Anal. Chem. 1994,66, 1620-1629
Determination of Alkyl Sulfates and Alkyl Ethoxysulfates in Wastewater Treatment Plant Influents and Effluents and in River Water Using Liquid Chromatography/Ion Spray Mass Spectrometry Darwin D. Popenoe,' Samuel J. Morris, 111, Paul S. Horn, and Kevin 1.Norwood The Procter and Gamble Company, Ivorydale Technical Center, 5299 Spring Grove A venue, Cincinnati, Ohio 452 17
A method has been developed for monitoring concentrations of alkyl sulfates and alkyl ethoxysulfates (AES) in environmental matrices using ion spray LC/MS to determine individual AES species. The ion spray LC/MS technique enablesinterferencefreequantitationof completely resolved (by alkyl and ethoxylate chain length) AES species at less than part-per-billion concentrations. This method was validated using spike and recovery and measuring concentrations of 36 AES species (ranging in size from 12 to 15 alkyl carbons and from 0 to 8 ethoxylate groups) in river water and influents and effluents from a municipally owned wastewater treatment plant. Two different spike concentrations were used for each matrix, and the total mass of AES recovered from each matrix was greater than or equal to 90%of the amount spiked, except for effluent at a high spike level, from which 75% was recovered. A mathematical model, which fits experimentally measured concentrationsto an expecteddistributionof AES components, enables prediction of total AES concentrations from measurements of a few selected components in cases where expected distributions are known with sufficient accuracy. This report presents a new, highly specific method for determination of alkyl sulfate (AS) and alkyl ethoxysulfate (AES, also known as alcohol ethoxysulfate or alcohol ethoxylate sulfate) in environmental matrices. These anionic surfactants are commonly used in consumer products (shampoos, hand dish washing liquids, laundry detergents, etc.). After such use, these chemicals are generally discharged into municipal wastewater treatment plants (WWTP), where they are extensively removed. After treatment, a residual quantity may enter surface water. Monitoring WWTP influents and effluents, as well as river waters, increases our understanding of the removal of these chemicals by WWTPs and of their exposure levels to aquatic organisms. Few data on environmental concentrations of AES components currently exist in the literature. Accurate monitoring requires a specificmethod that quantifies individual AES components without interference from non-AES species. The chemical structure of AES consists of an aliphatic hydrocarbon chain (the alkyl group) connected tooneor more ethoxylate groups and terminated by a sulfate group. The general formula for AES is CH~(CH~)ncl(OCH2CH2)n,OS03-*M+, where nc is the number of carbon atoms in the alkyl portion of the molecule, nEO is the number of ethoxylate 1620
Analyticai Chemistry, Vol. 66,No. 10, May 15, 1994
groups, and M+ is a counterion, frequently Na+ or NH4+. Commercially produced AES consists of a blend of individual AES species, encompassing a range of nc and nEO values. AES mixtures typically contain some level of AS (nEO = 0). In the United States, commercial AS and AES are produced via sulfation of fatty alcohols or alkyl ethoxylates (AE), respectively. The majority of AES blends manufactured commercially are produced using AE feed stocks which have alkyl chains in the range 12 I nc I 15 and a low degree of ethoxylation (i-e., average nEO I3).l Many methods have emerged for measuring trace surfactant levels in water, as described in recent however, previous measurements of environmental AES concentrations have relied upon nonspecific methods such as the methylene blue active substance (MBAS) methodlOJ1-or have utilized techniques which provide resolution by alkyl chain length, but not by both alkyl chain length and number of ethoxylate g r ~ u p s . ~ *AJ ~knowledge of the distribution of AES components in the environment will improve assessments of environmental safety, because aquatic toxicity varies with nc and nEo;'thus, a component-specific measurement technique is key to accurately assessing the environmental safety of AES. Such a method must be capable of separating and quantifying a large number of AS/AES homologues and must resolve AES by both nc and nEO. A combined liquid chromatography/ mass spectrometry (LC/MS) technique is an appropriate method for trace-level quantification of such charged, nonvolatile compounds. In fact, LC/MS has recently been applied (1) Environmental and Human Safety of Major Surfactants. In Final Report io the Soap and Detergent Association; Arthur D. Little Co.: Cambridge MA, February 1991; Vol. 1, Part 2. (2) Nubbe, M. E.;Adams, V. D.; Watts, R. J.;Clark, Y. R.Res. J. WaterPollur. Control Fed. 1991, 63, 338-361. (3) Shimoishi, Y . ;Miyata, H. Fresenius J . Anal. Chem. 1992, 338, 46-49. (4) Marcomini, A. Riu. Ital. Sostanze Grasse 1991, 68, 339-344. (5) Huber, W. Tenside, Surfactants, Deterg. 1991, 28, 106-110. (6) Kalbfus, W. Muench. Beitr. Abwasser-, Fisch.-Flussbiol. 1990.44, 195-204. (Umweltvertraeglichkeit Wasch-Reinigungsm.) (7) Matthijs, E.; Hennes, E. C. Tenside, Surfactants, Deferg. 1991, 28, 22-27. (8) Sakamoto, T. Jpn. J . Toxicol. 1990, 3, 223-230. (9) Huber, L.; Wagner, H. Muench. Beitr. Abwasser-,Fisch.-Flussbiol. 1991.45, 336-353. (Aktue. Chem. Biol. Wasser-Sch1ammanal.-Anwend., Ergeb. Deren Oekol. Bewertung). (10) Llenado R. A.; Jamieson R. A. Anal. Chem. 1981, 53, 174R-182R. (11) Llenado, R. A.; Neubecker T . A. Anal. Chem. 1983, 55, 93R-102R. ( 12) Neubecker T. A. Enuiron. Sci. Technol. 1985, 19, 12. ( 13) Fendinger, N. J.; Begley, W. M.; McAvoy, D. C.; Eckhoff, W. S . Enuiron. Sci. Technol. 1992, 26, 2493-2498. 0003-2700/94/0366-1620$04.50/0
0 1994 American Chemicai Society
widely to analysis of trace organic compounds in water,”17 and specifically to surfactants in water.18-22 The LC/MS method described here used a commercial triple-quadrupole mass spectrometer with an ion spray interface, which has been described previo~sly.2~-~~ Ion spray LC/MS is ideally suited for trace surfactant analyses and superior to other techniques we tested-including infusion MS techniquesand GC/MS analysis (using electron ionization and positive- and negative-ion chemical ionization) of various derivatives of AS/AES. Ion spray is generally described as a pneumatically assisted electrospray ionization source. The ion evaporation model, whereby a charged analyte is ejected from a microsized charged droplet by Coloumbic repulsion, is frequently cited as a prominent ion formation mechanism. The ion spray interface affords higher selectivity and sensitivity due to the lack of molecular fragmentation, while delivering efficient ionization of analyte in the MS. Fast atom bombardment MS has also been used without coupled LC28929 or coupled to capillary electrophoresis30 for environmental analysis of surfactants. Ion spray LC/MS has been used previously for analysis of AS;23however, to our knowledge, its use for quantitative analysis of AS/AES or other surfactants in environmental matrices has not been reported.
Table 1. Dbtrlbutlonr of AES Compomntr In the Two Standard Tml AES Bhnck Used for Spiking and StanUarde
~~z.~SEO~.~S blend Cl4-l~EOl.6S blend nm 0 1 2 3 4 5 6 7 8 sum of 0-8 ethoxylatee
%=12 21.00 11.68 7.02 3.71 1.90 1.15 0.95 0.60 0.42 48.43
“13 21.85 12.06 7.21 3.79 1.94 1.17 0.96 0.60 0.42 50.00
“14 20.06 7.33 7.08 5.49 3.97 2.75 1.54 0.94 0.62 49.78
~ 3 1 5 18.57 6.75 6.49 5.01 3.61 2.50 1.39 0.85 0.56 45.73
Tabulated values are masses of each component, with a given alkyl chain length (m) and number of ethoxylate oups (nm), expressed as percentage of the total mass of the AEfblend. @
species of interest for this work (12 Inc I15 and 0 < EO < 8). Both test substances were characterized for molar distributions of nE0 by Shell DevelopmentCo., using a combination of LC and GC techniques. The weight percents of each AES species in each blend (given in Table 1 for components up to EO = 8) were calculated from the nEocharacterizationdata,3l EXPERI MENTAL SECTION together with the alkyl distribution of typical alcohol feedstocks Standard Test Substances. The alkyl ethoxysulfatesused for the alkyl ethoxylates which were precursors to the AES for both quantitative standards and spiking were commercial blends used in this study. The average molecular weight of blends denoted by C12-13EO1.0S and C~Q-~SEOL.SS. This each material was determinedfrom measurements of hydroxyl nomenclature refers to the range of alkyl chain lengths and numbers of the AE precursor and was used to determine the the average degree of ethoxylation. Specifically, the percent activity of the standard test substances. C12-13EOl.oSstandard is the ammonium salt of a mixture Chemical Reagents. C2 solid-phase extraction (SPE) which contains Cl2 and C13 alkyl chain lengths and has an cartridges (Varian Associates), which contain 500 mg of average EO of 1.0. Similarly, the C1Q-lSE01.SS standard packing, were used to extract AES from the samples. All is the sodium form of a mixture containing C14 and C15 alkyl deionized (d.i.) water was purified by a NANOpure I1 system chain lengths with an average nEO of -1.5. Both were (Barnstead). The reagent grade methanol (MeOH) and produced by sulfation of Neodol alkyl ethoxylate precursors 2-propanol (2-PrOH) used were obtained from J. T. Baker. obtained from Shell Development Co. These blends were For HPLC mobile-phase preparation, HPLC-grade amchosen for this study because they contain all of the AES monium acetate (NHsOAc) and acetonitrile (CH3CN) were utilized (J. T. Baker). The internal standard (IS) used in this (14) Clark, L. B.; Rosen, R. T.;Hartman, T.G.;Alaimo, L. H.; Louis, J. B.; Hertz, work was pure CDs(CD2)110CH2CH20SO3Na,which was C.; Ho, C.-T.; Rosen, J. D. Res. J . Wafer Pollut. Control Fed. 1991, 63, 104-1 13. synthesized and purified at Procter and Gamble. The (15) Miles, C. J.; Doerge, D. R.;-Bajic,S. Arch. Emiron. Contam. Toxicol. 1992, materials used to check instrument sensitivity were sodium 22, 247-251. (16) Brown, M. A.; Stephens, R. D.; Kim, I. S. Trends Anal. Chem. 1991, 10, dodecyl sulfate (Mallinkrodt) and sodium dodecyl-d25sulfate 330-336. (Cambridge Isotopes). A solution of three different types of (17) Kim, I. S.; Sasinos, F. I.; Rishi, D. K.; Stephens, R. D.; Brown, M. A. J. Chromafogr. 1992,589, 177-183. poly(propy1eneglycol) (PPG) (Aldrich) was used to calibrate (18) Clark, L. B.; Rosen, R. T.;Hartman, T. G.; Louis, J. B.; Rosen, J. D. Int. J . the MS daily. This solution contained 0.4 g/L PPG 2000,O.l Environ. Anal. Chem. 1991, 45, 169-178. (19)Clark,L.B.;Roscn,R.T.;Hartman,T.G.;Louis,J.B.;Suffet,H.,II;Lippincott,g/LPPG 1000,0.014g/LPPG425,1 g/LCHsCN,O.l5g/L R. L.; Rosen, J. D. Int. J . Environ. Anal. Chem. 1992. 47, 167-180. NH~OAC,and 1 mL/L of formic acid in 5050 d.i. H20/ (20) Schrocder, H. F. DVGW-Schriffenr.. Wasser 1990, 108, 121-144 (Neue Technol. Trinkwasserversorg.). CH30H. (21) Schroeder, H. F. WaferSci. Technol. 1991. 23, 339-347. Samples. Sewagesamples used for method validation were (22) Schrocder, H. F. J . Chromatogr. 1991, 554, 251-266. (23) Conboy, J. J.; Henion, J. D.; Martin, M. W.; Zweigenbaum, J. A. Anal. Chem. obtained from the Clermont County Lower East Fork 1990,62,800. wastewater treatment plant. This treatment plant, which uses (24) Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem. 1987, 59, 2642. (25) Thomson, B. A.; Ngo, A.; Shushan, B. I. Advantages of an API Source for rotating biological contractor treatment, is located 5 km Analysis by Liquid Chromatography/Mass Spectrometry. In Instrumentarion from Milford, OH, and it receives primarily domestic for Trace Organic Monitoring; Clement, R. E., Siu, K. W. M., Hill, H. H., Eds.; Lewis Publishers: Boca Raton, FL, 1992; pp 209-217. wastewater. Sewage samples were either primary influent (26)Yergey, A. L.; Edmonds, C. G.;Lewis, I. A. S.; Vestal, M. L. Uquid (before clarification) (IF) or final effluent (EF). Samples of Chromafography/MassSpectrometry: Techniquesand Applications; Plenum Press; New York, 1990; pp 62-79. river water (RW), from the Lower East Fork of the Little (27) Allen, M. H.; Shushan B. I., LC-GC 1993, 11, 112. Miami River, were collected upstream of the WWTP. The (28) Ventura, F.; Fraisse, D.; Caixach, J.; Rivera, J. Anal. Chem. 1991,63,2095-
-
-
2099. (29) Borgerding, A. J.; Hites, R. A. Anal. Chem. 1992, 64, 1449-1454. (30) Brumley, W. C. J . Chromatogr. 1992, 603, 267-272.
(31) Private communication from L. Kravetz and S. Dubey of Shell Development Co., April 2, 1993.
Analytcal Chemistry, Vol. 66,No. 10, M y 15, 1994
1621
I F and EF samples were grab samples collected in a metal bucket and transferred into clean glass bottles via glass funnels. The samples were stirred and then preserved with 3% formaldehyde (added in the form of 8% (v/v) of a 37% formaldehyde solution) and spiked with AES (except for control samples). The entire procedure took 1 1 h from collection to spiking. River water samples were handled the same way-except that river water was collected directly into the bottle (after rinsing the bottle twice with the same water) and the time between collection and spiking was 1 2 h. Isolation and ConcentrationProcedure. Before extracting, each sample was shaken to ensure thorough mixing and suspension of particulate material-except the refrigerated preservation samples, which were allowed to reach room temperature and stirred with a magnetic stirrer. SPE cartridges were attached to a vacuum manifold, and the following procedure was performed in parallel on several cartridges. A filter paper (Fisher, fluted, 0.45 pm), a plastic funnel, and a 70-mL cartridge reservoir were connected in series on top of the cartridge. The SPE columns were preconditioned with 10 mL of 80% methanol/20% 2-propanol (v/v), followed by 10 mL of d.i. H2O. First, the appropriatevolume (100 mL for influent, 200 mL for effluent and river water) of sample was poured into the cartridge assembly graduallyand aspirated at 1drop/s. The assembly was then washed with 10 mL of d.i. HzO. The eluate from these steps was discarded. The AES was then eluted from the cartridge with 10 mL of 80:20 MeOH/2-PrOH. The eluate was collected in a 4-dram (- I5-mL) vial. A rinse of 3 mL of MeOH/2-PrOH was sprayed into the mouth of the assembly and added to the sample in the vial. The eluate was evaporated to dryness at room temperature under Nz. The residue was reconstituted with 5050 MeOH/ d.i. H20 (v/v) containing 2.5 mg/L internal standard. The reconstitution volume was 2 mL for IF and 1 mL for EF and RW, yielding a concentration factor of 50: 1 for influents and 200:l for effluents and river waters. Liquid Chromatography. The LC system consisted of a 600 multisolvent delivery system, a Model 600 system controller, and a WISP 715 autosampler (Waters). The chromatographic separation used a 4.6-mm-diameter X 250mm CS reversed-phase column, with 5-pm packing having pore size of 120 A (J. T. Baker). A linear gradient was used in the separation. Mobile phase A consisted of 0.3 mM NH40Ac in 20% CHsCN/80% d.i. H20 (v/v), and mobile phase B consisted of 0.3 mM NH4OAcin 80%CH&N/20% d.i. HzO. The gradient progressed from 80% A and 20% B at t = 0 min to 45% A and 55% B at t = 30 min. All 36 AES species eluted within the 30-min gradient. Following the separation, the % B was linearly increased to 100% between t = 30 and t = 34 min and held there for 36 min. This step was necessary to elute unknown compounds and provide reliable long-term operation of the LC column. The flow rate was held constant at 1.OmL/min, and the injection volume used for all samples and standards was 100 pL. Mass Spectrometry. The column effluent was split and -40 pL/min was diverted to the ion spray interface through a 100-cm length of 75-pm-diameter silica capillary tube. A Perkin-Elmer Sciex API I11 mass spectrometer equipped with
-
-
-
-
1622 Analytlcai Chemistry. Vol. 00, No. 10, May 15, 1994
an ion spray LC/MS interface was used for detecting and quantitating the AES species in the column effluent. The API 111, which is a triplequadrupole MS, was used because it contained the needed interface and was available to the authors; however, only a single quadrupole was used for collecting data. The MS was operated in negative-ion mode, using one of two types of mass monitoring. Most samples were analyzed in full-scan mode, scanning over the range of 200 5 m/z 1800. However, the reanalysis of low-spiked RW used selected-ion recording (SIR) of eight masses-one of which corresponded to the internal standard. The MS was set to scan with a step size of 1 amu and a dwell time of 1.68 ms. Each group of LC/MS runs was started on a separate day and preceded by a recycle period on the MS, tuning, and calibration. The recycle period (12 h long) is an automatic sequence of baking out, cooling, and pumping out the mass spectrometer. Calibration of the instrument was performed with PPG in positive-ion mode and on a commercial blend of Clz-15E012S AES in negative-ion mode each day prior to analyses. A solution of sodium dodecyl sulfate and sodium dodecyl-dz5 sulfate (Le., containing a perdeuterated alkyl chain) in methanol was analyzed in negative-ion mode as a sensitivity check and to verify the absence of mass discrimination. Quantitation. Calibration curves for each of the 36 AES species were generated by first injecting three different concentrations of a standard mixture which contained C12-13E01.&, C14-15E01.5S and internal standard. The IS concentration was held constant while the concentrations of theC12-13E01.& and C I ~ I S E O I .werevaried. SS All standards contained the same blends and thus the same relative distributions of AES components. The concentration of each component in each standard injection was determined using the known weight percentage distribution for the standard test materials. A new set of standard curves was made for each group of runs on the LC/MS and for each different matrix and spike level. The standard concentrations (in milligrams per liter in the injection vial) were approximately 9, 90, and 180 for IF; 9, 27, and 45 for EF; and 45, 90, and 180 for RW. Peak areas of standards and sampleswere integrated (using the MacSpec software from PE Sciex) and normalized against the IS ( m / z = 334). The IS was used to correct for any difference betweenvolumesof different LC injections. Leastsquares analysis to fit a straight line to concentrations vs normalized area yielded slopes and intercepts for each of the 36 AES species. Typically, R2 values were 10.99. The resulting equations (standard curves) for the AES species were used for quantitating levels of AES in environmental samples. All concentrations reported herein are corrected for the dilution and concentration steps of sample preparation and correspond to amounts in the original sample upon collection. THEORY A model was developed to predict total AES concentrations from measured concentrations of selected components, provided the distribution of components in the unknown sample is proportional to a known, “theoretical” distribution (e.g., the weight percentsof componentsin a standard test material).
The model fits the known AES distribution to the concentrations of the measured components and determines a scaling factor which yields total AES concentration. In this model, the theoretical distribution is a vector X of weight fractions of all J components comprising the total AES mass. The measured concentrations form a vector Y of I components, where the set of I components is a subset of the total J components. The particular I components may be chosen from the total set of J components based on the proportion of total AES mass to be measured or some other criterion. In any case, weight percent values of the corresponding I components must be known for X. The Model. We propose the following regression model: yi = j3xi
+ ti
where yi is the measured concentration of component i, xi is the weight fracllon of componenti in theknown ("theoretical") distribution, /3 is the (unknown) proportionality constant, and ti is an error associated withyi. The error terms are assumed to be distributed as mutually independent normal random variables with zero means, but with variances proportional to the weight fraction in the known distribution; symbolically,
The predicted total E is then the sum of the predicted concentrations over all J components,
cpj BZj J
J
P=
=
1-1
1-1
Since the total of weight fractions in the known distribution satisfies J
EXj= 1 1-1
where J is the total number of components in the mixture, then J
P=Epj=B 1'1
is the estimated total concentration of all components. Standard Error of the hedicted Total and Resulting Predictionhterval. The estimate of the parameter ts2,denoted s2, is derived from the reparameterization as follows:
The proposed model has the intuitive appeal that as the weight fraction in the test material increases (decreases), the absolute variability in the measured concentration also increases (decreases). Predicted Total Concentration. The original expression for the model,
may be reparameterized, to yield error terms that are identically distributed
where all sums are taken from i = 1 to I . The variance of the estimated total concentration is equal to
Var(S-7 = Vur(j3) I
(This reparameterization is used solely to estimate the unknown parameters j3 and u2. The x's and y's are original and are not redefined in theseor any subsequent calculations.) The least-squares estimate for 8, denoted 8, is therefore given by I
I
where the sums are taken over the I discrete measured components.32 The predictedconcentrationof each component j is given by
I
(The estimated variance of Ereplaces u2with s2 in the above formula.) The standard error of the predicted total is the square root of the estimated variance of the total concentration and is given by
The 95% prediction interval for the total concentration equals
(32) Sncdccor,G.W.; Cochran, W. G.Statistical Methods; Iowa State University Press: Am-, IA, 1980; p 174.
Analytical Chemistry, Vol. 66, No. 10, May 15, 1904
1623
Table 2. Observed Background Concentratlone and Amounts of AES Splked for Each Matrlx
concn spiked,' pglL low spike level high spike level
background concn,' pg1L influent effluent river water (full scan) river water (SIR)
cl2-13
cl4-16
total c12-15
c12-13
c14-16
c12-13
c14-16
224 12 60* 15 2.5 k 1.4
346 f 6 28 f 2 7.8 f 1.6 5.7 0.8
570 f 18 88 f 17 10.3 f 0.2
2257 113 113
2232 112 112 95
4514 226 1128
4464 223 1116
*
a All concentrations refer to anionic form (the mass of the cation is excluded). The error values for background concentrations are the sample standard deviation for duplicate measurements.
Table 3. Results of Splke and Recovery Expwlments
where to.o25,(r-i) is the 97.5 percentile of a Student's t distribution with I - 1 degrees of freedom. Proportional Change Model. This additional model can be used to assess whether significant differences exist between two distributions. In general, if xi and yi are values of AES component concentrations (divided by unit concentration) before and after some process, each value of In (yi) may be plotted as a function of In (xi); corresponding yi and xi values must each refer to the same chemical species. Then, the two sets may be related by In (y) = a + b In (x), where a and b are determined by fitting a straight line using linear least squares. If a set of AES concentrations possessinga particular distribution is scalled linearly without error between x and y , there will be no change in distribution and b = 1. The more b deviates from 1, the greater is the change in distribution. Of course, obtaining meaningful results requires a good fit of the line to the data-that is, a high linear correlation coefficient and absence of any trend in the residuals between the points and the line. If b = 1.O, then each yi represents a loss in mass of (1 - exp(a)) of the corresponding xi.
RESULTS AND DISCUSSION General Overview. The objectives of this work were to developa method for determining AES, resolved by individual components, and to demonstrate the applicability of this method to river water and to WWTP influent and effluent. Specifically, we sought the ability to quantify 36 components in the range 12 I nc I 15 and 0 I EO I 8 in I F and at least 10 species in EF and RW. That range of components is expected to encompass most of the mass of AES present in the environment. Overall recovery of spiked AES was used to evaluate the quality of the method and the degree to which it quantitatively measures AES. We also examined how the method recovery differed for different homologues and hence how the measurement would preserve or skew the actual distribution. Each matrix was spiked with the AES blends at two levels which were higher than the background concentrations. Table 2 summarizes the total observed background concentrations and the spike levels used for each matrix, grouped together for C12-13, C14-15, and C12-15. Separate totals for C12-13 and Cl4-1~ were evaluated because they represent the two different test substances, C12-13EOl.oS and C14-15E01.5S. A plot of spiked (i.e., expected) and recovered concentrations is shown later for each set of experiments, corrected for background levels. Background results represent the average of duplicate measurements; results for spiked samples are averages of three replicates for the full-scan data and four replicates for SIR 1624
Ana&ticalChemistry, Vol. 66,No. 70, M y 15, 1994
concn of AES comDonents. % matrix and spike level influent (n = 3) low spike high spike effluento (n = 3) low spike high spike river water (full scan) (n = 3) low spike high spike river water (SIR)(n = 4) low spike
totalCl2-16
cl2-13
c14-16
104f 5 106f4
88+ 2 90f6
96*3 98f5
99f8 85f6
95k44 65f6
97&24 75f6
96f4 98f5
87i12 85*7
92*8 92*6
90*6
The components ClzEO& and C&O& were excluded from the anal sis of effluent. Abnormally high background concentrations precfuded accurate recovery results for those two components.
data. Error bars represent sample standard deviations of replicate extractions. An additional plot for each matrix displays the percent recovery of every component measured. For all plots, lines joining adjacent data points serve only to guide the eye. Table 3 summarizes the overall percent recoveries for all matrices, calculated from the sums of the ratios of measured to expected values for only the components measured. Influent. Examples of raw data obtained appear in Figures 1 and 2, showing the total ion chromatogram (TIC) and extracted chromatograms for nominal m / z values for a standard and an extract of unspiked IF. The TIC plots cover the range 200 I m / z I 800, which includes the parent ions of all the components measured in this study. The largest peaks in theTIC of thestandard (Figure 1A) at 5.6,8.3,11.8, and 14.9 min correspond to the dominant AS componentsC12EO& through ClsEOoS, respectively. The peak at 7.6 min corresponds to the internal standard at m / z = 334. Extracted mass chromatograms corresponding to the homologues C12EOlS through ClsEOlS are shown in Figure 1B-E. Each homologue appears as a set of three peaks. The first two small peaks for each homologue are believed to represent AES derived from 2-alkyl-substituted alcohols, which are present in many commerical samples of alcohol ethoxysulfate. The major, last eluted, peak has a maximum at 8.1, 11.6, 14.8, and 17.7 min for ClzEOlS through C15EOlS, respectively. Comparing the extracted chromatograms to the TIC shows that the components with nE0 = 1 cannot be seen in the TIC except for the major C15EOlS peak at 17.7 min. The other three components are only shoulders under the peaks for C13EOoS through C15EOoS. This overlap shows that using the extraction procedure from this study without mass spectral
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Flgure 1. Mass chromatogram in fuii-scan mode of an injection of a solution of the standard AES blends containing 9.50 mglL C12-13E01.0S and 8.26 mglL CiC15E01.5S:(A) total ion chromatogram, 200 ImlZ 5 800; (B)mlz = 309 extracted, corresponding to ClgOIS; (C) mlz = 323 extracted, corresponding to C13E01S;(D) mlz = 337 extracted, corresponding to C14E01S;(E) mlz = 353 extracted, corresponding to ClsEOIS. Peak iabeis represent scan time measured in minutes from the point of injection.
detection would not resolve many of the AES components, even for the pure standard test material. The TIC of the I F sample (Figure 2A) includes large peaks from many non-AES species, which were not quantified. For example, the peaks at 6.2,9.6,13.1, 16.0,and 18.8 min are attributed to a homologous series of linear alkylbenzenesulfonates beginning at m/z = 297 (for the peak at 6.2min) andincrementing by 14m/zunits (aCH2group). Theinternal standard gives rise to the peak at 7.9 min. A few AES components are visible in the TIC; for example, the peaks at 12.1 and 15.2 min correspond to C14EOoS and ClsEOoS, respectively. Parts B-E of Figure 2 show extracted chromatograms for the components with ~ZEO= 1 (analogous to Figure 1). Retention times differ between Figures 1 and 2 because of slight matrix differences between sample extracts and standards. The extracted mass chromatograms for I F (Figure 2B-E) illustrate the excellent resolution and absence of interferences obtained from the LC/MS method-even for a sample as dirty as influent. Although the AES
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Figure 2. Mass chromatogram of unspiked influent: (A) total ion chromatogram, 200 I mlz I 800; (B) mlz = 309 extracted, corresponding to CIPEOiS; (C) mlz = 323 extracted, correspondlng to C 1 3 E O l S ; (D) mlz = 337 extracted, corresponding to ClfOlS; (E) mlz = 353 extracted, correspondingtoClsEO1S. Peak iabeisrepresent scan time in minutes.
components with ~ZEO= 1 appear as three peaks (similarly to the standard test material), no other ions overlap with the AES component peaks. This was true for all components measured. The group of peaks in Figures 1 B and 2B at 15 min arise from ClsEOoS containing a 34Satom, which results in the same mass as C12EOlS. The raw influent to the WWTP contained the highest background concentration of AES of the three matrices studied. This is expected because less opportunity exists for removal by biodegradation and adsorption in the pipe between the points of discharge and the WWTP than exists within the WWTP. Concentrations of 224 f 12 pg/L total C12-13 and 346 f 6 pg/L total C1615 were measured in the unspiked I F (Table 2). The measured background levels of each component are shown in Figure 3A. Because of these high background levels, I F also received the highest spike levels of the matrices. Table 2 shows the concentrations of spikes used for each matrix. The low spike level for I F was -2.2 mg/L of each of the C12-I3EOl,oSand C l ~ s E 0 1 . 5 blends, S and the high spike level was about twice this amount. Visual observation of the spiked
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Figure 3. Component-specific recovery results for influent. (A) AES concentrations measured in background (*) and concentrations and expected (bars) at the low spike level; (E) measured (0) concentrationsof AES components measured (0) and expected (bars) at the high spike level; (C) percent recovery of each AES component expressed as the ratio of measured concentration (after background subtraction) to spiked concentration for low spike (V)and high spike (A)levels. Error bars represent the sample standard deviation of replicate measurements (see text).
and recovered concentrations of individual components of IF, in Figure 3A and B, shows that overall EO distribution is essentially maintained for both matrices. There are no major differencesbetween the results for the two spike concentrations. Figure 3C displays percent recoveries for each component. The overall recovery from I F (Table 3) was 96 A 3% for the low spike and 98 f 5% for the high spike. Effluent. The background concentrations for effluent are shown in Figure 4A and Table 2. Background concentrations of C12E00 and C14E00 were anomalously high and were attributed to an experimental artifact. The measured concentrations of those components were 59 15 (this is beyond the upper ordinate limit of Figure 4A) and 27 f 2 pg/L, respectively, whereas background levels of almost all other components were less than 0.3 pg/L. Contamination with a coconut-type AS (i.e., a blend having primarily 12- and 14carbon linear alkyl chains) is probably the cause, although we
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1626 Analytical Chembtw, Vol. 66, No. 10, May 15, 1994
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Figuro 4. Component-speclfkrecovery results for efflwnt. Symbols are assigned as given in Figure 3.
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cannot be certain at which stage the contamination occurred. For effluent, the low-spike level was 112 pg/L of both the C12-13E01.0Sand the C14-lSEOl.SSblends and the high-spiked level was about twice this amount (Table 2). Parts A and B of Figure 4 show the concentrations of individual components recovered at the two spike levels. Since the large background amounts were subtracted from the measured concentrations, anomalously low values were observed for C12EOo and Cl4EOo recoveries (zero or near zero after background subtraction) for both spikelevels (Figure 4C). The overall recoveries calculated from all 36 measured components are 77 f 19% and 61 f 5% for the low and high spikes, respectively. Neglecting the Cl2EOo and C14E00 components, overall recoveries were 97 f 24% for the low spike and 75 f 6% for the high spike (Table 3). At the low-spikelevel, recoveries of C14-15components were near loo%, but the standard deviations of these components are very high. For some reason, one of the three replicates gave concentrations for C14-1~components that were consistently higher than the other two replicates. Possibly an excess of the C14-15E01.sSblend was spiked to that sample. This problem did not appear for the Cl2-13 components. Recoveries
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In the full-scan experiment, RW was spiked at 113 or 1120 pg/L with each of the two AES blends (Table 2). We chose this wide range of spiking concentrations partly to bracket the highest AES dose concentration to be used in a separate toxicity study. Visual observation of the spike and recovery data for RW (Figure 5 ) shows that the shape of the EO distribution was largely maintained for both spike levels-except that the ClzEOo component at low-spike appeared abnormally high. We do not know what led to the anomalously high concentration (33 pg/L recovered vs 24 pg/L spiked) for that component. Figure 5C shows percent recovery for individual AES components in RW. Recoveries at the high spike level (1 116pg/L total C14-15material spiked) fall below 80% for many of the C14 and CIScomponents; however, we note that the method was optimized to provide adequate recoveries over the entire family of components (212EO0 through C15EOg. Lower extraction efficiencies for the C14 and C15components in the SPE column may be the cause of the lower percent recoveries for those components, since increasing nc decreases the solubility of AES in the solvents used. For different applications, the sample preparation could be modified to improve recoveries of components with particular alkyl or ethoxylate chain lengths. Overall recoveries (Table 3) are 92 f 8% for the low spike and 92 f 6% for the high spike. River Water with SIR Mode. River water was also examined using selected-ionrecording mode, because the LOD of 0.5 pg/L for full-scanmeasurements appeared insufficient for measuring background levels in many samples. Using SIR, a trade-off exists between signal-to-noiseratio (and hence LOD), number of components measured, and length and difficulty of analyses. This occurs because when the data collection software is operated in SIR mode, groups of up to eight m / z values are monitored (one is needed for the IS). Monitoring additional groups of eight m / z values would reduce S/N ratios for all ions. Making an additional injection and recording additional masses for the additional ions alleviates the S/N degradation. Such an approach is the most thorough way to obtain concentrations for many components at subppb levels, but it requires much more analysis time for each sample. Since we had validated the method for the full grid of 36 components in full-scan mode and we expected the precision and percent recovery to be equal or better using SIR mode, we anticipated that validating the method with only seven components of C14-15E01.5S would reveal the performance characteristics obtained using SIR. In addition, since it appeared possible to model AES distributions and predict total concentrations (see below), measurements of only seven components offered a way to reduce LOD and still yield total AES values in subsequent studies. In this second RW experiment, the components C14EO0, C14E03, C&Og, CISE O O , C ~ ~ E O I , C ~ ~ Eweremeasured. O ~ , ~ ~ ~ CWechose ~~EO~ to measure species with nEO values of 0,3, and 8 because these give a reasonable picture of the overall distribution. In addition, the EO distribution of the standards peaks at EO = 0 (Table l), so it is important to include the AS component. The SIR data for RW background (Figure 6A and Table 2), unlike the full-scan data, significantly show the shape of the background RW distribution. This distribution is similar to the test material (bars in Figure 6A). The background
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Flgwe5. Component-specificrecoveryresultsfor river water measured in full-scan mode. Symbols are assigned as given in Figure 3.
fall below 80% for all of the C14 and CIScomponents at the high-spike level (223 pg/L total C14-15 material spiked) and for most of the C13 and some Clz components (Figure 4C). It should be noted that the method may underestimate concentrations in effluent containing more than 112 pg/L AES. Studies on additional effluents may shed light on whether this is a general phenomenon or instead particular to this effluent. River Water with Full-ScanMode. The background AES concentrations in RW analyzed using full-scan mode are shown in Figure SA and Table 2. Measurements were below the limit of detection (LOD -0.5 pg/L for RW and full-scan mode) for many components; those values are shown as zero. The LOD was estimated by calculating the concentration of a component which would give rise to a peak height three times the peak-to-peak noise on the mass chromatograms. It is expected that this LOD could be reduced by adopting larger sample volumes and/or smaller reconstitution volumes. Although concentrations of some components are reported slightlyabove the LOD, theuncertainty in thoseconcentrations is clearly large, and no conclusions can be made about the observed distribution in unspiked RW.
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Flgurs6. Component-speciflcrecoveryresultsfor river water measured in selected-ion recording (SIR) mode at the low spike level. (A) AES concentrations measured in background (*) and concentrations measured (0)and expected (bars) in spiked samples: (B) percent recovery of each AES component expressed as the ratio of measured concentration(after background subtraction)to spiked concentration. Error bars represent the sample standard deviatlon of replicate measurements (see text).
levels of AES measured in the SIR experiment are well above the detection limit, which is -0.01 pg/L for R W in SIR mode. This LOD is 50 times lower than in full-scan mode. (In terms of absolute mass, the LOD in SIR mode is -200 pg of a component injected onto the column for most of the components in the standards.) The distribution of spiked concentrations was maintained in the recovered concentrations for the seven components measured (Figure 6A). No trends appear in the percent recoveries for these components (Figure 6B), similarly to the full-scan data at low-spike level (Figure 5A). The total mass recovery (Table 3) of 90 f 6% agrees well with the recovery of the low spike obtained with full-scan (87 f 12% for the C14-15 components); this supports the assertion that the two scan modes are equivalent except for LOD. Preservation of Samples. Additional experiments examined how effectively samples are preserved by formaldehyde. Without any preservation treatment, AES concentrations in environmental samples decrease with time, due to biodegradation, enzymatic hydrolysis, e t ~ . 3We ~ examined the ability to prevent thesedegradation processesby adding formaldehyde to spiked samples (see Experimental Section) and storing them for different periods before extracting and determining the AES. Influent and effluent were preserved at least 42 days (the longest period studied) at room temperature (22 f 3 "C).At
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(33) Vashon, R. D.;Schwab, B.S.Enuiron. Sci. Technol. 1982, 16,433-436.
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42 days, the total concentration of Cl2-15AES measured in an IF sample was 114% of the amount measured at day 0, and an E F sample gave 101% of the amount measured at day 0. For comparison, Table 3 shows the amounts measured at day 0 compared to the spiked amount. For river water, on the other hand, a sample held at room temperature was not preserved well up to 42 days at room temperature, because the total AES recovered was only 43% of that at day 0. Concern over the ability to preserve RW led us to repeat and expand the preservation study for that matrix during the experiments on R W using SIR mode. For the latter study, samples were stored either at room temperature (22 f 3 "C) or under refrigeration (-4 "C). The 22 "C samples were extracted after 0 and 14days, whereas the 4 "C samples were sampled and extracted after 0, 3, 7, and 14 days. The sample preservation was limited to 14 days in the second experiment because, in any subsequent experiment or monitoring study, samples could be extracted within 2 weeks after collection. In the SIR study, replicate samples were measured to get information on precision and experimental error: two replicates for background, three for day 0, and two each for the other samples. The sample at 4 OC after 14 days exhibited 100 f 16% of the AES measured at day 0. On the other hand, recovery from the 22 "C sample was only 47 f 10%. Thus, weconclude that refrigeration up to 14days is an adequate way to preserve AES in R W samples and is more effective than keeping samples at room temperature. The effectivenessof formaldehyde combined with refrigeration for preserving R W is reinforced by the data at day 3 and day 7, at which times the recoveries were 97 f 10% and 100 f 14% respectively. More work is needed to examine why AES recovery from RW decreases after several weeks at room temperature. Prediction of Total AES Concentration. Obtaining an LOD lower than that afforded by full-scan data collection necessitates using SIR mode and limits the number of components measured in each analysis. Nonetheless, total AES concentrations can be obtained efficiently in some cases by measuring a limited number of components and then fitting them to an AES distribution which is expected to represent thedistribution in the measured sample. Although this approach requires validation, a pilot study can be used to validate the technique for a particular matrix before applying it to a large set of measurements. The simplest case is where AES in the sample has nearly the same distribution as the test material used for quantitative standards. This case occurs often-for example, in dosed systems where the dosing material is characterized and skewing of the distribution is expected to be negligible. When WWTPs are being monitored, on the other hand, the structure of EF and RW distributions might be obtainable from measurements of a large number of components of IF. This would require exhaustive analysis of E F and R W in a few test cases and comparison of the distributions for validation. In order to predict total AES concentrations, we developed a model (Theory section) by scaling a known distribution to the measured concentrations of selected components. As an example, the model was applied to selected C14-15components in R W at the low-spike level to provide a prediction interval for the total concentration of AES. The known distribution used was that of the (214-15test material (Table 1). Normally,
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Figure 7. Comparisonof measuredconcentrationsof AES components in river water at the low-spike level (0)to the Concentrations predlcted by scaling the known distrlbutlon of componentsto the predictedtotal (XI.
prediction of total AES concentrations is desirable only for SIR data; however, the following example uses the full-scan data for the recovery of 18 C14-15 components from a spike of 112 ppb of Clk15E01.5S (Figure 5A) in order to compare the measured total of 18 components with the total concentration predicted from only 6 of those components. The data for C12-13 components was not used, but those components could be treated similarly. This model requires confirmation that there is no skewing in the distributions between the two sets of AES concentrations used. Differences in extraction efficiency, adsorption, or ionization efficiency for different components might skew the EO distribution of a sample, even when the recovery of spiked AES is measured, as in this case. If any skewing exists, it normally exhibits a trend with nc or EO which would be revealed by the proportional change model (Theory section), a log-log model which compares twodistributions. Therefore, the function In (C,) = a + b In (C,) was fit by linear least squares to the data for the 18 components, where C, is the observed concentration of a component (divided by unit concentration) and C, is the expected concentration obtained by scaling the known distribution (Table 1). The resulting equation is In (C,) = -0.09 + 0.99 In (CJ and the linear correlation coefficient is R = 0.98. Visual observation of the plot did not reveal any trend in the residuals. The fact that the slope ( b = 0.99) of this plot is nearly unity strongly suggests that the distribution is not significantly skewed and that the regression model is applicable to these data; however, we did not rigorously determine how far the slope may deviate from unity without proscribing use of the model. The foregoing equation also yields an approximate percent recovery of exp(a) = exp(4.09) = 91% for the 18 C14-15components. This is consistent with the experimentally determined value of 87 f 12% recovery for the same components measured by full scan (Table 2). This example compares the total AES concentration predicted from only 6 selected components-C14EOo, (214EO3, C14EO8, ClsEOo, C15E03, and ClsEOs-to the total concentration of all 18 measured components. The six components simulate data generated in an SIR experiment. The measured concentrations yi of these six components are
plotted in Figure 7, together with the concentrations pi of these same components predicted from the model. The regression model was applied to these data (using I = 6) and it gave a 95% prediction interval of 88 f 12 ppb for the total AES concentration. Compared to the spiked concentration of 112 ppb total C14-15E01.5S,this predicted totalcorresponds to 79% recovery with a precision of f 11% recovery. For comparison, addition of the concentrations of all 18 measured C1k15components yields a sum of 93 f 13 ppb AES; the error here is the sample standard deviation of three measurements. This latter total is 87 f 12% recovery (Table 3)-from the expectedvalueof 107 ppb, rather than 112ppb. (Even though the sample was spiked with 112 ppb total AES, the 18 components measured comprise only 96% of the spiked mass, or 107 ppb.) Although the mean values for total concentration and percent recovery differ between the prediction from 6 components (comprising -50% of the total AES) and the measurement of 18 components (comprising 96% of the total AES), this difference is within the precision of either interval.
CONCLUSIONS A quantitative method for analysis of AES in aqueous environmental samples using ion spray LC/MS has been demonstrated and validated for three aqueous sample matrices-river water, sewage influent, and sewage effluent. This method is applicable to monitoring studies, to measurements of AES in toxicity tests, and to other studies requiring environmental analysis of AES. A single injection provided concentrations of 36 components down to ppb levels in fullscan mode. The same number of components can be measured at 50-fold lower concentrations by multiple injections and SIR mode. A subsequent monitoring study which used this approach will be published later. A mathematical regression model has been demonstrated which provides prediction of total AES concentrations from measurements of selected components, provided the distribution of components in the sample can be estimated with sufficient accuracy. This model provided useful predictions oftotal C ~ CAES ~ S in a subsequent experimental stream study, the results of which will be published later. More work is needed to probe the relationship between differences in distributions used and the accuracy and precision of the predicted total. At this time, applications of the model should be limited to laboratory situations where AES of a known distribution is introduced into a test system. ACKNOWLEDGMENT The following people at Procter and Gamble assisted with this work Bill Eckhoff and John Bowling (sample collection); Jim Guckert (data processing); A1 Marrer (statistics and models); Dave Wernery (LC/MS technique); and Scott Dyer, Nick Fendinger, Drew McAvoy, Dave Lawrence, and Steve Morrall (helpful discussions and comments). We gratefully acknowledge the assistance of Shell Development Co. in providing and characterizing AES samples and in helpful discussions of sample preservation and analysis. Received for review October 26, 1993. Accepted March 11, 1994.' ~~
~~
Abstract published in Aduance ACS Abstracts, April 1, 1994.
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