can probably be ascribed to the presence of xenobiotic sulfonated detergents in sewage sludge. Several low molecular weight (MW 1000) DOC fractions. Many analytical procedures have been reported on the GC (23-25) and HPLC analyses (26, 27) of low molecular weight aromatic and aliphatic (28)sulfonic acids. However, to our knowledge, no specific and quantitative analytical procedure has been reported for sulfonic acid groups present in macromolecular biomolecules and geomacromolecules. In-source platinum filament Py-MS was used as the principal techique in this study. It is a MS technique in which polymeric materials are thermally degraded into monomeric and oligomeric structural units inside the ion source. Py-MS has been used frequently for the structural characterization of synthetic polymers, biopolymers, and geopolymers (29). Advantages of this technique are the acquisition of general structural information (functional groups, major monomeric and oligomeric units), low compound losses due to in-source pyrolysis, temperatureresolved pyrolysis information (25-900 "C), small sample sizes (1-5 pg), and speed (analysis time 1-2 min). Due to the low E1 ionization energy (14-16 eV), fragmentation of aromatic compounds is low and the interpretability of the Py-MS spectra is improved. Disadvantages of this technique are that unambiguous structural assignments to mlz values are often not possible due to the overlap of isobaric compounds and their fragment ions, and consequently quantification of pyrolysis products often is a complicated task. Furthermore, ion source contamination occurs more rapidly compared to Py-GC-MS. Py-GC-MS was used as a supporting technique in this study. It is a hyphenated MS technique in which pyrolysis products are separated prior to MS detection. The pyrolysis process may be based on Curie-point pyrolysis, using reproducible and rapid heating of a ferromagnetic wire by radiofrequent energy to a fixed Curie-point temperature, or on filament pyrolysis in which a platinum 2388
Environ. Scl. Technoi., Vol. 27, No. 12, 1993
filament is resistively heated by a programable current. Py-GC-MS is a well-known technique for the structural characterization of synthetic polymers, biopolymers, and geopolymers (30,311. Advantages of Py-GC-MS are that detailed structural information on macromolecules, on at least the monomeric level, can be obtained reproducibly. The GC separation of pyrolysis products enables their unambiguous identification. In this paper the selective, highly sensitive and quantitative analysis of sulfonic acid groups present in macromolecular lignosulfonic acids and aquatic humic substances by Py-MS is reported, using the characteristic pyrolysis product SOz. This procedure is optimized and evaluated with respect to the specificity and spectral/ structural interferences, reproducibility, linear dynamic range, detection limit, reference materials, and quantification method. The optimized procedure is applied to several lignosulfonicacids isolated from pulp mill effluents and to dissolved organic matter (DOM) isolated from the Rhine River, from several relatively unpolluted tributaries of this river, and from drinking water. Experimental Procedures
Materials. Sodium lignosulfonate was obtained from Roth (Karlsruhe,FRG), cation-exchanged (Dowex 50x8100; Janssen Chimica), and lyophilized, yielding a saltfree reference material lignosulfonic acid. The sulfur content of this material was determined by elemental analysis to be 6.1496, corresponding to a sulfonic acid content of 192 mequiv/100 g. For this calculation, we assumed that all the sulfur in this lignosulfonic acid material is associated with sulfonic acid groups; we have no Py-MS evidence indicating otherwise (4). Chlorolignosulfonic acid was synthesized according to a procedure reported elsewhere (32). Poly(sodium 4-styrenesulfonate) (MW 4000) was purchased from Polymer Laboratories (Church Stretton, UK), cation-exchanged,and lyophilized. The sulfur content of this material was determined to be 13.996, corresponding to 434 mequiv1100 g. Poly@phenylene ether sulfone) was purchased from Scientific Polymer Products (Ontario, NY). Effluent samples were obtained from the following pulp mills which discharge lignosulfonic acids into the Rhine River: PWA Aschaffenburg (FRG), PWA Mannheim (FRG), Holtzmann (Karlsruhe, FRG), Stracel (Strasbourg, France), and Cellulose Attisholz AG (Switzerland). A reference pulp mill effluent sample was composed using the effluent samples listed above(32). Water samples were obtained from the following relatively unpolluted tributaries of the Rhine River: the Neue Rein (Austria), the Bregenzer Aach (Austria),the Aare (Switzerland),the Thur (Switzerland), and the Moselle (FRG). Drinking water samples were collected at several stages of the waterworks WRK (Nieuwegein, The Netherlands), which uses Rhine River water to produce drinking water, and in the city of Amsterdam (The Netherlands). High molecular weight DOC fractions were isolated from the water samples by microfiltration (0.45 Fm), XAD-8 adsorption chromatography (sample pH l ) , cation-exchange chromatography, and ultrafiltration (MW cutoff 1OOO) as reported elsewhere (32). The recovery of this procedure, as determined by DOC measurements, is 38% for pulp mill effluent DOC and 14% for Rhine DOC (32). This procedure yields saltfree materials in the acid form, which is essential for the quantitative conversion of sulfonic acid groups into SO2
during pyrolysis and prevents secondary pyrolysis reactions of these groups (4). Pyrolysis-Mass Spectrometry. Py-MS was performed on a Jeol (Tokyo, Japan) JMS-DX303 double focusing (EB) mass spectrometer equipped with an in-source platinum/rhenium (10% Rh) filament pyrolysis probe. PyGC-MS was performed using a homemade FOM 3-LX Curie-point pyrolysis injector (FOM-AMOLF, Amsterdam, The Netherlands), which was installed on a HewlettPackard (Palo Alto, CA) Model 5890 Series I1 GC connected to a Finnigan MAT (San J b e , CA) Incos 50 quadrupole MS. Data were acquired on a Finnigan DG10 data system running under Incos 50 software and were processed on a Sun Spark/IPC workstation running under Kratos Mach3 software. Py-MS conditions used were as follows: sample amount 0.1-10 pg dissolved in water; filament heating range 25920 "C; filament heating rate 16 OC/min (current ramp 1 A/min, full-scan mode) or 8 "C/min (current ramp 0.5 A/min, SO2 mode); source temperature 180 "C; electron impact (EI) ionization; electron energy 18 eV; acceleration voltage 3.0 kV; resolution 500; mlz range 20-1000 (fullscan mode) or 20-130 (SO2 mode); cycle time 1s (full-scan mode) or 0.5 s (SO2 mode); post-acceleration voltage -10 kV; electron multiplier voltage 1.1kV; and analysis time 120 s. The pyrolysis filament temperature was calibrated using an Ircon (Chicago, IL) pyrometer Model 300C. Analyses were performed in duplicate routinely. Relative standard deviations (RSD) were calculated using the g-1 formula, which corrects for a small number of replicates. The following Py-GC-MS conditions were used. The high-frequency pyrolysis energy (1 MHz, 1 kW) was supplied by a Fischer (Meckenheim bei Bonn, FRG) power supply. Curie-point pyrolysis wires (Philips, Eindhoven, The Netherlands) were inserted into low dead-volume (80pL) glass tubes, which were inserted into the heated pyrolysis injector. Sample size 40 pg (full analysis) or 20 pg (SO2 analysis); injector housing temperature 240 (full run; SO2 analysis/liner pyrolysis) or 30 "C (SO2 analysis/ Curie-point pyrolysis); glass liner temperature 180 (full run), 240 (SO2 analysis/liner pyrolysis), or 30 "C (SO2 analysislcurie-point pyrolysis);pyrolysis temperature 510 or 358 "C; pyrolysis time 5 s. GC conditions used were as follows: column DB-5 (J&W, Folsom, CA); L 25 m X i.d. 0.22 mm; film thickness 0.25 pm; carrier gas helium; linear gas velocity 27 cm/s; temperature program 30 "C (0 min) at 8 "C/min to 100 "C (0 min) at 4 "C/min to 325 "C (0 min) (complete analysis) or isothermal at 30 "C (SO2 analysis); GC-MS interface temperature 240 (full run) or 30 "C (SO2 analysis). MS conditions used were as follows: ion source temperature 180 "C; electron energy 70 eV; emission current 250 PA; rnlz range 47-400 (complete analysis) or 10-100 (SO2 analysis); cycle time 0.8 s. Optimization Procedures. An ionization potential curve of SOi+ and a fragmentation potential curve of SO+ were measured by Py-MS using an electron energy range of 10-30 eV. The specific Py-MS detection of SO2 at r n l z 64 was investigated by Py-GC-MS analysis of 40 pg of synthesized chlorolignosulfonic acids and 40 pg of Rhine humic substances. Interference by sulfone groups was investigated by PyMS analysis of 5 pg of poly@-phenylene ether sulfone). The linear dynamic range was investigated by Py-MS analysis of 0.1-5 pg of lignosulfonic acid and of macro-
Figure 1. Integration procedure for sulfonic acid (A) and sulfone (B) groups. The mass trace m/z 64, representing SO2+,,is shown.
molecular material isolated from the Rhine River. The detection limit was determined by Py-MS analysis of 0.1 pg of lignosulfonic acid.
Quantification Procedures. The polymeric sample, S, was pyrolyzed. A standardized sample amount, Q(S), of 3 pg was used. The total SO2 signal, Int(S), was quantified by integration of mass trace rnlz 64. The sulfonic acid group fraction F (0 I F I 1) of a specific sample was determined by Py-MS analysis, followed by gravimetric integration of the SO2 peak fractions (A, B) according to Figure 1and calculation of the sulfonic acid fraction F according to eq 11:
F = area (fraction A)/area (fractions A + B) (11) The sulfone fraction was calculated to be 1 - F. When performing standard addition analysis, first the sample was measured (5;3 pg/3 pL, n = 21, then the sample (3 pg/3 pL) combined with the standard addition of lignosulfonic acid (S + LSA; 3 pg/3 pL + 1pg/2 pL, n = 2) and finally the lignosulfonic acid standard alone (LSA; 1pg/2 pL,n = 2) was measured. The sulfonic acid response factor R, giving the SO2 signal per microgram of added lignosulfonic acid, is calculated using eq 111: R = [Int(S + LSA) - Int(S)l/Q(LSA)
[counts/pgl (111) The quantity of lignosulfonic acid equivalents in the analyzed sample, Q(LAE), is calculated using eq IV: &(LAW = [Int(S)FI/R [pgl (IV) Finally, the sulfonic acid content, SAC, of the isolated material is calculated by eq V; 192 represents the sulfonic acid content [mequiv/100 gl of the lignosulfonic acid reference material: SAC = [Q(LAE)/Q(S)I X 192
[mequiv/100 gl (V)
By substituting eqs I11and IV into eq V, eq VI is obtained, which is used for routine calculations: SAC =
Int(S)F Q(LSA) 192 Int(S + LSA) - Int(S) Q(S) [mequiv/100 gl (VI)
The pyrolysis yield (Y) of SO2 after standard addition (+LSA) to the sample (S) was calculated by eq VII:
Y = [Int(S + LSA) - Int(S)l X 100/Int(LSA)
[%I
(VI11 The contribution of sulfonic acid groups to the sulfur content of isolated macromolecular materials was calculated by multiplying the sulfonic acid content with the atomic weight of sulfur. Environ. Sci. Technol., Vol. 27,No. 12. lBB3 2389
\ IIM 39
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Figure 2. (A, top) Mass chromatograms of m/z 64, specific for SOn, and m/z 124, representative for guaiacol (2-methoxyphenoi), as produced during Py-MS analysis of ilgnosuifonic acid. The TIC trace (m/z 20-1000; not shown) essentially co-elutes with mass trace m/z 124. (B, bottom) Corresponding Py-MS spectrum of iignosuifonic acid, integrated over the total TIC trace.
Flgure 3. (A, top) Mass chromatograms of m/z 64, specific for SO2, and m/z 94, specific for phenol, as produced during Py-MS analysis of poiy(ppheny1ene ether sulfone). The TIC trace (m/z 20-1000; not shown) co-elutes with these mass traces. (B, bottom) Corresponding Py-MS spectrum of poiy@phenyiene ether sulfone), integrated over the total TIC trace.
Results and Discussion In this section, first the results on the development of an analytical procedure for sulfonic acid groups in macromolecules are reported, followed by the results on the application of this novel procedure to lignosulfonic acids and aquatic humic substances. The analytical part of this section is divided into the following paragraphs: (a) the specific detection of SO2 using Py-MS at rnlz 64; (b) the structural specificity of SOz, and (c) the quantitative aspects of the developed procedure. The application part consists of the following paragraphs: (a) identification of sulfonic acid groups and (b) quantification of sulfonic acid groups. Specific MS Detection of SO2. S O 2 can be sensitively detected by Py-MS at rnlz 64. However, the specific detection of SOz+ at this rnlz value is not obvious. Spectral overlap of compounds which are identified using Py-MS analysis at a specific r n l z value frequently occurs, and therefore, potential interferences have to be investigated by, e.g., Py-GC-MS. Generally, it can be observed that Py-MS spectra show low mass signals in the r n l z range around mlz 64 (mlz 60-65) (see Figures 2-4). Py-GC-MS analysis, using an electron energy of 70 eV, of several lignosulfonic acid samples and macromolecular materials isolated from the Rhine River (mass traces rnlz 64 not shown) illustrate that SO2 is by far the major contributing compound to rnlz 64. However, a minor amount (2-6% of total signal of mass trace mlz 64) of interference occurs in the Py-GC-MS analyses (not shown) and is caused by 1,2-dihydroxybenzene. As discussed below, this interference is probably suppressed by using a low electron energy (18 eV). No other compounds which show a fragment ion at mlz 64 are detected in the Py-GC-MS chromatogram to a significant extent (relative intensity >0.1% 1. The electron energy is known to play an important role in Py-MS analysis (29). The major fragmentation pathway of sulfur dioxide after electron impact ionization is the loss of an oxygen radical (33). This fragmentation reaction 2390
Envlron. Sci. Technol., Vol. 27, No. 12, 1993
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Flgure 4. (A, top) Mass chromatograms of m/z 64, specific for SO2 and m/z 124, representingguaiacol and other compounds/fragments, as produced during Py-MS analysis of macromolecular dissolved organic carbon (DOC) isolated from the Rhine River. The TIC trace (m/z 201000; not shown) essentially co-elutes with m/z 124. (B,bottom) Corresponding Py-MS spectrum of Rhine DOC, integrated over the total TIC trace.
leads to a loss of SOz+ ions and sensitivity and, therefore, is undesirable. Furthermore, it has been shown above that E1 fragmentation (70 eV) of 1,2-dihydroxybenzene, a pyrolysis product of oxidatedlignins, leads to a significant (relative abundance, 39 % ) fragment ion at rnlz 64 (33). Both fragmentation pathways can probably be suppressed by lowering the applied electron energy to 10-20 eV. For these reasons, the electron energy was optimized in the low eV range (10-30 eV). It appears that, at an electron energy of 18eV, only 10 % of the SOz'+ ions fragment into SO+, while adequate sensitivity is obtained (ionization potential curve of SOz*+and fragmentation potential curve
of SO+ are not shown). Because of the lack of a commercially available 1,2-dihydroxybenzene reference compound, no fragmentation potential curve of the interfering fragment ion at rnlz 64 could be measured. However, due to the relatively stable nature of this aromatic compound, it is quite likely that the formation of its fragment ion at rnlz 64 is suppressed using a low electron energy (15-20 eV). Therefore, an electron energy of 18 eV was used for the specific Py-MS detection of SO:! in further experiments. On the other hand, the use of low energy, low fragmenting E1 conditions will favor the spectral interference by compounds with a MW of 64. Only five compounds with a MW of 64 have been reported (33). Apart from SO:!,the other four compounds are synthetic compounds which will not interfere with the specific detection of SO:!at rnlz 64 in the bio- and geopolymers under investigation. Elemental sulfur, which is produced by pyrolysis of coal (35) and soil polluted by a coal gasification plant (36), shows an intense SZ+fragment at rnlz 64 (relative abundance, 100%)(33)under 70 eVEI conditions. Fortunately, sulfur ions &, x = 2-8; r n l z = 32x) were not detected in Py-MS spectra (mass chromatography on S,) of lignosulfonic acids and aquatic humic substances in this study. If interference by SZ+occurs in other sample matrices, pyrolysis-high resolution-massspectrometry (Py-HR-MS), Py-GC-MS (36)and Py-MS-MS, can be employed for the chromatografic or mass separation of SO:! and Sa. In our study, Py-MS was preferred over Py-GC-MS because of (a) specific detection of SO:! at rnlz 64 in our sample matrices, (b) shorter analysis times (15 versus 30 min), and (c) better reproducibility for lignosulfonicacid samples (see section on Quantitative Aspects). In conclusion,it appears that SO:! is detected specifically (>99%) in lignosulfonic acids and aquatic humic substances by Py-MS at rnlz 64 under low energy (18 eV) E1 conditions. Structural Specificity of SO2. SO:!can be pyrolytically produced from several sources, namely from (a) sulfonated macromolecules, (b) macromolecules containing sulfone structural units, and (c) inorganic sulfates (4). Sulfone groups were suspected to be present in macromolecules and to produce SO:! on pyrolysis, but no data were available. Obviously, pyrolytic characteristics have to be found to distinguish these SO:! sources in Py-MS analysis. Initial experiments showed that sulfonic acid groups pyrolyze at remarkably low temperatures (T C300 "C). Therefore, we investigated the SO:! production from these sources, with special attention to the pyrolysis temperatures. The Py-MS spectrum of lignosulfonic acid (see Figure 2; Table 11) shows the presence of an intense SO2 mass peak and a mass series of monomeric and dimeric guaiacyl (2-methoxyphenyl)derivatives. These guaiacyl pyrolysis products of lignosulfonic acids have been identified confidently by Py-GC-MS (4,37)and represent monomeric structural units of softwood and hardwood lignins. The specific mass trace of SO:! (mlz 64; see Figure 2) shows that SO2is produced at relatively low temperatures (Table I; Tmax,soz = 168 "C). Tmax,SOz is defined as the pyrolysis temperature at which maximum SO:! formation occurs. It must be noted that T m = , s o z depends on the heating rate; a higher heating rate results in higher T m = , s o z values because of the shorterhnadequate heat-transfer times. It thus appears that sulfonic acid groups are quite thermo-
,, Values of Sulfonic Acid Groups, Sulfone Table I. T Groups, and Aromatic Monomeric Units in Various Sample Types sample
Tmar,so: ("(2)
Tmaxmon"
("C)
monomeric unit
168 168 196
240 260
2-methoxyphenol 2-methoxyphenol styrene
360
376
phenol
>600b 184 184
260 260
m/z 124c mlz 124c
lignosulfonic acid reference effluent polystyrene sulfonic acid poly @-phenylene ether sulfone) sodium sulfate Rhine DOC Moselle DOC
288
a Tm, is the pyrolysis temperature at which the maximum formation of a specific pyrolysis product occurs; heating rate 8 "C/ min. Heating rate 16 "C/min. In complex humic samples, m/z 124 represents a mixed mass peak and contains guaiacol.
1 TIC
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Figure 5. Illustration of liner pyrolysis (LPy; liner temperature 240 "C) and Curie-point pyrolysis(Cub; 5 10 OC) of sulfonic acid groups present in lignosulfonicacid. The liner pyrolysis phenomenondemonstratesthe thermolability of these groups. The Py-GC-MS traces show the TIC, the mass trace m/z 64 (SO,',) and the co-eluting mass trace m/z 48
(SO+).
labile, which is in agreement with the high leaving group ability of sulfonate anions. Furthermore, the sulfonic acid group pyrolysis temperature is significantly lower than that of guaiacol (2-methoxyphenol), a major monomeric unit of lignosulfonic acid (see Figure 2 and Table I; Tm=,guaiacol = 240 "C). Lignosulfonic acids isolated from pulp mill effluent and polystyrene sulfonic acid both show that the Trnax,soz is significantly lower than the TmuaOnomer (see Table I), Furthermore, a characteristic nearly linear decay of the SO:! signal, after the signal has reached its maximum, is observed for all the sulfonated macromolecules (see Figure 2; Py-MS data on reference pulp mill effluent and polystyrene sulfonic acid not depicted but show similar linear decay). The higher T m a x , s o 2 of polystyrene sulfonic acid (196OC, see Table I) can probably be explained by the higher bond energies of aromatic sulfonic acid groups compared to their aliphatic analogues; the sulfur-carbon bond is inductively stabilized by the aromatic ring. The thermolability of sulfonic acid groups is remarkably illustrated by the fact that sulfonic acid groups partially pyrolyze in the heated (240 "C) glass liner of the pyrolysis injector of our Py-GC-MS system (see Figure 5 ) . Here, two SO:! pyrolysis fractions are observed, namely, induced by (a) the heated liner (here defined as 'liner pyrolysis') and (b) the Curie-point pyrolysis process. This liner pyrolysis is quite exceptional, again pointing to the characteristic low pyrolysis temperatures of sulfonic Environ. Sci. Technol., Vol. 27, No. 12, 1993 2391
Table 11. Tentative Identifications of Pyrolysis Products As Detected by Pyrolysis-Mass Spectrometric Analysis of Lignosulfonic acid, Poly(ppheny1ene ether sulfone), and DOC Isolated from the Rhine Rivers
m/z
sample
compound
origin
28 LR carbon monoxide, ethene, etc. Af 44 LR carbon dioxide Fg 48 P methyl thiol Fg 78 P benzene AP 64 LRP sulfur dioxide Fg 94 R phenol Lg, AP 108 LR methylphenol isomer Lg, AP 110 L 1,2-dihydroxybenzene Lg 122 R dimethylbenzene isomer AP 124 LR guaiacol (2-methoxyphenol) Lg 138 LR 4-methylguaiacol Lg 150 L 4-vinylguaiacol Lg 162 L 4-CsH3-guaiacol Lg 164 L 4-propenylguaiacol isomers Lg 170 P diphenyl ether AP 178 L 4-propenalguaiacol, etc. Lg 180 L 4-(propan-2-one)guaiacol,etc. Lg 272 L l,2-diguaiacylethene Lg 284 L dimeric guaiacyl derivative Lg 310 L dimeric guaiacyl derivative Lg 402 P 1,l-diphenylether sulfone AP "The corresponding Py-MS spectra are shown in Figure 2-4. Supporting Py-GC-MS evidence for the identifications listed here has been reported (4,32,37). Abbreviations used: L, lignosulfonic acids; P, poly@-phenylene ether sulfone);R, Rhine River; Lg, lignins; Ap, aromatic polymers; Fg, functional group; Af, aspecific fragment.
acid groups. Usually, effective pyrolysis occurs in the Curie-point temperature range of 400-500 OC. It has been reported that pyrolysis at 358 "C usually only results in the evaporation of adsorbed low molecular weight compounds (36). It is very well possible that Curie-point pyrolysis of biopolymers and geopolymers at 358 "C will result in the specific pyrolysis of sulfonic acid groups, leaving sulfone groups intact (see the section Further Research). Very little is known on the presence of sulfone groups in bio- and geopolymers. Considering the lack of data on the presence of sulfur-containing groups in these geomacromolecules, it cannot be excluded that sulfone substructures are present in aquatic humic substances. No biological reference materials with a known content of sulfone groups are commercially available. We therefore investigated a synthetic poly(ppheny1ene sulfone ether) by Py-MS. Major identified pyrolysis products (seeFigure 3; Table 11) are SO2 (mlz 64), benzene (mlz 78), phenol (m/z9 4 , diphenyl ether (mlz 170),and 1,l-diphenylether sulfone (mlz 402). The Py-MS spectrum clearly demonstrates that SO2 can also be formed from sulfone groups in polymers. A schematic representation of the pyrolysis reaction is shown in eq VIII: R,-SO,-R,
PYr +
SO2
+ R1' + R2'
(VIII)
Thus, a structural interference by sulfone groups in the Py-MS analysis of sulfonic acid groups is possible. However, the lack of humic substance reference materials with a known content of sulfone groups limits the evidence on the pyrolytic behavior of sulfone groups in aquatic humic substances. The mass chromatograms of SO2 (mlz 64) and phenol (m/z94),the major aromatic pyrolysis products of the synthetic sulfone polymer, are shown in Figure 3. The Tmax,soz of poly@-phenylene ether sulfone) is 360 "C (seeTable I). This relatively high temperature shows that 2392
Envlron. Scl. Technol., Vol. 27, No. 12, 1993
the sulfone-phenyl bond is quite thermostabile, which is probably caused by the large inductive stabilization of these bonds by the two neighboring phenyl groups. Another important feature of Figure 3 is the simultaneous pyrolytic production of SO2 and phenol. This probably reflects the fact that sulfone groups, as well as many monomeric units, have to break two linkages of average bond strength before they are released into the gas phase, leading to comparable pyrolysis temperatures. The thermal coproduction of SO2 and major monomeric units is proposed as an important diagnostic feature of sulfone groups. Inorganic sulfates produce minor amounts of SO2 at higher pyrolysis temperatures (T >600 "C, heating rate 16 OC/min) ( 4 ) . A pyrolysis reaction is proposed in eq IX:
-
PYr
sod2- so2+ 0;-
(IX) Sulfonic acid groups generally pyrolyze at temperatures below 300 OC (see Table I, heating rate 8 OC/min). Consequently, these two SO2 sources can be discriminated easily. In addition, the use of salt-free samples eliminates this interference and is strongly recommended for optimum pyrolysis results. Quantitative Aspects. Now that the structural significance of the SO2 pyrolysis product has become clear, the development of a quantitative procedure and its evaluation becomes meaningful. The relative standard deviations of the Py-MS measurements are less than 5 % ( n = 2) in most cases. Linear calibration curves (0.1-5 pg; r = 0.998-0.999) of lignosulfonic acid and aquatic humic substances (not shown) are obtained routinely. The detection limit is 30 ng of lignosulfonic acid (S/N ratio lo), corresponding to 60 pequiv of sulfonic acid groups. By using Py-MS combined with SIM detection (mlz 641, the detection limit can probably be lowered by a factor of 10-20. The procedure provides ample sensitivity to determine sulfonic acid groups at trace levels in low microgram quantities of lignosulfonic acids and aquatic humic substances. Lignosulfonic acid and polystyrene sulfonic acid were tested for use as reference materials for quantification. Lignosulfonic acids are industrially modified biomacromolecules which contains aliphaticallybound sulfonic acid groups (2). On the other hand, polystyrene sulfonic acid is an anthropogenic polymer which contains aromatically bound sulfonic acid groups. These macromolecular characteristics suggest on beforehand that lignosulfonic acid is a more representative material for the quantification of sulfonic acid groups in biomacromolecules and geomacromolecules. I t is demonstrated in the section Quantification of SulfonicAcid Groups that the measured sulfonic acid content of polystyrene sulfonic acid is significantly lower than that calculated with the elemental sulfur content. Obviously, the SO2 production from polystyrene sulfonic acid is not quantitative, and lignosulfonic acid became the reference material of choice. In general, quantification of target compounds can be performed by external or internal quantification. Internal quantification can be performed using an internal standard or standard addition procedure. Standard addition quantification was selected in this study to correct for the pyrolysis efficiency of sulfonic acid groups. The pyrolysis efficiency is defined in this study as the fraction ( % ) of a specific structural unit in the macromolecules under
investigationwhich reaches the detector following pyrolysis and separation. In general, these pyrolysis efficiencies are low and not quantitative. For example, pyrolysis efficienciesof milled wood lignins of 16-18 wt % (38)have been reported. The pyrolysis efficiency of sulfonic acid groups is discussed in the following paragraph. Important advantages of this standard addition quantification procedure are the correction for this efficiency and the correction for the effect of the sample matrix on the pyrolysis efficiency of the target structure. Because of the correction, quantitative conversion of sulfonic acid groups into ,302 is not required any more. Additional advantages of the standard addition procedure are corrections for variations of (a) the instrumental sensitivity and of (b) the SO2 loss due to electron impact fragmentation. Standard addition of lignosulfonic acid was performed at a relatively low sample concentration level of 25 % (3pg of sample, 1pg of lignosulfonic acid) in order to change the pyrolysis matrix as little as possible,probably resulting in reasonably accurate measurements. In summary, considering the uncertainties in the pyrolysis efficiency of sulfonic acid groups and the effect of the sample matrix on this efficiency, standard addition quantification is probably the most accurate procedure. Identification of Sulfonic Acid Groups. The identification of sulfonic acid groups in lignosulfonic acids has already been reported (4)and is described in the paragraph Structural Specificity of SOz. The Py-MS spectrum of macromolecular material isolated from the Rhine River (see Figure 4; Table 11)shows the presence of intense SO2 (mlz64) and CO2 (mlz44) signals,originating from sulfonic and carboxylic acid groups, respectively. The presence of sulfonated macromolecules in the Rhine River is not unexpected, and these presumably are lignosulfonic acids. The intense CO2 signal is in agreement with the high carboxylic acid contents of aquatic humic substances (19). Several phenol derivatives were identified tentatively in this Py-MS spectrum and were identified confidently by Py-GC-MS as reported elsewhere (32). The frequently occurring overlap of isobaric molecular ions and fragment ions hinders the unambiguous identification of many ions as observed in this complex Py-MS spectrum. Figure 4 shows the mass chromatograms mlz 64 and mlz 124, representing the formation of SO2 and guaiacol (and other compounds/fragments) during pyrolysis of macromolecules isolated from the Rhine River. Interestingly, a shoulder superimposed on a linearly decaying SO2 signal can be observed at higher pyrolysis temperatures. This SO2 shoulder pyrolyzes simultaneously with guaiacol. Considering the pyrolysis characteristics of the sulfonic acid and the sulfone group as described above, the SO2 shoulder probably represents sulfone groups. It thus appears that Py-MS enables the identification of sulfonic acid and, in addition, sulfone groups in aquatic humic substances. This probably is the first report on the presence of sulfone substructures in aquatic humic substances. These substructures can possibly be ascribed to (a) oxidated methionine amino acid structural units (contain an in-chain sulfur atom) (44) in aquatic humic substances and to (b) sulfonated detergents which have undergone condensation reactions with these substances. It appears from Py-MS spectra of aquatic humic substances (not shown),isolated from relatively unpolluted river systems, that they surprisingly contain a significant amount of sulfonic acid groups. To our knowledge, the
presence of sulfonic acid groups in aquatic humic substances has never been reported before. Their presence in highly oxidized aquatic humic substances (39) can be explained by the oxidation of end-chain sulfur atoms, presumably derived from cysteine and homocysteineamino acid structural units (44), to sulfonic acid groups. In addition, if the river system receives domestic wastewater, the sulfonic acids may partially be introduced by condensation or adsorption reactions of anthropogenic sulfonated detergents with dissolved humic Substances. Model experiments on the introduction of sulfonic acid and sulfone groups into aquatic humic substances via sulfonated detergents are of interest. The presence of sulfonic acid groups in aquatic humic substances sheds new light on their adsorption behavior on XAD-8 resin under acidic sample conditions. It has been reported that, at a sample pH of 2, XAD-8 adsorption of these geopolymers has not yet reached a maximum level (32,42). If all the acidic groups present in aquatic humic substances were carboxylic acids, at pH 2 an effective neutralization of these groups (pK, 4) should be achieved and maximum DOC adsorption should be reached. The fact that maximum DOC adsorption has not yet been achieved at pH 2 or 1(32,42)can be explained quite well by the presence of strongly acidic [pK, 0.5 (43)l sulfonic acid groups. It is possible that at low-sample pH values (1-2), the ion strength influences the distribution coefficients (KD)of aquatic humic substances onto XAD-8 resin, and the interpretation of the pH effect would become less clear. Dedek et al. demonstrated that the effect of saturated NaCl of the adsorption efficiency of polar pesticides on Y77 resin (comparable to XAD-4 resin) is small in general (45). However, since it is unclear to what extent these results can be applied to the adsorption behavior of aquatic humic substances on XAD-8, additional experiments regarding the effect of the salt concentration (at a constant sample pH, e.g., pH 2) on the XAD-8 adsorption efficiencyare necessary. A consequence of the sulfonated nature of aquatic humic substances is that sample acidification to, e.g., pH -0.5 would be necessary to achieve 90% neutralization of the sulfonic acid groups, thus obtaining maximum XAD-8 adsorption. Obviously, this extreme pH value would lead to chemical degradation of DOC and the adsorption resin and is not practical any more. Thus, the presence of sulfonic acid groups fundamentally limits the isolatability of aquatic humic substances on XAD-8 resin and probably on other hydrophobic resin types. Quantification of Sulfonic Acid Groups. The quantitative results of the sulfonic acid analysis are given in Table 111; the results are discussed in the order of appearance in this table. No sulfonic acid groups were detected in the blank sample, showing that our isolation procedure does not introduce sulfonated material. The pyrolysis yield (Y;see eq VI1 and Table 111) of the standard addition of lignosulfonic acid quantifies the matrix effect of the sample on the pyrolysis of lignosulfonic acid and visa versa. I t appears that the average pyrolysis efficiency is 102% (RSD 9 % ,n = 15; polystyrene sulfonic acid sample excluded). A pyrolysis efficiency lower than 100% indicates a decreased SO2 yield of the added lignosulfonic acid, possibly due to a denser sample matrix. A pyrolysis efficiency of over 100% indicates a higher SO2 yield of the sample, probably due to an opening up of the sample matrix by the addition of the less dense lignosul-
-
-
Envlron. Sci. Technol., Vol. 27, No. 12, ID93 2383
Table 111. Quantitative Results of Sulfonic Acid Group Analysis sample
sulfonic acid content (SAC) (mequiv/100 g)
blank reference materials lignosulfonic acid chlorolignosulfonic acid polystyrene sulfonic acid pulp mill effluents refererence effluent PWA Aschaffenburg PWA Mannheim Holtzmann Stracel Cellulose Attisholz AG tributaries of Rhine Bregenzer Aach Thur Aare Moselle Rhine and drinking water WRK inlet WRK outlet drinking water Amsterdam
sulfonic acid fraction (F)( % )
relative yieldo
Sbd
(Y) (%)
(% wt/wt)
192d 155 173 (434)
100 100 100
100 88 139
6.14 -
100 179 55.1 110 77 111
96 95 95 100 100 100
115 112 99 99 100 105
3.20 5.73 1.76 3.52 2.46 3.55
5 16.2 5 24.7
100 85 100 85
107 112 97 113
0.16 0.52 0.16 0.79
43.6 14.7 25.9
83 55 76
81 99 95
1.40 0.41 0.83
ndc 4.96 5.54 (13.9)
a Pyrolysis yield (Y) of the standard addition of lignosulfonic acid. Sulfur content, calculated from the determined sulfonic acid content. nd, not detectable. Underlined data are derived from elemental sulfur analyses.
fonic acid. This average pyrolysis yield indicates that, apart from minor interactions between the sample and the lignosulfonic acid, the pyrolysis efficiencies of the samples are quite comparable and probably nearly quantitative. The approximately quantitative pyrolytic formation of SO2 from lignosulfonic acid can be explained as follows. Sulfonic acid groups in lignosulfonic acid are released before pyrolysis of the lignin matrix starts (see Figure 21, and the SO2 formed can probably escape from the unpyrolyzed lignin matrix quantitatively. A sufficiently low heating rate, e.g., 8 OC/min, probably offers the sulfonic acid groups sufficient time to pyrolyze before the pyrolysis of the lignin macromolecules starts. Simultaneous pyrolysis of the sulfonic acid groups and the lignin skeleton would increase the risk of (a) secondary reactions of sulfonic acid groups with lignin pyrolysis products (especially methyl and hydrogen radicals, ref 4)and of (b) capture of SO2 in the pyrolysis residue. It appears that the chlorolignosulfonic acid (see Table 111)synthesized from lignosulfonic acid has lost a fraction of its sulfonic acid groups during the chlorination process. This can be explained by cleavage of the propyl-type side chains, to which the sulfonic acid groups are attached, from the lignosulfonic acid macromolecules during chlorination (3). The measured sulfonic acid content of polystyrene sulfonic acid is much lower than the value obtained by elemental sulfur analysis. It thus appears that the pyrolysis of sulfonic acid groups present in polystyrene sulfonic acid is not quantitative. The underlying mechanism of this phenomenon is not yet clear but possibly may be related to condensation reactions of sulfonic acid groups in the polystyrene melt. In conclusion, the procedure presented in this paper is not suitable to determine sulfonic acid contents of polystyrene sulfonic acids. As expected, the macromolecularmaterials isolated from sulfite pulp mill effluents all possess high sulfonic acid contents. Virtually no sulfone groups are detected in these materials. It thus appears that the procedure is well suited 2394 Environ. Scl. Technol., Vol. 27, No. 12, 1993
to determine the sulfonic acid content of lignosulfonates and chlorolignosulfonates specifically and accurately. An advantage of the sulfonic acid analysis is that the associated sulfur content can be calculated directly. The sulfur contents of the materials isolated from the Rhine tributaries are 0 . 2 4 8 % (average 0.574, n = 4, see Table 111). This agrees rather well with the sulfur contents of aquatic humic substances isolated from relatively natural rivers of 0.2-1.7% [average 0.5%,RSD 90%, n = 8 (4013, strongly suggesting that sulfonic acid groups are a major sulfur-containing structural unit in aquatic humic substances. Additional sulfonic acid and elemental sulfur analyses on identical aquatic humic substance samples are necessary to investigate this relationship more closely. Thus, the structural speciation of dissolved organic sulfur (DOS) can partially be revealed, offering new information on aquatic humic substances. The sulfonic acid contents of relatively natural aquatic humic substances are relatively high (5-25 [mequiv/100 gl; see Table 111)and cover a wide range. It is, therefore, not possible todetermine a generally valid background sulfonic acid content of aquatic humic substances. Therefore, the contribution of lignosulfonic acid groups to the sulfonic acid content of Rhine River DOC cannot be determined with confidence. Table I11 suggests that most of the SO2 in aquatic macromolecules originate from sulfonic acid groups and not from sulfone groups. However, quantitative information on the contents of sulfone groups in macromolecules cannot be derived from Table III'because the pyrolysis yield of this group is unknown and may not be quantitative and because of the lack of representative reference materials. It is remarkable that the Thur and the Moselle Rivers, which contain higher sulfonic acid contents than the other two tributaries, also display significant levels of sulfone groups. The presence of these sulfone substructures may be explained by higher organically bound sulfur or sulfate contents of soils in the Thur and Moselle drainage basins, for instance, due to specific agricultural activities which lead to soil leachates with higher sulfonic acid and sulfone contents. However,
additional research on the presence and mechanisms of formation of sulfonic acid and sulfone substructures in aquatic humic substances is necessary to obtain a better insight. The water purification plant WRK removes 60% ( n = 1)of the sulfonic acid group content of DOC during their water treatment processes (coagulation with FeC13 and rapid sand filtration). This is surprising since sulfonated macromolecules are expected to be highly water soluble. Eberle however showed that the high molecular weight (MW >1000) DOM fraction of Rhine River water is completely precipitated by FeCl3 coagulation (411, while the low molecular weight (MW C1000) fraction was only partially removed. This was explained by the stronger aggregation properties of high molecular weight DOM fractions. The WRK water purification plant also employs FeCl3coagulation. Therefore, the decrease of the sulfonic acid content by 60% can probably be explained by the water purification of a large macromolecular and sulfonated DOM fraction. This sulfonic acid removal however cannot be ascribed to the removal of lignosulfonic acids unambigiously. So, the sulfonic acid analysis enables the monitoring of water purification processes of sulfonated DOM, and additional measurements are necessary to investigate these processes more closely. The purified WRK water is transported to the dunes for natural sand filtration and then purified additionally by the waterworks of the City of Amsterdam. The sulfonic acid content of DOM isolated from the drinking water of Amsterdam is higher than that of the purified WRK water ( n = l ) , which can be explained by the sampling of two different water bodies or by the input of additional sulfonated DOM during the dune filtration processes. It is possible that sulfonated river DOM has accumulated in the dunes used for filtration over an extended period of time and that these sulfonated macromolecules are delivered back to the water during the natural sand filtration processes. Therefore, it is of interest to monitor the water quality before and after the natural sand filtration process on a realistic time scale.
Further Research Preliminary experiments on the liner pyrolysis phenomenon (see also the section Structural Specificity of S02) show that at a liner temperature of 180 "C only 2 % of the sulfonic acid groups pyrolyze, while at a liner temperature of 240 "C a pyrolysis yield of 68 % is obtained. Assuming a sigmoid curve fit for these two data points, it can be estimated that quantitative conversion of sulfonic acid groups will occur using a liner temperature in the range of 300-350 "C. By analogy, it is very likely that a Curie-point pyrolysis temperature of 358 "C, which is commercially available as a Curie-point wire, will give quantitative pyrolysis of sulfonic acid groups. We tested this hypothesis by pyrolyzing 20 bg of lignosulfonic acid at 358 and 510 "C. Surprisingly, the SO2 signal at 358 "C was significantly higher than at 510 "C, suggesting that at the former temperature quantitative pyrolysis already occurs and that sample losses occur at 510 "C. A Curiepoint pyrolysis temperature of 358 "C still is quite low and will not give a general pyrolysis of the biopolymer/ geopolymer matrix (36). In addition, considering the relative thermostability of sulfone groups, it is likely that a Curie-point temperature of 358 "C will not lead to
significant pyrolysis of sulfone groups, and a selective pyrolysis of sulfonic acid groups will be obtained. For example, we determined that the Curie-point (358 "C) SO2 production from poly@-phenylene ether sulfone) was negligible, providing evidence for the specific pyrolysis of sulfonic acid groups at 358 "C. If the specific analysis of sulfonic acid groups alone is desired, this specific pyrolysis approach is promising. Instead of the Curie-point pyrolysis procedure at 358 "C, a pyrolysis procedure using a conventional splitsplitless injector and an injector temperature of 300 "C seems feasible. The lower pyrolysis temperature in the injector compared to Curie-point pyrolysis at 358 "C can be compensated by a longer pyrolysis/heat transfer time. The technical concept of this injector can be described as follows. The sample can be introduced by opening the split-splitless injector at the top, followed by inserting the sample in a metal cup into the heated glass liner. The use of a metal cup will prevent sample losses, which obviously occur during Curie-point pyrolysis [RSD ca. 25 % ( n = 2) for several lignosulfonic acid samples]. It is recommended to vent the injector before opening, in order to remove the injector cap safely and to prevent the blowing away of the sample by the carrier gas. The introduction of air onto the GC column does not present a risk since all column temperature zones can be maintained at room temperature. In fact, the GC column is only used for flowinjection of SO2 into the SO2 detector (MS or sulfurselective detector). This injector pyrolysis system is expected to result in a simple, reliable, specific, sensitive and quantitative GC(-MS) procedure for the routine determination of the sulfonic acid contents of bio- and geopolymers.
Conclusions A novel analytical procedure, based on temperatureresolved Py-MS detection of sulfur dioxide, has been developed for the selective, sensitive, and quantitative analysis of sulfonic acid groups in macromolecular (MW >1000) lignosulfonic acids and aquatic humic substances. Sulfur dioxide is a characteristic pyrolysis product of sulfonic acid groups, sulfone groups, and inorganic sulfates. These three sulfur-containing groups can be separated thermally by determining the pyrolysis temperature range using in-sourcepyrolysis-mass spectrometry. Sulfonicacid groups pyrolyze at relatively low (T600 "C) temperatures. Sulfonic acid groups can be analyzed quantitatively, while sulfone groups can only be identified. In this paper, the analysis and presence of significant amounts of sulfonic acid groups, and in some cases sulfone groups, in aquatic humic substances are demonstrated for the first time. It appears that sulfonic acid groups bound to macromolecules contribute significantly to the dissolved organic sulfur (DOS) content of river water. The structural speciation of DOS can partially be revealed by the combination of sulfonic acid and elemental sulfur analyses. The high and variable background concentrations of sulfonic acid groups in aquatic humic substances preclude the specific assignment of the detected sulfonic acid groups in Rhine River water to lignosulfonic acids alone. Envlron. Scl. Technol., Vol. 27, No. 12, 1993
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Acknowledgments T h i s work is part of the research p r o g r a m of the Foundation for F u n d a m e n t a l Research on Matter (FOM) and was made possible b y the Dutch Organization for Scientific Research (NWO). The financial and practical s u p p o r t of this research project b y the Water T r a n s p o r t C o m p a n y Rijn-Kennemerland ( W R K ) is greatly appreciated.
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Abstract published in Advance ACS Abstracts, September 15, 1993. 8
