Solid-Phase Microextraction Coupled with High-Performance Liquid

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Anal. Chem. 1996, 68, 1521-1529

Solid-Phase Microextraction Coupled with High-Performance Liquid Chromatography for the Determination of Alkylphenol Ethoxylate Surfactants in Water Anna A. Boyd-Boland† and Janusz B. Pawliszyn*

Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1

Nonionic surfactants of the alkylphenol ethoxylate class are relatively nonvolatile analytes that are typically analyzed by LLE and HPLC or GC after derivatization. Solidphase microextraction (SPME) has seen many GC applications, but recently the ability to hyphenate SPME with HPLC was investigated. In this paper, a new SPME/ HPLC method for analysis of Triton X-100 and other alkylphenol ethoxylates is described. Normal-phase gradient elution with detection by UV absorbance at 220 nm was used for the analysis. Several new coated phases, currently in the developmental stage, were evaluated. New Carbowax/template resin and Carbowax/divinylbenzene coatings allowed successful analysis of alkylphenol ethoxylates with a linear range of 100-0.1 mg/L. These coatings provided the best agreement between the ethoxamer distribution of surfactants after extraction and that in the original surfactant. Limits of detection of the individual ethoxamers were determined to be in the low microgram per liter to submicrogram per liter range. Some applications of the method have been demonstrated for sewage sludge samples. It has been estimated that the growth in nonionic surfactants will outpace the growth of surfactants overall in the detergent market in the next 10 years.1 Of the nonionic surfactants used in Europe, alkylphenol ethoxylates and alkyl ethoxylates constitute 80% of the market.2 It is expected that alkyl ethoxylates will increasingly be used as substitutes for alkylphenol ethoxylates that have been scheduled for phasing out of industrial and institutional (I&I) cleaning products by the year 2000.1 This phasing out has occurred as a result of concerns that alkylphenol ethoxylates may not biodegrade sufficiently and the possible links with human health problems.3 Despite these concerns, Union Carbide recently reported that the U.S. sales of nonylphenol ethoxylates in I&I and household cleaning products had continued to grow.1 The reason for their popularity is the ease with which they can be formulated into liquid cleaning products, which are more popular than the conventional powders. The determination of alkylphenol ethoxylate surfactants is of particular relevance in the light of evidence linking them to † Department of Chemistry, University of New England, Armidale, NSW 2351, Australia. (1) Ainsworh, S. J. Chem. Eng. News 1995, 73, (Jan 23), 30. (2) Clunie, J. S. Biodegradation of Detergents. The Chemical IndustrysFriend to the Environment? Royal Society of Chemistry: London, 1992. (3) Soto, A. M.; Justicia, H.; Wray, J. W.; Sonneschein, C. Environ. Health Perspect. 1991, 92, 167.

0003-2700/96/0368-1521$12.00/0

© 1996 American Chemical Society

estrogen mimicking.1,3 These surfactants are often found in influent and effluent streams of sewage treatment plants, and consequently in river waters and sediment.4-9 In Canada alone, in 1990, 4.1 kton of nonylphenol ethoxylates was consumed,9 mostly by the textile and pulp and paper industries. Liquid-liquid extraction (LLE) and solid-phase extraction (SPE) methods are generally combined with chromatographic methods for the separation and resolution of nonionic surfactants into their ethoxamers.4-9 The limitations of these current methods for surfactant analysis include the need for concentration of analytes after extraction to achieve the required sensitivity, the large volumes of often toxic solvents that are required for LLE methods, low breakthrough volumes of analytes in SPE methods, and the plugging of SPE cartridges by particulate matter that is often present in wastewater. Solid-phase microextraction (SPME) methods have been increasingly applied to the analysis of volatile and semivolatile analytes as a means of overcoming many of these disadvantages.11-23 Until recently, the application of SPME has almost exclusively relied on the effective combination of SPME and GC. However, many environmental pollutants, such as surfactants, are not sufficiently volatile to enable GC analysis without prior derivatization. Attempts have been made to incorporate derivatization reagents in the SPME device, but these methods are in the developmental stages. A simple alternative for many applications (4) Kubeck, E.; Naylor, C. G. J. Am. Oil Chem. Soc. 1990, 67 (6), 405. (5) Ahel, M.; Giger, W. Anal. Chem. 1985, 57, 2584. (6) Allen, M. C.; Linder, D. E. J. Am. Oil Chem. Soc. 1981, 58, 950. (7) Aserin, A.; Frenkel, M.; Garti, N. J. Am. Oil Chem. Soc. 1984, 61, 805. (8) Crescenzi, C.; Di Corcia, A.; Samperi, R.; Marcomini, A. Anal. Chem. 1995, 67, 1797. (9) Scilia, D.; Rubio, S.; Perez-Bendito, D. Anal. Chem. 1995, 67, 1872. (10) Lee, H.-B.; Peart, T. Anal. Chem. 1995, 67, 1976. (11) Zhang, Z.; Yang, M. J.; Pawliszyn, J. Anal. Chem. 1994, 66, 847A. (12) Chai, M.; Arthur, C. L.; Pawliszyn, J.; Belardi, R. P.; Pratt, K. F. Analyst 1993, 118, 1501. (13) Arthur, C. A.; Pratt, K.; Motlagh, S.; Pawliszyn, J. Environ. Sci. Technol. 1992, 26, 979. (14) Zhang, Z.; Pawliszyn, J. J. High Resolut. Chromatogr. 1993, 16, 689. (15) Buchholz, K. D.; Pawliszyn, J. Anal. Chem. 1994, 66, 160. (16) Potter, D. W.; Pawliszyn, J. Environ. Sci. Technol. 1994, 28, 298. (17) Buchholz, K.; Pawliszyn, J. Environ. Sci. Technol. 1993, 27, 2844. (18) Boyd-Boland, A. A.; Chai, M.; Luo, Y. Z.; Zhang, Z.; Yang, M. J.; Pawliszyn, J. B.; Gorecki, T. Environ. Sci. Technol. 1994, 28, 569. (19) Boyd-Boland, A. A.; Pawliszyn, J. B. J Chromatogr. 1995, 704, 163. (20) Magdic, S.; Pawliszyn, J. J. Chromatogr., in press. (21) Boyd-Boland, A. A.; Magdic, S.; Pawliszyn, J. Environ. Sci. Technol., submitted. (22) Eisert, R.; Levsen, K.; Wiinsch, G. J. Chromatogr. 1994, 683, 175. (23) Eisert, R.; Levsen, K. Fresenius J. Anal. Chem. 1995, 351, 555.

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Figure 1. A typical alkylphenol ethoxylate, Triton X-100, where n )10.

is the use of HPLC and SPME, which was recently reported by Chen and Pawliszyn.24 SPME eliminates the separate concentration step from the SPE and LLE methods because the analytes diffuse directly into the coating of the SPME device and are concentrated there. The device is then transferred to the injection port of the HPLC, via a specially designed interface, where all the analytes are desorbed in the eluent stream and deposited at the head of the HPLC column. The modifications to the HPLC system that enable SPME to be used with HPLC are minimal and are described in the Experimental Section and the Results section of this paper. This paper presents the first application of HPLC/SPME to the analysis of nonionic surfactants, specifically, the alkylphenol ethoxylates. The new SPME/HPLC interface described by Chen and Pawliszyn24 has been modified somewhat and used for the development of a new method with normal-phase gradient HPLC and UV detection. Several developmental coated fibers were evaluated for the analysis of a reference nonionic surfactant, Triton X-100 (Figure 1). It is important to ensure that any method for the analysis of surfactants maintains the same oligomer distribution in extracted samples as is present in the original water sample. For many LLE methods, this is not the case. The evaluation of the SPME methods included investigation of the distribution of ethoxamers obtained from injection of a stock solution of the surfactant with the distribution found after extraction of the same surfactant by the different coated fibers. The effect of addition of salt to the water matrix was investigated as a means of improving the extraction efficiency and the ethoxamer distribution. These experiments formed the basis for selection of the optimal coating. The linear range, limit of detection, and precision data are given for the optimal coating. The new method is shown to be ideally suited for the analysis of different types of alkylphenol ethoxylates of varying ethoxylate chain length. After it was demonstrated that the method could be used for different types of alkylphenol ethoxylates, sewage sludge known to contain alkylphenol ethoxylates or their degradation products, the corresponding alkylphenols, was analyzed by the new method. EXPERIMENTAL SECTION Instrumentation. A gradient programmable HPLC (Eldex, Model 9600; Napa, CA), coupled with a Supelcosil LC-NH2 column (25 cm × 4.6 mm × 5 µm; Supelco, Bellefonte, PA) and a UV detector (Toso Haas, TSK 6041; Philadelphia, PA), was operated at 1.5 mL/min, from 3 to 53%B, where A is 90/10 hexane/2propanol and B is 90/10 2-propanol/H2O. The optimal wavelength for detection was found to be 220 nm,25 rather than the 270 nm used by others.5 Data collection was provided by a PC interfaced to the detector using Star software (Varian, Palo Alto, CA). The GC/MS system used for the identification of ethoxamers consisted of a Varian Model 3400 GC coupled with a Varian Saturn (24) Chen, J.; Pawliszyn, J. Anal. Chem. 1995, 67, 2530. (25) Boyd-Boland, A. A.; Crisp, P. T.; Eckert, J. M. J. Am. Oil Chem. Soc., submitted.

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ion-trap mass spectrometric detector. A 15 m × 0.25 mm × 0.10 µm SPB-1 column (Supelco) was used with temperature programming as follows: 50 °C, hold for 2 min, then to 350 °C at 15 °C/ min, hold for 30 min. The injector was temperature programmed from 50 °C to 300 °C at 250 °C/min, hold for 35 min. The transfer line was held at 300 °C and the ion-trap manifold at 280 °C. The mass spectrometer was tuned to FC-43 (perfluorotributylamine). The electron multiplier and automatic gain control target were set automatically. The mass range scanned was 50-650 amu. The detector was turned off for the first 300 s to prevent overloading the electron multiplier from the solvents used in preparation of the composite standard. HPLC Interface Design. The interface (depicted in Figure 2) consisted of a regular six-port injection valve (a) (Rheyodyne, Model 7161; Cotati, CA), in which the injection loop is replaced by a fiber desorption chamber (b). The injection loop is usually connected between injection ports 3 and 6, but in this configuration it is replaced by the fiber desorption chamber. The fiber desorption chamber consists of a three-way tee (Valco Tee, supplied through Supelco, 5-8283), in which two outlets are connected to the injection valve and the third houses the SPME device. The solvent flows from the injection port to the tee (c) via a 0.75 mm i.d. stainless steel tube (Supelco) connected with stainless steel nuts (d), and the flow from the tee back to the injection port is via poly(ether ether ketone) (PEEK) tubing (e), connected to the tee by a finger-tight PEEK union (f) (PEEK tubing and unions from Sigma-Aldrich, Milwaukee, WI). The tee is oriented so that the flow from the injection port is forced up through the bottom of the tee and out through the side; the SPME device (g) resides in the top part of the tee, with the fiber exposed directly into the flow path. The stainless steel tubing (d) used in this interface design is somewhat larger than that used by Chen and Pawliszyn.24 It was observed that, for some of the coated phases being investigated, the solvents caused significant swelling that resulted in the coated phase being stripped from the silicon rod by the narrow tubing. Increasing the diameter to 0.75 mm eliminates the problems, even for coatings of 100 µm thickness. To provide a sufficiently strong seal around the SPME device and minimize damage to the fragile fiber, the configuration of the third port of the tee was slightly modified from the design described by Chen and Pawliszyn.24 In the original design, the fiber was exposed to the narrow part of the tee without any guide, and the seal was made around the inner needle of the SPME device, while the outer needle remained above the top of the desorption chamber. In the new design, the PEEK tubing and finger-tight nut were replaced by a larger diameter Teflon tube (h), inside a Slipfree SFE connector (i) (Keystone Scientific, Bellefonte, PA), which rested on a regular 0.4 mm i.d. M-2B GC ferrule (j) (Supelco). The Teflon tubing has an inner diameter just large enough to accommodate the outer needle of the SPME device, in contrast to the tubing used by Chen and Pawliszyn,24 which could only accommodate the inner tubing of the SPME device. The SPME device is inserted into the Teflon tubing until the outer needle comes to rest on the ferrule. The ferrule was beveled slightly to act as a guide for the needle. Once the needle has come to rest, the plunger is depressed, and the fiber slides through the center of the ferrule. The distance required to expose the fiber to the desorption chamber and leave the inner needle in the ferrule can easily be estimated by assuming that the ferrule is less than 0.5 cm in length and depressing the plunger so that

Figure 2. Modified SPME/HPLC interface in the fiber desorption (a) and insertion (b) modes.

the fiber and 0.5 cm of the inner needle are exposed. Once the fiber is through the ferrule, and the inner tube is inside the ferrule, closing the Slipfree connector causes the ferrule to constrict around the inner needle and provide a seal that can withstand the high pressures required for HPLC analysis. Alkylphenol Ethoxylate Standards. Triton X-100 (500 mL, VWR Scientific, West Chester, PA) stock solutions, 500 mg/L, were prepared every 2 weeks and diluted as required. Triton X-100, shown in Figure 1, is an octylphenol ethoxylate with an average of 10 units in the ethoxylate chain. Nonylphenol ethoxylates of varying average ethoxylate chain lengths, Rexol 25/4, 25/9, 25/10, and 25/15 (Hart Chemicals,

Toronto Canada), were prepared at 500 mg/L and diluted as required. Solvents. Hexane, 2-propanol, and dichloromethane (BDH, HPLC grade) were used as supplied. Water for HPLC and standard preparation was NANOpure water (Barnstead ultrapure water system, Barnstead/Thermodyne, Dubuque, IA). Reagents. A trimethylchlorosilane with BSTFA (TMCS/ BSTFA) derivatization reagent (Sylon BFT Kit, Supelco) was purchased to prepare the trimethylsilyl derivatives of ethoxamers for GC/MS. Fractions are collected from the HPLC, evaporated to dryness, redissolved in 0.5 mL of dichloromethane, and allowed to react with 200 µL of the derivatizing reagent at 70 °C for 15 Analytical Chemistry, Vol. 68, No. 9, May 1, 1996

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min in a sealed vial. The derivatized Triton X-100 species have the general formula C(CH3)3CH2C(CH3)2C6H4O(CH2CH2O)nSi(CH3)3, where n is the number of units in the ethoxylate chain (i.e., the ethoxamer number). External Standard. Anisole (99%, Aldrich, Milwaukee, WI) was dissolved in hexane and diluted as required. The external standard was used to determine the limit of detection of ethoxamers by the following reasoning. Calibration of anisole was achieved by injecting three replicates at seven different concentrations and finding the slope and intercept when the amount injected was plotted against the area counts. The correlation coefficient for the line of best fit with an intercept of 0 was 0.998, and the slope was 2 390 000. A typical noise reading of the HPLC system was 150 µV, and an area of 450 µV was selected to calculate the limit of detection. From the anisole calibration curve, it was found that 0.188 ng of an ethoxamer produced a peak with an area count of 450 µV. The amount of Triton X-100 ethoxamers extracted ranges from 0.5 to 5%, with the average amount extracted being 3%. Therefore, the concentration of a 4 mL sample in which 0.188 ng corresponds to 3% of the amount of the ethoxamer in the sample is the limit of detection. The volume of 4 mL is chosen because all extractions in this method were performed from 4 mL samples. The average limit of detection of an individual ethoxamer is 6.27 ng/4 mL, which is 1.57 µg/L. Sewage Sample. Air-dried sewage sludge (from Leslie Sewage Treatment Plant, supplied through CCIW, Burlington, Canada) was suspended in water (1 g/4 mL) and the slurry exposed the SPME device for analysis. Coated Phases for SPME Fibers. The commercially available phases, poly(acrylate) and poly(dimethylsiloxane), were conditioned as recommended (Supelco). Several experimental coated fibers were supplied by Supelco for the investigation: Carbowax/template resin (CWAX/TR), Carbowax/divinylbenzene (CWAX/DVB), poly(dimethylsiloxane)/template resin (PDMS/ TR), poly(dimethylsiloxane)/divinylbenzene (PDMS/DVB), and β-cyclodextrin. All the developmental coated phases are of unknown thickness and were conditioned as recommended by the supplier, typically at around 250 °C for 3-4 h. SPME Procedure. Aliquots of 4 mL of standard solutions or samples were extracted from 4.6 mL vials sealed with hole caps and Teflon-lined septa. After addition of the aliquot and a stirrer bar to the vial, approximately 0.5 mL of headspace is left, into which the needle is placed to prevent wicking of the sample during extraction. The fiber is then exposed to the aqueous phase for 60 min with stirring and at room temperature. After extraction, the fiber is placed in the desorption chamber of the HPLC, the solvent loop is switched to the inject position, and the fiber is exposed to the eluents for 1 min. RESULTS AND DISCUSSION Solid-phase microextraction is an equilibrium process in which analytes partition between the sample matrix and a polymeric stationary phase that is exposed to the sample for a predetermined period of time. The commercially available coated phases act like the solvents in a LLE method and absorb analytes from the matrix. Extraction occurs when the analytes distribute between the two phases according to their partition coefficients, K. At equilibrium, the amount extracted by the stationary phase can be calculated 1524

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from the following equation:

ns )

KVsVaqCaq0 KVs + Vaq

(1)

where ns is the amount extracted by the fiber coating, K is the partition coefficient, Vaq and Vs are the volumes of the aqueous and stationary phases, respectively, and Caq0 is the initial concentration of analytes in the aqueous phase. Once equilibrium has been established, the stationary phase is removed from the sample and usually placed in a hot GC injection port. The extracted analytes are thermally desorbed and focused at the head of the GC column before being separated and analyzed. For nonvolatile or thermally unstable analytes, it was proposed that HPLC combined with SPME would provide a simple alternative to LLE/HPLC or SPE/HPLC methods and eliminate the need for derivatization before SPME/GC can be used. The extraction process used for SPME and HPLC is exactly the same as that described for GC analysis. It is only the desorption technique that must be modified for HPLC analysis. In SPME/GC, the analytes are thermally desorbed in a conventional GC injection port, whereas for SPME/HPLC, they are desorbed by the eluent solvents of the HPLC system. To ensure complete desorption of the analytes by the solvent, it is essential that the coated fiber is exposed directly into a solvent flow. This is necessary because SPME is an equilibrium process; thus, exposing the coating to a stagnant solvent will result in an equilibrium, like the one described above, being established between the coating and the solvent. Alternatively, if the coating is exposed to a flowing system, the concentration gradient remains large, and analytes will continue to diffuse from the coating until all of them have desorbed. The same principle is in operation in the SPME/GC system, where a high linear flow rate of the carrier gas along the fiber is essential to ensure complete desorption of the analytes.26 Conventional HPLC injection ports are unsuitable for SPME but can be easily modified by replacing the injection loop with a specially designed desorption chamber. The chamber is shown in Figure 2, and described in the Experimental Section. The simplicity of the design allows for simple replacement of the injection loop when the HPLC is required for solvent calibration or conventional analyses. When SPME analysis is performed, the regular six-port valve is used to control the solvent flow through the desorption chamber. Switching the injector to the “inject” position starts the flow through the desorption chamber and hence across the coated phase of the SPME device. Figure 2a shows the flow path through the regular six-port injection valve when it is in the “inject” position. The mobile phase flows via the desorption chamber to the column. When the injection valve is switched to the “load” position, injection ports 1 and 6 are internally connected; similarly, ports 5 and 4 and ports 2 and 3 are connected (Figure 2b). The mobile phase then flows directly to the column, not passing through the desorption chamber, and the desorption chamber is at ambient pressure. This allows for insertion or removal of the SPME device. A further advantage of this system is that the injection valve can still be used for solvent injections while the SPME desorption chamber is in place. When the valve is in the “load” position, (26) Gorecki, T.; Pawliszyn, J. Anal. Chem. 1995, 67, 3265.

Figure 3. Desorption (a) and extraction (b) time profiles for ethoxamers of Triton X-100. The legend refers to the number of units in the ethoxylate chain.

injection of a sample or solvent into the injection valve deposits it into the desorption chamber. Switching the injector back to the “inject” position flushes the sample or solvent through the chamber and onto the column. Thus, small quantities of a “modifier” can be injected into the chamber to facilitate the desorption of analytes. The two commercially available coated phases [poly(acrylate) and poly(dimethylsiloxane)] and several experimental coatings (listed in the Experimental Section) were investigated to determine if they were able to extract the reference nonionic surfactant at the 100 mg/L level. Both unsalted and salted (25% NaCl) solutions of Triton X-100 were investigated. Samples were left to extract overnight before analysis. The two commercial coatings were unable to extract the ethoxamers to any significant extent. They were thus eliminated from further consideration. All of the experimentally developed coatings did provide extraction of the Triton X-100 at levels that warranted further investigation. The desorption time profile for Triton X-100 using one of the experimental coatings, a Carbowax/template resin-coated fiber (CWAX/TR), is shown in Figure 3a. From the figure, it is apparent that most of the ethoxamers have completely desorbed in the first minute. Although it appears that slightly more analytes are extracted with a 50 min desorption time than are extracted with a 1 min desorption time, only analytes that have retention times less than the desorption time being tested can be affected

by the length of the desorption process. This effect may be due to the changing gradient of the HPLC program, which provides an increasingly polar eluent mixture. This may lead to better solubility of the longer ethoxamers (i.e., later-eluting analytes), which are more polar. In this way, a desorption time of 50 min, when compared with one of 20 min, can increase the amount desorbed of the ethoxamers that elute after 20 min. This hypothesis was tested by injecting small amounts (between 20 and 40 µL) of polar solvents into the desorption chamber via the injection port using a 1 min desorption time. Three solvents of different polarity were tested: methanol, acetone, and dichloromethane. In no case did the addition of a modifier significantly improve the desorption of the more polar ethoxamers. Another consideration of the desorption process is the bandwidth of the peaks in chromatograms obtained from extraction of the analytes by SPME compared with direct injection. If analytes were slowly being desorbed as the gradient changed, then the focusing effect of the gradient would be lost and peak broadening would result. This effect would also be observed if the volume of the interface was too large to produce a small injection band. Direct comparison of the bandwidths of peaks obtained from injection of Triton X-100 and the bandwidths of those from SPME extraction with 50 min and 1 min desorption times is shown in Table 1. Typically, the peak width at half-height is slightly less Analytical Chemistry, Vol. 68, No. 9, May 1, 1996

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Figure 4. Effect of addition of salt on extraction of Triton X-100.

Table 1. Bandwidths of Peaks from Ethoxamers in Triton X-100 by Extraction and Injection peak width at half-height (s) retention time (min)

50 min desorptiona

1 min desorptiona

20 µL injectionb

3.359 4.238 5.627 7.709 10.516 13.986 17.838 22.451 26.649 29.722 32.516 35.05 37.321 39.451 41.421

8.4 8.8 10.4 12.3 13.7 14.3 15.3 26.0 17.6 16.6 15.8 15.5 14.8 16.1 15.8

9.2 9.5 10.2 11.7 13.2 14.2 15.1 22.3 19.7 19.6 15.5 15.3 14.7 15.8 15.9

5.2 7.2 9.8 13.0 16.4 19.4 22.8 23.6 22.0 21.3 18.3 16.5 16.1 16.4 17.8

a Extraction of 100 mg Triton X-100/L samples with addition of 20% NaCl, for 50 min. b Injection of 20 µL of a 600 g (Triton X-100)/L (dichloromethane) solution. All values are averages of duplicates.

for the peaks in the extracted chromatograms than for the injection of 20 µL of the standard. Furthermore, the bandwidths of the peaks in the chromatograms from the 1 min and 50 min desorptions are the same. This means that the desorption process must be completed before the gradient can influence the mobility of analytes through the column, and in fact, this must occur within the first minute. Chen and Pawliszyn also found that the desorption chamber did not affect the bandwidth of isocratically eluted PAHs.24 A final consideration of the desorption process is the presence of carryover. If the desorption process is not complete, analytes that are left in the coated phase may be subsequently desorbed, giving rise to false signals in blank analyses. The carryover could also influence analysis of samples containing the target analytes if the extraction time is significantly less than the equilibrium time. In this case, subsequent sampling with the same fiber may lead to overestimation of the concentration of an unknown because analytes that are present in the sample may be able to achieve 1526 Analytical Chemistry, Vol. 68, No. 9, May 1, 1996

their equilibrium concentrations more rapidly. This would occur because the amount extracted in the given time period would be a sum of the extraction occurring during that time and the amount already in the coating. Most often, extraction times are chosen that are close to, if not greater than, the equilibration time, so this effect is not obvious. In the case of the HPLC analysis of the alkylphenol ethoxylates, all the analytes have desorbed to at least 90% by the end of the desorption period, giving rise to, at worst, a 10% carryover. When a 1 min desorption time was used, no significant increase in peak areas was observed for subsequent extractions, probably because the extraction time was selected at a time close to the equilibration time. However, it is recommended that fibers be conditioned between analyses by either desorption in a large volume of solvent or in the HPLC injector for 2 min at the end of the gradient. The extraction time profiles obtained for Triton X-100 extracted with the poly(dimethylsiloxane)/divinylbenzene (PDMS/DVB) coating are shown in Figure 3b. Equilibration times of the individual ethoxamers ranged from 20 to 60 min, with more than 90% reaching equilibrium before 60 min. The HPLC separation is 50 min in duration, thus a 50 min extraction time ensures optimal time efficiency for the SPME method. Figure 4 shows a comparison between unsalted and salted (13% NaCl) solutions from which the ethoxamers were extracted by the Carbowax/template resin (CWAX/TR)-coated phase. The addition of salt at each concentration generally caused the amount of the longer ethoxamers extracted to increase slightly and the amount of the shorter ethoxamers extracted to decrease slightly. The results obtained with each of the other experimental coatings showed similar trends. This effect is also observed in many LLE methods, which require “salting out” of the surfactants to obtain the correct distribution of ethoxamers in the solvent. An important consideration in the analysis of surfactants is the integrity of the ethoxamer distribution found after extraction. In some LLE methods, smaller ethoxamers are better extracted than larger ones, producing a skewing of the apparent distribution in the extracted species. It is important to compare the distribution found in samples extracted by the new SPME method with that found for injection of the surfactant standard. The most important

Figure 5. Comparison of ethoxamer distribution between direct injection of a Triton X-100 standard and extraction of Triton X-100 using different coated fibers: (a) CWAX/TR, (b) PDMS/TR, (c) PDMS/DVB, and (d) CWAX/DVB.

factor influencing the extraction process in SPME is the coated phase; similarly, in LLE methods, it is often the choice of solvent. An optimal coating would thus be one that achieves an extracted species that has the same ethoxamer distribution as the standard surfactant. Figure 5 shows the comparison between the ethoxamer distribution found by extraction of Triton X-100 from salted solutions with each of the coatings. The ethoxamer distribution obtained by injection of 20 µL of a 1000 mg/L solution of Triton X-100 is indicated by the solid line above the graph. Salted solutions were chosen because the overall effect of salt was to improve agreement between the ethoxamer distribution of the extracted species and that of the standard. It is evident that the agreement is poor for the PDMS/DVB and PDMS/TR coatings, moderate for the CWAX/DVB, and very good for the CWAX/TR coating. On the basis of this experiment, the CWAX/TR coating was selected for further method development. The identity of each of the ethoxamers present in Triton X-100 was determined by collecting fractions from the HPLC, derivatizng the compounds to their trimethylsilyl analogs, and analyzing the resultant solution by GC with detection by ion-trap mass spectrometry. The limitations of GC/ITMS for the analysis of these compounds is the molecular weight range that can be analyzed. The derivatized ethoxamers of Triton X-100 range from n ) 0 to at least n ) 16, and have masses ranging from 278 to 982 amu. The GC/ITMS system was only capable of determining masses in the range 50-650 amu. This limits the method to determining

ethoxamers of up to 10 units because the mass of the parent ion for n ) 10 is 718, but the base peak of 647 can still be detected. Therefore, three ethoxamers that eluted early from the HPLC were selected to determine the identity of all the ethoxamers. The peaks eluted from the HPLC column at 10, 13, and 15 min and were the sixth, seventh, and eighth peaks eluted after the solvent front. They were identified as the ethoxamers n ) 6, 7, and 8, respectively. These results agree with previous assignment of the ethoxamers of Triton X-100 found under the same HPLC conditions and GC with detection by a quadrupole MS.25 In both cases, the ethoxamer with the greatest area counts was n ) 8. The numbers of units in the ethoxylate chain of each of the ethoxamers found in Triton X-100 are labeled on the chromatogram in Figure 6a. An advantage of the SPME method over LLE methods is the absence of a solvent peak in chromatograms obtained after extraction by SPME. In the case of the nonionic surfactant analysis, the first peak that is detected by solvent injection is the ethoxamer with one ethoxy unit. However, many nonionic surfactant mixtures also contain the n ) 0 fraction (unreacted alkylphenol), and many real samples metabolize to the corresponding alkylphenol.10 Figure 6a clearly shows the presence of a large quantity of the nonylphenol fraction present in Triton X-100, but which cannot be seen in the injection of Triton X-100 because it elutes with the solvent front. Analytical Chemistry, Vol. 68, No. 9, May 1, 1996

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Figure 6. HPLC chromatograms of the extracted alkylphenol ethoxylates: (a) Triton X-100, (b) Rexol 25/4, and (c) Rexol 25/15. Peak assignment in (a) refers to number of units in the ethoxylate chain.

The precision of the method was investigated for a set of seven replicates using the CWAX/TR fiber. The ethoxamers of Triton X-100 were extracted with precision ranging from 2 to 15%. Even for ethoxamers that are not very well extracted, the precision is much lower than that required by the U.S. EPA (i.e., 30%). Therefore, the precision of the new method can be considered very good. The linearity of the method for Triton X-100 has been investigated over the range 100-0.1 mg/mL for both the PDMS/ DVB and CWAX/TR fibers. The correlation coefficients were 0.990 or better. The limit of detection of an individual ethoxamer was calculated from extraction of a 0.1 mg/L Triton X-100 solution by comparison with an external standard, anisole. Anisole has the same chromophore as alkylphenol ethoxylates but has the advantage of not containing a range of analytes like Triton X-100. From the calibration curve, the area counts of Triton X-100 ethoxamers were converted to amounts extracted. The limit of detection of an individual ethoxamer is defined as the concentration of an individual ethoxamer in a sample from which a sufficient quantity will be extracted by the coated phase to produce a chromatographic peak that has a signal-to-noise ratio of 3. The amount of anisole injected into the HPLC to produce a 1528

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peak with an area 3 times the noise level was found to be 0.188 ng. Therefore, 0.188 ng of an ethoxamer must be extracted from a sample to be detected. To calculate the limit of detection of the ethoxamer in the original sample, the amount (%) extracted of each ethoxamer must first be determined. This is calculated by comparing the amounts extracted by the CWAX/TR coated fiber with the amount of each ethoxamer present in the original solution. The latter is calculated after determining the ratio of each ethoxamer in the stock solution of Triton X-100 by injection and comparison with the anisole calibration. The average limit of detection of an individual ethoxamer is 6.27 ng/4 mL, which is 1.57 µg/L. The 4 mL volume was chosen because it is the volume used for all experimental work. This limit of detection enables detection of alkylphenol ethoxylates in sewage influent and effluent waters, which can contain alkylphenol ethoxylates in the concentration range from low ppb to ppm levels.10 Extraction of 3% of each of the Triton X-100 ethoxamers from a 4 mL sample of a 100 mg/L solution results in 12 µg of Triton X-100 being transferred to the HPLC column. To achieve the same quantity of Triton X-100 reaching the HPLC column by injection, 120 µL of the 4 mL sample needs to be injected.

Injection of such large quantities of samples is often impractical, particularly when real samples are analyzed, and can lead to unwanted band broadening and loss of resolution. It would also inhibit the use of microcolumn HPLC, which requires injection of very small volumes of sample. The peak widths at half-height for the SPME injection were better than or equal to those of a 20 µL injection, enabling application of this technique to microcolumn LC. After the successful extraction of Triton X-100 by the CWAX/ TR, other nonionic surfactants were investigated. Figure 6 shows the extraction of 100 ppm solutions of Triton X-100 and two octylphenol ethoxylates (Rexol 25/4 and 25/15) that contain, respectively, an average of 4 and 15 units in the ethoxylate chain. The octylphenol ethoxylate with 15 units in the ethoxylate chain, Figure 6c, was separated with a slightly different gradient than the other two surfactants: 3 to 67%B over 50 min, instead of the usual 3 to 53%B over 50 min. The gradient was modified to ensure that the polarity of the final mixture was sufficient to elute all the ethoxamers. The resolution of up to 18 individual ethoxamers is achieved by SPME extraction and HPLC with UV detection. Sewage treatment plant (STP) sludge that had been air-dried was analyzed for the presence of nonylphenol ethoxylates. The samples were suspended in water and analyzed as a qualitative experiment by the new procedure. The amount of analytes in the sample was estimated by assuming that all the analytes present had desorbed from the sludge and then calculating the amount present in the sample by comparison with a calibration curve obtained from anisole. This was only undertaken to provide a comparison between the amounts detected by this method and those found by Lee and Peart10 using SFE and acetylation. The resolution of different nonylphenols is not achieved by the current HPLC method; however, determination of the total concentration of nonylphenols is achievable. The chromatogram obtained from extraction of the sewage sludge contained only one peak, with the same retention time as the n ) 0 ethoxamer found in Triton X-100 extractions. The peak was thus assigned to the alkylphenols known to be present in the sewage sludge. The concentration of the alkylphenols in the original sewage sludge sample was then estimated by assuming that 3% was extracted from the sewage sludge (i.e., the average amount of an ethoxamer extracted from water by this method). However, this estimation does not account for any matrix interference that may occur. This estimation was close to the value Lee and Peart10 found for octylphenol ethoxylate in the same sample (i.e., 10.0 µg/g by this method, and 9.2 µg/g by Lee and Peart); however, they also found a large quantity (450

µg/g) of nonylphenol ethoxylate which was not determined by this method. This suggests that there are some matrix interferences occurring that should be investigated by spiking the sewage sludge samples with known quantities of the surfactant before they are air-dried. In view of this, this method was used simply to qualitatively determine the presence of alkylphenols in the sewage sludge. CONCLUSIONS This paper describes the successful development of an SPME/ HPLC method for the analysis of nonionic surfactants of the alkylphenol ethoxylate type. Several different fiber coatings were evaluated for extraction of the reference nonionic surfactant, Triton X-100. The addition of salt was investigated and found to improve the amount extracted. The coated fiber that produced the best agreement between the distribution of ethoxamers before and after extraction was that coated with Carbowax/template resin. The linear range of the method using this fiber was determined over the range 100-0.1 mg/L. The limits of detection for individual alkylphenol ethoxamers are at the low ppb level. The new method can be applied to the analysis of many different samples. The analysis of commercial products containing alkylphenol ethoxylates can be achieved due to the wide linear range of the method. Although the linear range was only investigated up to 100 mg/L, it is expected to extend beyond that with the use of the UV detector and the coated phase. Commercial products can contain surfactants at up to 30% by weight for dry products and even 50% for liquid cleaning agents. Appropriate dilution of these liquid formulae will enable quantitative determination; however, qualitative determination of the types of ethoxamers present can be performed on the pure sample. ACKNOWLEDGMENT The authors acknowledge support from Varian, Supelco, and the Natural Science and Engineering Research Council of Canada. Tom Peart, from CCIW (Burlington, Ontario), is thanked for providing the sewage sludge sample for analysis. The HPLC pump was donated by Eldex Laboratories, Inc. Received for review September 6, 1995. February 6, 1996.X

Accepted

AC950902W X

Abstract published in Advance ACS Abstracts, March 15, 1996.

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