Anal. Chem. 1987, 59, 1709-1715
1709
Simultaneous Determination of Linear Alkylbenzenesulfonates, Alkylphenol Polyethoxylates, and Nonylphenol by High-Performance Liquid Chromatography Antonio Marcomini’ a n d Walter Giger*
Swiss Federal Institute for Water Resources and Water Pollution Control ( E AW A G ) , CH-8600 Dubendorf, Switzerland Linear 4-alkylbenrenesutfonates (LAS), 4-alkylphenol polyethoxylates (APEO) (Le., 4-octylphenol polyethoxylates (OPEO) and 4-nonylphenol polyethoxylates (NPEO)), and 4-nonylphenol (NP) are slrnultaneously determlned by reversedphase hlgh-performance Uquld chromatography (HPLC) uslng octylslllca columns and waterlacetonltrlle gradlent elutlon. LAS are separated accordlng to thelr alkyl chain lengths and coelutlon of NPEO and NP Is observed In contrast to the separation of OPEO and NPEO. The determlnatlon of ollgomer distrlbutkns of APEO and quantltatlon of NP, NPlEO, and NPPEO requlre additional lnformatlon attainable by normal-phase HPLC. Recoveries of 85-100% were found for LAS, APEO, and NP Isolated by Soxhlet extractlon under basic condltlons from laundry detergent powders, sewage sludges, sludgeamended soils, and river sedments. Relative standard deviations for all analytes In the different matrlces dld not exceed 6%. Detectlon llmlts with UV fluorescence detection are 95 ng for NP, 80 ng for LAS, and 65 ng for NPEO with respect to injected amounts.
Linear alkylbenzenesulfonates (LAS) and alkylphenol polyethoxylates (APEO) are very widely used synthetic anionic and nonionic surfactants, respectively. In particular, LAS are major surfactants in laundry detergents, whereas APEO are important surfactants in institutional and industrial surface cleaners (I,2). Linear alkylbenzenesulfonates are synthesized by Friedel-Crafts alkylation of benzene followed by sulfonation of the aromatic ring (predominantly at the para position). The technical LAS products are mixtures containing homologues with alkyl chain lengths usually varying from 10 to 14 carbon atoms (Figure 1). The phenyl group may be attached to the alkyl chain a t any carbon atom except at the end position (so-called phenyl positional isomers). The alkylphenolic nonionic surfactants are manufactured from highly branched 4-nonyl- and 4-octylphenols by reaction with different amounts of ethylene oxide leading to mixtures of oligomers with varying lengths of the polyethoxy chain. The most utilized APEO have nonyl alkyl chains (NPEO, Figure 1) and are complex mixtures of isomers with differently branched alkyl substituents ( 3 ) . LAS and APEO are of environmental concern because of their high abundance in municipal wastewaters ( 4 , 5 ) . NPEO is biotransformed during wastewater and sewage treatment partially forming more persistent and more toxic metabolites with only one (NPlEO) or two (NPZEO) ethoxy units as well as N P (6). Linear alkylbenzenesulfonates have been detected worldwide in many different environmental compartments (7,8). Unaltered LAS accumulate in the range of 0.3-1.2% of the total dry matter in sewage sludges (9). Colorimetric analytical procedures based on complex formation with dye cations (10, II), followed by photometric detection, are routinely used to measure levels of anionic surfactants in various environmental samples (12-14). Howl On leave from the University of Venice, Department of Environmental Sciences, 1-30123 Venice, Italy.
ever, the need to obtain information on specific surfactants, as well as on individual homologues and phenyl positional isomers of LAS, prompted the application of gas chromatography (15) or gas chromatography/mass spectrometry (GC/MS) (16). For these two techniques, the surfactant molecules had to be transformed by desulfonation to yield alkylbenzenes (8) or by derivatization to sulfonyl chlorides (16)or sulfonate esters (17). The determination of LAS without any derivatization has also been carried out by reversed-phase HPLC (18-23). APEO were also largely determined in environmental samples by nonspecific methods (24-26)that measure total contents of nonionic surfactants. These methods fail to differentiate between APEO and linear alcohol polyethoxylates which are also widely used nonionic surfactants. Methods capable of specific determination of APEO employ GC/MS using chemical ionization (27)or both reversed- and normal-phase HPLC (5, 28-30). We present here the first report on procedures for the simultaneous determination of the aromatic surfactants of the LAS and APEO types by using reversed-phase HPLC. In addition, the important refractory metabolite N P can also be measured, if corroborated by normal-phase HPLC analysis. EXPERIMENTAL SECTION Reagents and Materials. The two commercial surfactants Marlon A and Marlophen 810 were supplied by Chemische Werke Hiils AG, Marl, Federal Republic of Germany. Marlon A is a Cl0-Cl3 LAS mixture and Marlophen 810 contains NPEO oligomers with an average of 11 and a range of 1-18 ethoxy units. Imbentin-N/7A, a mixture of 4-nonylphenol mono-, di-, and triethoxylates, was received from W. Kolb AG, Hedingen, Switzerland. Synperonic OPlO was obtained from Imperial Chemical Industries, Petrochemical Division, Middlesbrough, U.K. The latter is an octylphenol-basedproduct with an average of 10 and a range of 2-15 ethoxy units. Sodium dodecyl sulfate (SDS) was purchased from BDH, Poole, U.K., and recrystallized in ethanol/water. 1-Octylbenzenesulfonateand l-nonylbenzenesulfonate (l-CB-and l-C,-LAS) were synthesized by direct sulfonation at 70 “C of n-octylbenzeneand n-nonylbenzene (Fluka AG, Buchs, Switzerland; ref 31). The reaction products were recrystallized in water/ethanol. 3-Tetradecylbenzenesulfonate (3-Cl,-LAS) and 3-pentadecylbenzenesulfonate (3-Cl,-LAS), supplied by Unilever, Port Sunlight, U.K., were used without further purification. Technical 4-nonylphenolwas employed as received from Fluka. Sodium chloride, sodium perchlorate, and sodium hydroxide were all analytical grade from Merck, Darmstadt, FRG, and were used without further purification. The cellulose thimbles (22 X 80 mm) were purchased from Schleicher and Schull, Dassel, FRG and were Soxhlet extracted with methanol for 2 h before use. All solvents were HPLC grade (Mallinkrodt, St. Louis, MO) and the water for HPLC elution was “carbon free” grade (Mallinkrodt). Samples, Extraction, and Sample Preparation. Laundry detergent powders were obtained from commercial sources in Switzerland. Sewage sludges were collected in the treatment plants of Altenrhein and Ziirich-Glatt. The sludges were conserved by the immediate addition of 1% (v/v) of formaldehyde (37%, analytical grade, Merck). After vigorous shaking, aliquots of 500 mL were dried at 60 OC and the resulting solids finely ground. When not immediately dried, the sewage sludges were stored in
0 1987 American Chemical Society 0003-2700/87~0359-1709$01.50/0
1710
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13,JULY 1, 1987
Linear
Alkylbenzenesulfonates :
Homologues : Isomers Alkylphenol
C n -LAS ( n = 8-15) 2 - , 3 - ,4-1 5 - 1 6 - ,7- C n -LAS
Polyethoxylates : APEO
n = 0
NP
n = 1
NPlEO
n - 2
NPPEO OPEO
Flgure 1. Constitutions and acronyms of linear alkylbenzenesulfonates, alkylphenol polyethoxylates, and alkylyhenols: LAS, mixture of homologues and phenyl positional isomers; APEO, 2-[(4-alkylphenyl)poly(oxyethylene)oxy]ethanol; NPEO, 2-[(4-nonylphenyl)poly(oxyethylene)oxy]ethanol;NPPEO, 2-[2-(4-nonylphenyloxyethylene)oxy]ethanol; NPlEO, 2-(4-nonylphenoxy)ethanol;NP, complex mixture of isomers with differently branched nonyl substituents; OPEO, 2-[ 4( 1,1,3,3-tetramethylbutyI)phenyl)poly(oxyethylene)oxy] ethanol.
the dark a t 4 "C. Sludge-amended soil samples were obtained from the Swiss Federal Research Station for Agricultural Chemistry and Hygiene of the Environment a t Liebefeld, dried a t 60 "C overnight, and then pulverized. Sediment samples from the surface (0-3 cm) and from a deeper layer (9-12 cm) of a bottom core of the Rhine River a t Village Neuf (6 km downstream from Basle) were obtained as dried powders. Sediment dating was based on measurement of 137Cs(32). Typically, 500 mg of detergent powder, 2 g of dried sewage sludge, and 20 g of dried soil or sediment were transferred into a preextracted paper thimble. Amounts of the internal standards 1-C8- and 3-C15-LAS,according to those used in the standard solutions (see below), and solid NaOH (20% (w/w)) were added and allowed to equilibrate for 15 min. The thimbles were covered with preextracted cotton in order to prevent removal of fine powder from the thimble into the extracting solvent. The samples were extracted in a Soxhlet apparatus with 80 mL of methanol for 30 min for detergents, 4 h for sludges, and 1 2 h for soils and sediments. The extracts were evaporated to 20 mL (detergents and sludges) or 5 mL (soils and sediments) and then diluted to 50 mL (detergent and sludges) or 20 mL (soils and sediments) with water containing 5 X lo-' M SDS and acetone in such a way that a final composition of methanol (MeOH), water (H,O), and acetone of 1:1:2 was attained. The mixtures were finally centrifuged and aliquots of 20-80 pL were injected into the reversed-phase HPLC columns. The addition of SDS prevented adsorption losses of the analytes in the chromatographic system (20). The MeOH/H,O/acetone mixture was centrifuged in order to precipitate the fine suspended material (formed after addition of water) and to get a clear solution which could be injected without filtration. Portions (200-500 pL) of these mixtures, as well as, separately, aliquots of NP, NPlEO, and NP2EO standard solution in MeOH/H,O (1:l) (NP, 0.1 pg; NPlEO, 0.17 Fg; NP2EO,O.O2 pg), were diluted in a narrow neck volumetric flask to about 20 mL with doubly distilled water to which solution 500
mg of NaCl and 1 mL of n-hexane were added. The resulting suspensions were centrifuged for 15 min and 20-50-pL aliquots of the n-hexane phase were injected into the normal-phase HPLC column. In order to obtain the oligomeric distribution of APEO by normal-phase HPLC, 1g of detergent powder was extracted by stirring or Soxhlet with 100 mL of n-hexane, a solvent which is perfectly compatible with the applied mobile phase. A sample of raw sludge was extracted in a continuous steam-distillation/ solvent extraction apparatus as described elsewhere (30),in order to compare the recovery of NP, NPlEO, and NP2EO from the steam-distillation and the Soxhlet extraction. High-Performance Liquid Chromatography (HPLC). The HPLC system consisted of a Perkin-Elmer Series 4 solvent delivery system equipped with a Rheodyne syringe loading sample injector (Model 7125). Detection was performed either by the spectrophotometers Kontron SFM22 and Perkin-Elmer LS3 or by the UV-vis absorption detector Kratos Spectroflow 773 UV-VIS. Prepacked bonded-phase octylsilica, octadecylsilica, and aminosilica columns were supplied by Knauer, Berlin, FRG. In the reversed-phase mode the following gradient elution programs were used. When the irregularly shaped 10-pm LiChrosorb RP8 octylsilica column (100 mm X 4 mm i.d.1 equipped with a 30 mm x 4 mm i.d. precolumn of the same packing material was employed, first 6.5 min of 5% 2-propanol (IP), 40% HzO, 55% acetonitrile/H20 (ACN/H20,45/55) + 0.02 M NaClO, were applied, followed by a 0.4 convex elution gradient for 10 min leading to 5% IP, 80% ACN/H20 (45/55) + 0.02 M NaClO,, 15% ACN. An 8-min linear gradient reestablished initial conditions. The HPLC was operated with the column at ambient temperature and a flow rate of 1.2 mL min With the spherically shaped 3-pm ODS I1 octadecylsilica column (250 mm X 4 mm i.d.), 4 min of 3% IP, 12% H20,85% ACN/H20 (45/55) + 0.02 M NaC104were followed by a 0.3 convex gradient elution of 23 min leading to 25% ACN and 75% ACN/H20 (45/55) + 0.02 M NaClO,. An 8-min linear gradient was used to return to the starting eluent composition. The flow rate was 0.8 mL min-', and the column was held at ambient temperature. The composition of the mobile phase in the reversed-phase mode yielded a neutral pH (7.2). In normal-phase HPLC, an aminosilica 3-pm column (Hypersil APS, 100 mm X 4 mm i.d.) was employed according to Ahel and Giger ( 5 ) . For determination of NP, NPlEO, and NPZEO, an isocratic elution with an n-hexane/2-propanol mixture (H/IP, 98.5/1.5) was used at a flow rate of 1.5 mL min-', whereas for the determination of the APEO in laundry detergents, 2 min of 100% H/IP (98/2) was followed by a 25-min linear elution gradient leading to 50% H/IP (98/2) and 50% H/IP (98/2), at a flow rate of 1.5 mL min-'. A 5-min linear gradient restored the initial conditions. UV fluorescence detection was achieved at an excitation wavelength of 225 nm (Kontron SFM 22, slit width 10 nm, flow cell volume 30 pL) or 230 nm (Perkin-Elmer LS3, slit width 10 nm, flow cell volume 15 pL) and an emission wavelength of 295 nm. Reversed-phase HPLC with UV fluorescence detection or with UV absorption detection a t 225 nm was used; the work in the normal-phase separations was carried out with absorption detection at 277 nm. In order to obtain the relative molar absorptivities of NPEO with one and two polyethoxy units a t the mentioned fluorescence conditions, the commercial product Imbentin-N/7A was preparatively chromatographed by normal-phase HPLC ( 5 ) . Aliquots of the collected oligomers were weighed by a Cahn microbalance, gently evaporated, and then made up to 1 mL with MeOH/H,O (1/1)before injection into HPLC with an octylsilica column. Quantitation. Two sets of calibration curves were routinely used in the reversed-phase mode, depending on the samples analyzed. For commercial laundry detergents and sludges, standard solutions were prepared by dissolving in a mixture of MeOH/H,O (1/1)with SDS ( 5 X M), 0.040-0.400 mg/mL LAS, 0.020.500 mg/mL of Marlophen 810 or Synperonic OP10, or 0.010-0.500 mg/mL NP. Since Marlon A contains only Cl0-Cl3 chain length LAS (whereas in detergent formulations C9- and Cl,-LAS were also found), additional standards of l-CS-LAS (0.005-0.060 mg/mL) and 3-C14-LAS(0.006-0.110 mg/mL) were prepared. 1-C8-LAS(0.07 mg/mL) and 3-C15-LAS(0.09 mg/mLe), which were used as recovery standards and as internal quantitation
'.
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
1711
a Cn
0 0
B
12 10
24 mL 5 20 rnin
0 0
29
IO 10
39 rnL 30 min
Figure 3. Reversed-phase (A, B) and normal-phase (C, D) high-performance liquid chromatograms of mixtures of technical surfactants: (A) Marlon A and Marlophen 810, (B) Marlon A and Synperonic OP10, (C) Marlophen 810, (D) Synperonic OP10; HPLC columns, 100 mm X 4 mm i.d., LiChrosorb RP8, 10 p m for A and B; 100 mm X 4 mm i.d., Hypersil APS, 3 p m for C and D; Detection, UV fluorescence (A, B), UV absorption (C, D). Key: M, Marlophen 810; S, Synperonic OP10; Clo, CI1,C,, CI3, IS,,and ISpas in Figure 2. Peak numbers in C and D refer to numbers of ethoxy groups.
mal-phase HPLC analyses. The curves were calculated for each APEO oligomer as well as for NP.
RESULTS AND DISCUSSION HPLC Separation. Nakae and co-workers (21) obtained
C
0
0
a
12
I
1
10
20
24 mL 1
-
30 min
Figure 2. Reversed-phase high-performance liquid chromatograms of Marlon A (A), a laundry detergent (B), and a digested sludge extract (C): HPLC column, Spherisorb ODs I I , 3 pm; detection, UV absorption; Cl0, C,,, Cl2, and C13,LAS homologues. The numbers above the LAS peaks indicate the position of the phenyl group on the alkyl chain; IS,,
l-octylbenzenesulfonate; IS,, 3-pentadecylbenzenesulfonate.
standards, were added to each standard solution. Internal calibration curves were calculated for each LAS homologue, as well as for Marlophen 810, Synperonic OP10, and nonylphenol. Following this procedure, recovery was automatically being corrected. For the analysis of sludgeamended soils and sediments, standard solutions with concentrations 1 order of magnitude lower than the previous ones were used. Marlophen 810 (0.015-7.5 mg/mL), Synperonic OPlO (0.010-0.650mg/mL), nonylphenol (0.005-0.200 mg/mL), and Imbentin-N/7A (0.008-0.28 mg/mL) mixture were also separately dissolved in n-hexane/2-propanol (80/20) in order to obtain external calibration curves for nor-
an excellent HPLC separation of the many LAS components (homologues and isomers) by using an octadecylsilica column a t 40 "C with isocratic elution using an aqueous acetonitrile solution containing 0.1 M sodium perchlorate. However both the application of internal standards with alkyl chain lengths of 8 and 15 carbon atoms and the determination of LAS in complex sample matrices such as sewage sludges and sediments make it necessary to apply gradient elution. Good results were achieved by increasing the initial water content of the reported eluent mixture (21) followed by elevated acetonitrile percentages for the elution of the more retained compounds. Figure 2 shows the chromatograms of a commercial LAS surfactant, an extract of a granular laundry detergent, and a sewage sludge. Three-micrometer octadecylsilica columns a t room temperature and absorption detection at 225 nm were used. In addition to the LAS peaks, a broad peak, not shown in the figure, was detected around a retention volume of 34 mL. It was suspected that the latter peak could be assigned to APEO which are the most important aromatic surfactants other than LAS in laundry detergents and which are biotransformed during wastewater and sludge treatment to form more biorefractory intermediates with one and two ethoxy units as well as the not ethoxylated product N P (7). We collected a fraction corresponding to the broad peak and subsequently performed an extraction using nhexane. The collected fraction was then identified as APEO in laundry detergents and as NP, NPlEO, and NPPEO in the sewage sludge extracts by normal-phase HPLC employing an aminosilica column with elution conditions reported by Ahel and Giger (30). The octadecylsilica column was not satisfactory for the simultaneous and routine determinations of LAS, APEO, and NP because of the long analysis time and the band broadening of the APEO/NP peak. The chromatograms of Figure 3A,B were obtained by employing a 10-pm octylsilica column in combination with a higher initial water content of the eluent
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
Table I. Extraction Yields of LAS and APEO from Commercial Laundry Detergent Powders 70 yield, Soxhlet
extraction
% yield, stirring
solvent
extraction time, min
LAS
APEO
extraction time, min
LAS
APEO
60 180
43 101
95 101
40
100
102
60
102
101
30
101
103
60 180
71 101
95 102
30
102
101
ethanol + NaOH
60
101
100
20
100
101
n-hexane
12 24
94 100
30
methanol methanol + NaOH ethanol
_.
100
Table 11. Precision and Recovery of the Determination of LAS, NPEO, NP, NPlEO, and NP2EO in Detergents and Environmental Samples relative standard deviation,” %
LAS sample
C,
Cl0
C1,
laundry detergent digested sewage sludge sludge-amended soil surface river sediment
1.0 -
1.5 4.1
1.1 2.5 5.2 5.6
-
-
-
-
-~ C,* CI3 0.7 1.8 3.2 4.6
2.0 2.1 3.4 4.3
% recoveryb
NPEO 1.7 -
-
NP
NPlEO
NP2EO
LAS
NP
3.1 5.3 4.8
2.8 4.9 4.9
3.6 5.6 5.2
100 95 85 91
100 99 93 94
Triplicate determinations. -, not determinable. *After addition of LAS and NP in amounts of the same order of magnitude as those previously determined for each sample. The recovery was calculated by subtractingthe previous values from the amounts measured in the spiked samples. mixture and a successively sharper and higher increase of the acetonitrile content, in the presence of 0.02 M NaCIOa and 5% 2-propanol. The technical surfactants Marlon A, Marlophen 810, and Synperonic OPlO were used as standard materials containing LAS, NPEO and OPEO, respectively. Linear alkylbenzenesulfonates were separated according to their alkyl chain length; the phenyl isomers with the same alkyl chain length coeluted. The NPEO compounds of Marlophen 810 eluted under two peaks (see Figure 3A), which were tentatively attributed to two groups of alkyl isomers with differently branched nonyl side chains (28). Coelution was observed for the OPEO oligomers of Synperonic OPlO (peak S in Figure 3B). The minor NPEO peak had the same retention volume as the OPEO peak, which would preclude separate determination of NPEO and OPEO in reversed-phase HPLC. Nonylphenol eluted with same retention volume as NPEO. This suggests that the difference of the length of the ethoxy chains does not influence the partition of APEO between the mobile and stationary phases under the chromatographic conditions employed here. Figure 3C,D shows the oligomeric distributions of Marlophen 810 and Synperonic OPlO in the normal-phase mode. Extraction a n d Recovery. Both LAS and APEO can be quickly and completely extracted from laundry detergent powders either by vigorous stirring of an ethanolic or methanolic suspension or by Soxhlet extraction with the same solvents. When only APEO are to be determined by injection into normal-phase HPLC, 20 min of stirring in n-hexane is sufficient to completely extract APEO. Table I gives the extraction yields obtained for LAS and APEO from a granular laundry detergent by different extraction procedures. The yields of the internal standards l-CB-LASand 3-CIS-LAS added before the extraction were the same as those of LAS mixture contained in the detergent. The same yields were obtained by attributing 100% recovery to the extracted amounts which did not increase any more with longer extraction times. Both ethanol and methanol extract APEO more quickly than LAS. Moreover, in some detergents the
basic properties of the extracts improved the chromatographic separation of the LAS homologues. In normal-phase HPLC the APEO yield which did not increase any more with longer extraction times was taken as 100%. Four hours of Soxhlet extraction with methanol is sufficient to almost completely extract the LAS contained in sludges but only extract around 50% of the NP,NPlEO, and NPZEO. The recoveries from sludge shown in Table I1 were achieved after the addition of solid NaOH (20% (w/w)) to the dried sludge powder before the extraction. Since NP, NPlEO, and NP2EO coeluted in reversed-phase HPLC, determination of individual compounds was possible only by normal-phase HPLC (30). Therefore, 200-5OO-lL aliquots of the MeOH/ H20/acetone mixture of raw sludge, diluted with water, was centrifuged after addition of NaCl and 1 mL of n-hexane. Aliquots of a saturated solution of NP, NPlEO, and NP2EO were processed in the same way. Moreover, the same raw sludge previously Soxhlet extracted was also extracted in a continuous steam-distillation apparatus. Steam distillation extraction provided recoveries of 9 5 9 8 % for NP, NPlEO, and NP2EO (30). The latter chemicals contained in the aliyuots of the standard solution were efficiently recovered (95100%) into the n-hexane layer as was evident from comparison of the added and found concentrations. A further evaluation of the enrichment efficiency was carried out by comparing the results obtained from steam distillation and from Soxhlet extractions (Table 111). The values of NP, NPlEO, and NP2EO found by the two extraction methods were very similar indicating an exhaustive transfer of the analytes into n-hexane. Sample Preparation. In order to achieve the high recoveries reported in Table IT, the solvent composition of the samples must be adjusted before injection onto the reversed-phase column. The injection of the methanol solution from Soxhlet extraction gave poor HPLC results for LAS, Le., broad and distorted peaks. It is suspected that intramolecular interactions of LAS in pure methanol interfere with the chromatographic performance. For detergent powders better
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
Table 111. Normal-Phase (A, B) and Combined Normal- and Reversed-Phase (C) HPLC Determinations of NP, NPlEO, and NP2EO in Raw Sewage Sludge Using Steam-Distillation/SolventExtraction (A) and Soxhlet Extraction (B, C )
concentration,Omg/kg dry weight NP NPlEO NPZEO A
B C
0.47 & 0.02 0.48 f 0.02 0.46 f 0.02
0.69
* *
0.1 0.70 f 0.1 0.66 0.1
*
0.04 0.01 0.05 f 0.01 0.04 0.01
*
Arithmetic means and standard deviations of triplicate determinations. results were obtained when methanol extracts were partially evaporated and diluted with a t least 30% water containing 2x M SDS. When the same solvent composition was used for the sludge extracts, chromatographic separations were satisfactory, but considerably lower amounts of higher chain length LAS, as well as NP, NPlEO, and NPZEO, were recovered. The use of two internal standards braketing the elution volume of the LAS helped to clarify this point. It was observed that the methanol/water mixture led to a recovery of the first internal standard between 75 and 90% and to a recovery of the second internal standard between 40 and 70% (in the case of sludges). Even lower recoveries were found for soils and sediments. The low recovery was not due to incomplete extraction from the matrices but to interactions between the emulsified material formed by the addition of water to the methanol extract. When the mixture included acetone (methanol/water/acetone, 1/1/2), the extent of emulsification decreased and the recoveries of both internal standards were 85-95% as NP, NPlEO, and NPSEO. Detection and Quantitation. Absorption maxima of LAS and APEO mixtures such as Marlophen 810, Synperonic OP10, and NP in the solvent mixtures used for reversed-phase are at 225 nm with similar molar absorptivities. Additionally, APEO and N P also display an absorption maximum approximately 5 times less intense around 280 nm, the wavelength usually exploited for HPLC detection (28-30). The emission of UV fluorescence for LAS is at 270-320 nm, that of APEO and NP is at 280-350 nm. The fluorescence maxima are as follows: LAS, 295 nm; OPEO, 302 nm; NPEO and NP, 306 nm. Because LAS elute under as many peaks as the number of homologues, unlike the elution of single peaks of OPEO, NPEO, and NP, the conditions best suited for the detection of LAS were used in reversed phase, i.e., 225 nm as excitation wavelength and 295 nm as emission wavelength in fluorescence and 225 nm in absorption. In the normal-phase mode APEO and N P were detected by absorption at 277 nm. Since LAS with alkyl chain lengths greater than or equal to six have the same molar absorptivity (33),peak area ratios of LAS correspond to molar ratios of the LAS components. Thus, the average molecular weight of LAS mixtures can easily be calculated. Quantitation of LAS was carried out in the fluorescence detection mode by using the internal standards l-CB-LASand 3-C15-LAS.The calibration curves used for each homologue were all linear ( r = 0.9982-0.9991) in the concentration range 0.004-0.400 mg/mL of total LAS. In the normal-phase mode, the APEO oligomers and N P were quantitated through calibration curves (r = 0.9974-0.9995) of Synperonic OP10, Marlophen 810, and Imbentin-N/7A. The summed APEO oligomers as well as N P were also quantified in reversed-phase HPLC with the internal standards l-CB-LAS and 3-C15-LAS. The calibration curves were linear ( r = 0.9988-0.9994) in the concentration range 0.001-0.500 mg/mL. Since the molar extinction coefficients for APEO are independent of the ethoxylate chain length (34) (extinction coefficient for N P l E O to NP16EO at 230 nm was between
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8.9 and 10.6 x lo3), the quantitation in reversed-phase HPLC based on the total areas of the APEO standards implies the availability of standards with APEO oligomeric distributions similar to those of the examined environmental samples. Due to the coelution of NP, NPlEO, and NP2EO in reversed-phase HPLC, their individual quantitation in the reversed-phase mode needed the knowledge of NP, NPlEO, and NP2EO percentages and of their molar absorptivities. For this purpose, the fraction of eluent corresponding to their peak or aliquots of the Soxhlet extract were diluted with water, NaCl and n-hexane were added, and the mixture was centrifuged. The n-hexane extract was analyzed in normal-phase HPLC, and from the knowledge of the response factors (30), the percentages of NP, NPlEO, and NP2EO were calculated. After collection by preparative HPLC from the standard Imbentin-N/7A, N P l E O and NP2EO displayed in the reversed-phase solvents mixture molar absorptivities 1.5 times higher than NP. Table I11 shows that the concentrations of NP, NPlEO, and NP2EO determined by the combined normal- and reversed-phase HPLC are in quite good agreement with those obtained in the normal-phase mode. Accuracy, Precision, and Sensitivity. Recovery and reproducibility of the described determination method for different samples are reported in Table 11. Compared to commercial detergents and sewage sludges, the recoveries of LAS in sludge-amended soils and river sediments were lower with almost two times higher relative standard deviations. Fluorescence detection at 230/295 nm after separation on an octylsilica column gave detection limits ( S I N = 5) for APEO and LAS mixtures and N P of 95, 80, and 65 ng injected, respectively. When laundry detergents and sewage sludges were analyzed by reversed-phase HPLC, it was possible to employ either fluorescence or absorption detection. For sludge-amended soils and sediments only detection by fluorescence provided the necessary selectivity to reliably quantify LAS. However, NP, NPlEO, and NP2EO in sludge-amended soils and sediments could not be accurately measured by reversed-phase HPLC, even by using fluorescence, because of interferences in the resulting chromatograms. The detection in normal-phase HPLC was made by absorption a t 277 nm, since the response factors of APEO and N P were already available from the literature (5). However, for sediments and soils, fluorescence detection a t 225/306 nm or absorption a t 225 nm is recommended for their higher sensitivity. Applications. The described analytical method was used to determine LAS, APEO, and N P in laundry detergents, sewage sludges, sludge-amended soils, and river sediments. Typical results obtained by reversed-phase HPLC on a 10-pm octylsilica column are presented in Figure 4. The high concentration of hydroxy ions of the Soxhlet extracts caused a shift toward lower retention volumes of LAS, APEO, NP, and particularly of the first internal LAS standard (l-octylbenzenesulfonate). The same shift was displayed by standard solutions containing the similar hydroxy ion concentrations. Many analyses of extracts of detergents and sludges did not deteriorate the HPLC column when careful maintenance was performed, such a s cleaning of the precolumn filters, replacement of the precolumns, and regeneration of the column after approximately 60 runs. The LAS analysis on the 10-wm octylsilica column provides only the homologues distributions, compared with the 3-wrn octadecylsilica column by which both homologues and partial phenyl positional isomer distributions can be obtained. Substantial variability of the LAS phenyl positional isomers distributions was found in granular laundary detergents (35), whereas phenyl positional isomers distributions were observed to be remarkably constant in sludges. Therefore, the 10-pm
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
Table IV. LAS,OPEO, NPEO, NP, NPlEO, and N2EO Concentrations in Granular Laundry Detergents and Environmental Samples
laundry detergent" laundry detergent"
digested sewage sludgeb*' sludge amended soild,' river sedimentd*/
LAS
OPEO
NPEO
8.6 2.4
2.7
3.9 0.2
LAS
NP
NPlEO
NP2EO
7.3 15.0
1.2 1.6
0.07
5.6
0.9
0.22 0.40 0.80
0.03
0.70
"Values are percent w/w. *Values are g/kg dry weight. 'Grab sample from the treatment plant of Altenrhein, Switzerland. dValues are mg/kg dry weight (ppm). e Grab sample from the Swiss Federal Research Station for Agricultural Chemistry and Environmental Hygiene at Liebefeld. fSurficia1sediment (0-3 cm) from the River Rhine at Village Neuf (6 km downstream of Basle).
C
IS4
0 0
1.5
10
30 20
mL min
Figure 5. Normal-phase high-performance liquid chromatograms of commercial laundry detergents containing NPEO (A) and OPEO (B), respectively. HPLC conditions are described in Figure 3. Peak numbers refer to the numbers of ethoxy units. 0 0
I2
io
24 20
3jjrnL 30min
v lo
24 20
mL min
Flgure 4. Reversed-phase highperformance liquid chromatograms of two commercial laundry detergents containing LAS and APE0 (A, B), and digested sludge (C), a sludge-amended soil (D), and surficial (E) and deep (F) sections of a river sediment core: HPLC conditions as described for Figure 3; NP, NPlEO, NPPEO, NPEO, OPEO as in Figure 1; TBS, tetrapropylenebenzenesulfonate;Cia, C,,, C12,C,,, C,,, IS,, IS, as for Figure 2.
octylsilica column is particularly well suited for routine analyses of LAS, and the octadecylsilica column may be used as an ancillary technique when insights into phenyl isomer distributions are needed. As shown in Figure 4, the LAS homologue distributions vary remarkably between extracts from detergents, sewage sludges, sludge-amended soils, and sediments. The higher homologues are more abundant in sludges, soils, and sediments. This is of ecotoxicologicalsignificance since an increase in the length of alkyl chain has been associated with an increase in the toxicity toward various aquatic species (36). The chromatograms E and F of Figure 4 reflect the alkylbenzenesulfonate mixtures found on the surface (0-3 cm) and in a deeper layer (9-12 cm, corresponding to the years 1965-1970) of a dated river sediment core (32). The oligomer distribution in the surface sample is typical for the LAS, whereas in the deeper layer the tetrapropylene-derived branched alkylbenzenesulfonates (TBS) appear (21). The latter surfactants were of environmental concern because they caused foaming in sewage treatment plants and receiving waters. The quantitative
determination of TBS would be possible by using the methods discussed here and a TBS standard. However, it would not be feasible to analyze for LAS and TBS occurring in the same sample because of insufficient chromatographic separation efficiency. Figure 5 displays typical chromatograms obtained by normal-phase HPLC of two granular laundry detergents, one containing NPEO and the other OPEO. In the reversed-phase mode (Figure 4A,B) no split of the NPEO oligomers contained in the granular powder detergents (35)was observed. From the comparison of the chromatograms of Figure 3C and Figure 5A, it appears that the peaks of Marlophen 810, which was used as an NPEO standard mixture, are broader than those of NPEO recorded from the granular detergents. Broadening of the peaks in normal-phase HPLC and splitting into two peaks in reversed-phase HPLC were observed only in Marlophen 810. Therefore, separate determinations of OPEO and NPEO by reversed-phase HPLC were possible. The majority of the commercial powder detergents we examined were found to contain NPEO with up to 17 ethoxy units (35). The results from quantitation of APEO in the reversed- and normal-phase HPLC agreed within k8% suggesting that Marlophen 810 and Synperonic OPlO are good standards for determining APEO in granular laundry detergents by reversed-phase HPLC. Table IV presents the concentrations of LAS, APEO, and NP typically found in environmental samples. The environmental occurrence of these contaminants in sewage sludge, amended soils, and sediments depends on the rate of appli-
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
cation of sludge to the land and on the rate of degradation in the soil. Similarly, the distance of the river sediments from a sewage treatment plant outflow and microbial transformation in the sediments determine the residual LAS levels in sediments. An investigation into the occurrence and fate of LAS, NP, and the NPEO during sewage treatment and after sludge application to land is currently being carried out in our institute using the methods presented in this paper.
CONCLUSIONS The presented analytical procedure employing reversedphase HPLC is particularly suited for routine analyses of large numbers of samples because of its detection selectivity, simplicity, and short analysis time. Quantitation of individual APEO oligomers and NP requires additional information attainable by normal-phase HPLC. Therefore the combined use of reversed- and normal-phase HPLC provides detailed information on the concentrations and structural identities of LAS, APEO, and N P in a broad range of solid matrices including commercial detergents, sludges, soils, and aquatic sediments.
ACKNOWLEDGMENT We are grateful to M. Ahel, P. Brunner, J. McEvoy, and C. Schaffner for helpful discussions. We thank Unilever, Port Sunlight, U.K., for supplying LAS standard compounds, H. Bolliger for drafting the figures, and E. Heyerdahl for helping to prepare the manuscript. Registry No. NPEO, 9016-45-9; NP, 104-40-5; OPEO, 26636-32-8; SDS, 151-21-3; l-CS-LAS, 17012-98-5; l-CS-LAS, 47019-68-1;3-CI4-LAS,65186-01-8;3-Cl,-LAS, 107514-80-7;Marlon A, 25155-30-0.
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RECEIVED for review September 18, 1986. Accepted March 9,1987. This project was partly funded by the Swiss National Science Foundation (Nationales Forschungsprogramm 7D, research project on “Organic Contaminants in Sewage Sludges”). Additional support was obtained from the project COST 641 “Organic Micropollutants in the Aquatic Environment” in the framework of the European Cooperation for Scientific and Technical Research.
