Anal. Chem. 2004, 76, 3878-3886
Evaluation and Optimization of an On-Line Admicelle-Based Extraction-Liquid Chromatography Approach for the Analysis of Ionic Organic Compounds Francisco Merino, Soledad Rubio, and Dolores Pe´rez-Bendito*
Department of Analytical Chemistry, Facultad de Ciencias, Edificio Anexo Marie Curie, Campus de Rabanales, 14071-Co´ rdoba, Spain
Admicelles-based solid-phase extraction (SPE) was online coupled with liquid chromatography/electrospray ionization/ion trap mass spectrometry, and it was proposed for the extraction of ionic organic compounds based on the formation of surfactant-analyte ion pairs. The approach was illustrated by studying the preconcentration of quaternary ammonium herbicides (quats) on sodium dodecyl sulfate (SDS) admicelles produced on alumina. Optimization of the parameters affecting SPE were studied on the basis that admicelles are dynamic entities in equilibrium with the aqueous phase. Some general guidelines could be established for method development from the results obtained. Factors influencing on-line operation were elucidated. On-line regeneration of the sorbent in each run was easily achieved by disruption of SDS admicelles with methanol and posterior coating of the alumina with SDS. The recovery of quats from drinking water samples were found quantitative for paraquat, diquat, and difenzoquat and above to 70% for chlormequat and mepiquat. Concentration factors of about 250, using sample volumes of 50 mL, were achieved. The detection limits ranged from 10 to 30 ng/L. The approach developed permits compliance with the directives of the European Community for drinking waters (100 ng/L) and goes deeply into the basis of solid-phase extractions that use surfactant-coated mineral oxide as sorbents. Hemimicelles and admicelles, although largely studied by physical chemists,1-3 have been scarcely explored for the extraction/concentration of organic compounds.4 These sorbents are formed by the adsorption of ionic surfactants on metal oxides such as alumina, silica, titanium dioxide, and ferric oxyhydroxides. Hemimicelles consist of monolayers of surfactants adsorbing head down on the oppositely charged surface of the oxide. The formation of admicelles, which have the structures of bilayers, * Corresponding author. Fax: 34-957-218644. E-mail:
[email protected]. (1) O’Haver, J. H.; Harwell, J. H.; Lobban, L. L.; O’Rear E. A. In Solubilization in Surfactant Aggregates; Christian, S. D., Scamehorn, J. F., Eds; Dekker: New York, 1995; Chapter 8. (2) Goloub, T. P.; Koopal, L. K.; Bijsterbosch, B. H.; Sidorova, M. P. Langmuir 1996, 12, 3188-3194. (3) Valsaraj, K. T. Sep. Sci. Technol. 1989, 24, 1191-1205. (4) Rubio, S.; Pe´rez-Bendito, D. Trends Anal. Chem. 2003, 22, 470-485.
3878 Analytical Chemistry, Vol. 76, No. 14, July 15, 2004
occurs after surface saturation by the adsorbed surfactant. The hydrophobic interaction of the nonpolar chain groups of the surfactants is the driving force for their formation. Admicelles have the structure of bilayers, being that the hydrophobic interaction of the nonpolar chain-groups of the surfactants is the driving force for their formation. So, the outer surface of the hemimicelle and admicelle is hydrophobic and ionic, respectively, providing a 2-fold mechanism for retention of organics. On the other hand, the number of surfactants commercially available is very high, so both the degree of hydrophobicity and the charge of the sorbent can be easily modified according to the nature of analytes. Therefore, hemimicelles and admicelles are very good candidates to extend the use of surfactant aggregates in analytical separations.5-7 To our knowledge, two applications involving extraction/ concentration of organic compounds on hemimicelles/admicelles have been developed. These applications deal with the concentration of phenols,8 based on hydrophobic interactions, and benzalkonium surfactants,9 based on the formation of mixed aggregates. Benefits obtained with the use of these sorbents were high extraction yields, easy elution of analytes, high breakthrough volumes, and/or high flow rate for sample loading.9 However, more extensive studies must be undertaken in order to establish the real scope of application of hemimicelles/admicelles to the solid-phase extraction (SPE) of organic compounds. From a theoretical point of view, little attention10-12 has been paid in the development of analytical methods to the fact that both hemimicelles and admicelles are dynamic entities in equilibrium with surfactant monomers in solution. Since SPE operation involves percolation of aqueous sample volumes between 0.1 and 1 L, the continuous partition of the micellar aggregates to the (5) Armstrong, D. W. Sep. Sci. Technol. 1985, 24, 213-304. (6) Ordered Media in Chemical Separations, ACS Symposium Series 342; Hinze, W. L., Armstrong, D. W., Eds.; American Chemical Society: Washington, DC, 1987. (7) Hinze, W. L.; Pramauro, E. Crit. Rev. Anal. Chem. 1993, 24, 133-177. (8) Saitoh, T.; Nakayama, Y.; Hiraide, M. J. Chromatogr. A 2002, 972, 205209. (9) Merino, F.; Rubio, S.; Pe´rez-Bendito, D. Anal. Chem. 2003, 75, 6799-6806. (10) Hiraide, M.; Sorouraddin, M. H.; Shemirani, F. Talanta 1995, 42, 11511155. (11) Hiraide, M.; Shibata, W. Anal. Sci. 1998, 14, 1085-1088. (12) Manzoori, J. L.; Sorouraddin, M. H.; Shabani, A. M. H. Microchem. J. 1999, 63, 295-301. 10.1021/ac049736v CCC: $27.50
© 2004 American Chemical Society Published on Web 05/21/2004
aqueous phase has consequences on both the manner in which the optimization process must be carried out and the way in which the SPE method must be applied. Therefore, studies providing the necessary knowledge and guidelines for the selection of the more SPE relevant parameters when hemimicelles/admicelles are used as sorbents should be undertaken. From a practical point of view, it is interesting to investigate the potential of hemimicelles/admicelles for trapping polar and ionic organic analytes, which constitute a considerable percentage of pollutants in environmental waters13 and for which simple and robust SPE methods are lacking.14 On the other hand, on-line applications have not been developed, even though the regeneration of the mineral oxide surface can become easy by tuning the pH of the solution,15 that causes desorption, or by using organic solvents, that disrupts the micellar aggregates. This paper deals with the optimization and application of a new analytical method based on the extraction/concentration of quaternary ammonium herbicides (quats, Table 1) on sodium dodecyl sulfate admicelles, coupled on-line to a liquid chromatography/electrospray ionization-ion trap mass spectrometry (SPELC/(ESI-IT)MS) system. The analytes selected as models, quats, are widely used in the management of crops (chlormequat, CQ, and mepiquat, MQ, are both plant growth regulators, difenzoquat, FQ, is a selective herbicide and paraquat, PQ, and diquat, DQ, are nonselective, quick acting herbicides). The physicochemical properties of quats make these pollutants intractable from an analytical point view.16 Thus, their positive charge, log Kow values (0.238 (pH 7), -1.58, -2.82, -4.6 (pH 7), and -4.7 (pH 7) for FQ, CQ, MQ, DQ, and PQ, respectively) and solubility in water (about 700 g/L for FQ, PQ, and DQ and >1000 g/L for CQ and MQ) makes it very difficult to apply concentration processes prior to quats determination. The SPE methods developed for quats have been recently revised.16 They are based on cation-exchange resins, silica sorbents, and apolar phases. Ion-pair formation using apolar SPE is the preferred method for isolating quats, and it has been proposed by the Environmental Protection Agency (EPA) as the reference method for DQ and PQ determination.17 However some serious problems still remain (e.g. accuracy and/or precision are very poor and the methods are often too time-consuming and tedious) which make it difficult to optimize the methods for monitoring large number of samples.16 On the other hand, methods for the determination of the most recently commercialized quats (FQ, MQ, CQ) have been scarcely developed. All these reasons make quats very good candidates to investigate the real applicability of hemimicelles/admicelles for the concentration of ionic organic compounds. The aim of this paper was 3-fold: (a) to provide guidelines for method development based on the knowledge of the surfactant adsorption isotherms in order to avoid that method development can become a largely empirical, labor intensive, and time(13) Trace Determination of Pesticides and their Degradation Products in Water; Barcelo´, D., Hennion, M. C., Eds; Elsevier: Amsterdam, 1997. (14) Hennion, M. C. J. Chromatogr. A 1999, 856, 3-54. (15) Smith, J. S.; Valsaraj, K. T. Sep. Sci. Technol. 1998, 147-159. (16) Pico´, Y.; Font, G.; Molto´, J. C.; Man ˜es, J. J. Chromatogr. A 2000, 885, 251271. (17) Hodgeson, J. W. Method 549: Determination of Diquat and Paraquat in Drinking Water by Liquid-Solid Extraction and High Performance Liquid Chromatography with UV detection; EPA: Cincinnati, OH, 1990; p 101.
consuming trial and error process; (b) to investigate the applicability of hemimicelles/admicelles to the SPE of highly watersoluble ionic organic compounds; and (c) to study the coupling of hemimicelles/admicelles to liquid chromatography and the reusability of the sorbent during operation. EXPERIMENTAL SECTION Chemicals and Materials. All reagents were of analytical reagent-grade and were used as supplied. The surfactants sodium dodecyl sulfate (SDS), sodium tetradecyl sulfate (STS), sodium alkyllbenzene sulfonates (LAS), and sodium dioctyl sulfosuccinate (Aerosol OT) were obtained from Aldrich (Milwaukee, WI). Herbicides were purchased from the following sources: diquat (DQ), mepiquat (MQ), and difenzoquat (FQ) from Riedel de Hae¨n (Seelze, Germany) and paraquat (PQ) and chlormequat (CQ) from Aldrich. Ethyl viologen dibromide (EV), used as internal standard, was also obtained from Aldrich. Stock solutions of the herbicides were prepared in bidistilled water and were stored in plastic containers in order to prevent their adsorption. HPLC-grade acetonitrile and methanol were supplied by Panreac (Sevilla, Spain), and heptafluorobutyric acid (HFBA) was supplied by Aldrich. Alumina (γ-form, for column chromatography) was supplied by Sigma (St. Louis, MO). The physical properties of this mineral oxide were as follows: surface area, 155 m2/g; point of zero charge, pcz, 8.5; particle diameter range, 50-200 µm; mean value, 100 µm; mean pore size 58 Å; density, 3.97 g/cm3. Sample Treatment. Fortified water samples (tap and mineral water) were collected in polyethylene bottles. The spiked concentration for each quat ranged between 0.1 and 1.8 µg/L. Methyl viologen (1 µg/L) was added as internal standard. The pH of the solution was fixed to 2. The samples were filtered through 0.45 µm filters (Whatman GF/F, Osmonics, France) in order to remove suspended solids and stored at 4 °C under light protection conditions until analysis. On-Line Admicelles Extraction and Regeneration of the Sorbent. The on-line quats enrichment system used (Figure 1) consisted of the following: a Minipuls 3 speed-variable peristaltic pump (Gilson, OH), silicon rubber pumping tubes (1.14 mm i.d., 2.82 mm o.d., Omnifit, Cambridge, U.K.), a Rheodyne 7125 NS injection valve with a 200 µL sample loop (Scharlab, Barcelona, Spain), a Teflon precolumn (35 mm length, 0.4 mm i.d.) packed with 80 mg of γ-alumina (cotton was placed on either end to support the alumina), and a Rheodyne 7040 six-port switching valve with a 200 µL sample loop (Scharlab, Barcelona, Spain). The general scheme for the on-line preconcentration of quats and regeneration of the sorbent was as follows. In the first step (Figure 1a), a 25 mL solution containing 16 mg of SDS at pH 2 was passed through the precolumn at a flow rate of 2.5 mL/min in order to form admicelles over the alumina surface. Then 50 mL of water sample containing the analytes, the internal standard (1 µg/L), and 1.6 × 10-3 M of surfactant (in order to keep constant the amount of admicelles on the alumina) were passed through the precolumn at the same flow rate. Meanwhile, the 200 µL sample loop of the injector was loaded with the elution solution (0.1 M HFBA in methanol), and the analytical column was equilibrated with the mobile phase (solvent A: 15% acetonitrile/ H2O, 75:25. Solvent B: 25 mM HBFA). In the second step (Figure 1b), the injection valve was switched to the inject position, and the enriched quats were eluted from the precolumn with 200 µL Analytical Chemistry, Vol. 76, No. 14, July 15, 2004
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Table 1. Chemical Structure, Common Name, and Typical Ions and Their Relative Abundance Obtained under ESI (+)/MS and MS/MS Analysis of Quats
a
Cutoff mass: 27% of ion parent; CID time: 40 ms.
of the HFBA methanolic solution and carried to the sample loop of the six-port switching valve by distilled water at a flow rate of 3880
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2.5 mL/min for 15 s. In the third step (Figure 1c), the switching valve was set at the inject position, and the chromatographic
Figure 1. Schematic diagram of the operation mode of an on-line admicelle-based SPE-LC system.
gradient was simultaneously started. The switching valve was returned to the load position within 30 s. Meanwhile the injection valve was moved to a load position, and the alumina surface into the precolumn was regenerated by successively passing 1 mL of methanol, that disrupts the admicelles and removes the surfactant, and 5 mL of 0.01 M HCl, to recover the initial charge density on the alumina surface. Liquid Chromatography/Mass Spectrometry. The analytes were separated, identified, and quantified by using a liquid chromatography/electrospray/ion trap mass spectrometry system (1100 series LC/MSD, Agilent Technologies, Waldbronn Germany), coupled on-line with the SPE system above-described (Figure 1). The stationary-phase column was a 25 cm Nova Pack C8 column, with 3.5 mm i.d. and 5 µm particle diameter, from Waters (Milford, MA). Acetonitrile/water (75:25) and heptafluorobutyric acid (25 mM) were used as eluent solvents at a flowrate of 0.6 mL/min. The elution gradient program was as follows: 0-20 min (linear gradient from 15% to 35% of acetonitrile: water); 20-25 min (linear gradient to 100% of acetonitrile/water); 25-32 min (isocratic conditions with 100% of acetonitrile/water). The diver valve was programmed to send the mobile phase containing the most polar matrix compounds (salts) to waste. So, only 12 min after the initiation of the elution gradient program, the eluted components were sent to the ESI source. Quats analysis was carried out in the ESI (+) mode. The set of parameters used is shown in Table 2. They were optimized by chromatographic analysis of a mixture of quats and the internal standard ethyl viologen (50 ng each) under different working conditions for the ESI source and the ion trap analyzer. Full scan data acquisition was performed from 105 to 255 m/z, with the target mass fixed to the following m/z values: 114 for MQ, 122 and 124 for CQ, 183 and 184 for DQ, 185 and 186 for PQ, 249 for FQ, and 212 and 213 for ethyl viologen (see Table 1). Herbicides
Table 2. ESI Parameters Set for the Analysis of Quats quats[ESI(+)] parameter
CQ
MQ
DQ
capillary voltage (kV) capillary exit voltage (V) skimmer (V) trap drive source temperature (°C) drying gas (L/min) nebulizer gas (psi) maximal accumulation time (ms)
-1.0 50 10 35
-1.0 50 10 35
-3.5 -3.5 30 30 20 20 40 40 350 10 80 100
a
PQ
I.S.a
FQ
-3.0 40 30 35
-1.0 40 20 40
I.S. internal standard.
were quantified in all cases from the corresponding peak areas of the extracted ion chromatograms. Smooth chromatograms were obtained by using the Gauss function (width ) 5 points, cycles ) 1). Calibration curves were constructed using the whole procedure, i.e., with the same experimental conditions as selected for the analysis of water samples. Calibration solutions consisted of variable amounts of quats, ethyl viologen (1 µg/L), and surfactant (1.6 × 10-3 M). The curves obtained were linear in the concentration ranges 0.06-8 µg/L for CQ and MQ; 0.1-4 µg/L for DQ and PQ; and 0.04-8 µg/L for FQ (n ) 9). The determination coefficients, r2, were higher than 0.994 for all the target compounds. Structural identification was performed by MS/MS experiments after the parent ion was isolated and fragmented by using the ion trap mass spectrometer. The isolation width was set to 4 m/z units. The resonance excitation value used for each quat, the fragment ions obtained, and their relative abundance are shown Analytical Chemistry, Vol. 76, No. 14, July 15, 2004
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in Table 1. Quantification and identification of quats was performed in the same chromatographic peak. RESULTS AND DISCUSSION Admicelle-Based SPE. The capability of both hemimicelles (apolar sorbents) and admicelles (ion-pair and ion-exchange sorbents) for retention of quats was investigated. Because of the cationic nature of analytes, an anionic surfactant (SDS adsorbed onto alumina) was selected for the formation of the micellar aggregates. Adsolubilization of quats on the apolar surface of hemimicelles should be primarily governed by hydrophobic interactions, whereas their solubilization on admicelles should be greatly favored owing to the formation of quats-SDS ion pairs. Below, the main results obtained in the optimization of the parameters that affect the SPE sequence and some general considerations on hemimicelles/admicelles-based SPE methods are discussed. (a) General Considerations on Method Development. Method development involving hemimicelles/admicelles is related, like other SPE methods, to the properties of analytes of interest, the required trace level concentration, the nature of the matrix, the type of chromatography involved in the separation step, and the detection mode. However, the correct optimization of the parameters affecting the SPE sequence (e.g. type and amount of sorbent, pH, composition and volume of the elution solution, etc.) is only possible after a thorough knowledge of the surfactant adsorption isotherms under the different experimental conditions investigated. This knowledge is also essential to understand the mechanisms of adsolubilization of analytes. Figure 2 illustrates a representative example of the implications that factors affecting surfactant adsorption isotherms (e.g. pH) have on the optimization of the hemimicelles/admicelles-based SPE processes. This figure shows the experimental isotherms obtained for the adsorption of SDS on alumina at pH 2 (Figure 2A, data from ref 9) and 6 (Figure 2B, data obtained using the procedure previously described9). The regions where hemimicelles and admicelles coexist in equilibrium with aqueous surfactant monomers, which are suitable for SPE methods, have been pointed out. The region corresponding to aqueous surfactant concentrations above the critical micellar concentration (cmc) is not suitable for SPE owing to the partition of analytes between aqueous micelles and admicelles. So, premicellar conditions must prevail in the aqueous phase. Figure 2 shows that the total amount of surfactant adsorbed on the alumina surface, the surfactant concentration at which hemimicelles and admicelles are formed, and the concentration of aqueous surfactant monomers in equilibrium with hemimicelles/admicelles vary greatly with the pH since this parameter dramatically affects the charge density on the surface of alumina (pcz 8.5). This behavior hinders that the study of the pH influence on the SPE process can be carried out in a traditional way (e.g. keeping constant all variables except the pH), because changes in the pH of the solution also produce changes in the type of sorbent and/or its capacity. In general, factors affecting the cmc of aqueous micelles (e.g. electrolyte concentration, length of the hydrocarbon chain of the surfactant, etc.) will cause a shift in the adsorption isotherm of surfactants in the same direction and about the same rate as the cmc. The knowledge of these changes and their magnitude is 3882 Analytical Chemistry, Vol. 76, No. 14, July 15, 2004
Figure 2. Experimental adsorption isotherms for SDS on alumina at pH 2 (A) and 6 (B).
essential for method development involving hemimicelles/admicelles-based SPE. (b) Sorbent Selection. The adsolubilization of quats on SDSalumina at different surfactant concentrations and two pHs (2 and 6) was investigated in order to determine which kind of surfactant aggregate, hemimicelles, mixed hemimicelles/admicelles, or admicelles, was the best sorbent for their retention. Experiments were carried out in the batch mode by adding 500 mL of an aqueous solution containing 80 mg/L of each quat to the mixture SDS and alumina (0.5 g) in a 25 mL aqueous solution. The total amount of anionic surfactant added was varied to give adsorbed surfactants concentrations comprised in the hemimicellar, mixed hemimicellar/admicellar, and admicellar regions. The solution was vigorously stirred for 5 min, and then it was centrifuged at 4000 rpm for 5 min, and the concentrations of quats were determined in the supernatants by LC/(ESI-IT)MS, as specified in the Experimental Section. At the quat concentrations used in this experiment (1.6 mg/L) percentages of adsolubilization up to 99% can be determined by direct measurement of the remaining herbicide in the supernatants. Figure 3 depicts the main results obtained. As it was expected, the adsolubilization was greater when admicelles were present on the alumina owing to the formation of quats-SDS ion pairs, the existence of which was chromatographically confirmed. The presence of two quaternary nitrogen in the molecular structure of PQ and DQ permitted the formation of ion pairs of stoichiom-
Figure 3. Effect of the amount of SDS on the sorption of quats at pH 2 (A) and 6 (B): ([) CQ, (+) MQ, (b) DQ, (2) PQ, and (9) FQ. Operation in the batch mode. Amount of alumina: 0.5 g.
etry 1:2 (PQ:2SDS, DQ:2SDS) that were hydrophobic enough to achieve their quantitative adsorption. The less hydrophobic CQ: SDS and MQ:SDS were adsolubilized at percentages of about 7075% and required maximal concentration of admicelles to achieve this adsolubilization level. Therefore, the admicellar region is recommended for preconcentration of quats. In the hemimicellar region, where adsolubilization of quats should be primarily governed by hydrophobic interactions, log Kow was shown to be of limited help for predicting the SPE recoveries of quats because of the formation of ion pairs of different stoichiometry and because it has been proved that there is poor correlation between retention and log Kow for very polar analytes.14 No differences were found for the maximal solubilization of quats into SDS admicelles at the two pH values investigated (Figure 3), which seems logical considering that formation constants for the quats:SDS ion pairs are similar at these pH values and that, under the experimental condition investigated, the amount of SDS adsorbed on alumina (expressed as milimoles) was in a great excess over analytes concentration (i.e. about 70 and 500 times at pH 2 and 6, respectively). (c) Sorbent Amount. The amount of SDS admicelles formed on alumina depends on the charge density of the mineral oxide surface, and, therefore, it is a function of both, the amount of alumina and the pH of the SDS-alumina aqueous suspension. In off-line SPE, the amount of sorbent rarely is a limitation, but in on-line techniques, typical amounts of sorbents in precolumns are in the range 20-100 mg. For this reason, the amount of quats adsolubilized into the SDS admicelles retained on 80 mg of alumina was investigated at pH 2 and 6. Experiments were carried out in the batch mode in a similar way to that described in the above section. The amount of SDS added was fixed to produce maximal amount of admicelles (i.e.
200 and 70 mg of SDS added/g of alumina at pH 2 and 6, respectively). The total concentration of quats ranged to give SDS/ quats molar ratios between 1 and 20. The results obtained indicated that a SDS/quats molar ratio of about 10 was required at the two pH values investigated to achieve maximal adsolubilization of quats (i.e. that observed in Figure 3). Since the concentration of admicelles at pH 2 was about 7-fold greater than that at pH 6 (SDS adsorbed was 176 and 26 mg/g of alumina, respectively), a greater amount of analytes was retained at pH 2 for a fixed amount of alumina, and therefore this value was the recommended. (d) Consequences of the Admicelles: Monomers Equilibrium for SPE Operation Mode. Admicelles are in equilibrium with surfactant monomers in the aqueous phase (Figure 2). When admicelles are used in off-line and on-line SPE applications, the surfactant in the admicellar phase will continuously partition to the aqueous phase during percolation of the sample. As a result, the concentration of admicelles will progressively decrease during operation and the partition constant for analytes, and therefore their retention will change. We checked that the retention of quats on SDS alumina drastically decreased as the operation changed from batch mode (e.g. results in Figure 3) to off-line or on-line mode (e.g. maximal percentage of quats retention was about 17%, 20%, 32%, 39%, and 73% for MQ, CQ, DQ, PQ, and FQ, respectively, when 25 mL of sample were percolated). Therefore, a continuous supply of surfactant was necessary during percolation of the sample in order to ensure that the concentration of admicelles in the mineral oxide surface remained constant. Figure 4 shows the effect of adding different amounts of SDS to samples containing quats (pH 6) before their percolation through the sorbent. The experiment was carried out under conditions at which the bed of alumina was initially saturated (i.e. 26 mg of SDS/g of alumina, Figure 2B). The results obtained Analytical Chemistry, Vol. 76, No. 14, July 15, 2004
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Table 3. Percentage of Desorption of Quats as a Function of the Composition of the Eluent composition of eluenta
CQ
MQ
DQ
PQ
FQ
methanol acetonitrile HBFA (0.1 M) acetonitrile/HBFA (0.075 M) acetonitrile/HBFA (0.1 M) methanol/HBFA (0.1 M) NaOH (0.05 M) NaOH (0.1 M) NaCl (0.25 M)
18 61 32 59 100 99 45 77 100
35 74 30 78 98 100 47 83 92
59 50 37 83 99 100 11 6 50
63 58 36 89 100 100 5 0 45
95 89 10 83 100 100 0 0 5
a
Figure 4. Effect of the amount of SDS added to samples, before percolation through the SDS admicelle-based SPE, on the recovery of quats: ([) CQ, (+) MQ, (b) DQ, (2) PQ, and (9) FQ. Operation in the off-line or on-line mode. Amount of alumina, 80 mg.
(Figure 4) indicated that the retention of quats increased when the amount of SDS supplied during percolation of the sample increased, and it was only similar to that achieved in batch experiments as the SDS concentration supplied during operation permitted to keep saturated the bed alumina with admicelles. Similar results were obtained at pH 2. Therefore, to guarantee a unique partition constant for analytes it is recommended a supply of SDS that keeps constant the initial conditions selected. The amount of SDS that must be added can be easily inferred from the surfactant adsorption isotherms, where the surfactant in the aqueous phase is specified. Only in the case that the concentration of monomers in the aqueous phase is negligible (e.g. in the hemimicellar region for SDS at pH 2), it is possible to use the corresponding micellar aggregates in SPE without supply of the surfactant during operation.6 (e) Influence of the Surfactant Structure on Quats Retention. The influence of changes in the length and degree of branching of the hydrophobic moiety of the surfactant forming the admicelle on the adsolubilization of quats was investigated. Adsorption experiments were carried out in the batch mode using a similar procedure to that described for sorbent selection. Surfactants selected for this study were sodium tetradecyl sulfate (STS) and sodium dioctylsulfosuccinate (Aerosol OT). Investigations were restricted to surfactant concentrations corresponding to the admicelar region. It was found that the percentages of quats retained onto STS and Aerosol OT admicelles scarcely changed with respect to those obtained for SDS. Thus, the retention for PQ, DQ, and FQ was quantitative, and it was about 73-78% for CQ and MQ. Therefore, the increase in the hydrophobicity of the surfactant (CMC for SDS, STS, and Aerosol OT were 5.4 × 10-3, 2 × 10-3, and 1.6 × 10-3 M, respectively) scarcely influenced the partition constant for the most polar analytes (i.e. CQ and MQ). (f) Desorption Solution. Various solvents and mixtures expected to cause desorption of analytes from the admicelle and 3884 Analytical Chemistry, Vol. 76, No. 14, July 15, 2004
Elution volume, 2 mL.
to be compatible with the mobile phase (acetonitrile and the ionpair reagent heptafluorobutyric acid, HBFA) were tested. The elution volume used was 200 µL. Table 3 shows the most significant results. Organic solvents such as acetonitrile and methanol caused disruption of the admicelles and therefore elution of analytes. However, the eluted SDS-quats ion pairs were only partially substituted by the HFBA-quats ion pairs through the chromatographic process. As a result, the chromatogram showed peaks corresponding to both types of ion pairs. It was necessary the addition of HFBA to the elution solvent, at concentrations about 0.1 M, to produce complete destruction of the SDS-quats ion pairs and, as a result, quantitative recovery of analytes. The use of only aqueous HFBA did not achieve complete elution of quats. Sodium hydroxide, that causes disruption of SDS admicelles by desorption of the surfactant (pcz of the alumina 8.5), was also investigated as an eluent. Low recoveries were obtained for the most apolar analytes probably due to their high retention on the negatively charged surface of the alumina. Sodium chloride solutions were not effective in destroying the SDS:quats ion pairs from DQ, PQ, and FQ. According to these results, acetonitrile/ HFBA (0.1 M) or Me/HFBA (0.1 M) could be used as desorption solutions of quats. (g) Sorbent Regeneration. Because of the high capacity of admicelles to solubilize matrix components, the regeneration of the sorbent will usually require the complete removal of the surfactant bilayer from the alumina surface. Acetonitrile and methanol were investigated for this purpose. Acetonitrile was found not effective in removing SDS because of the low solubility of the surfactant in this organic solvent. Methanol completely removed SDS from the alumina, so it was also the solvent used for elution of analytes. After removal of the surfactant, the alumina surface was conditioning with HCl (0.01 M) before generation of a new admicellar phase. It was checked that the extraction precolumn worked within a run for at least 20 cycles without deterioration. (h) Breakthrough Volume. The breakthrough volume for each quat was estimated by preconcentrating samples of increasing volumes, each containing the same amount of analyte and then measuring the peak areas eluted from the sorbent.14 Breakthrough was considered to occur when the amount eluted decreased about 5%. Because of the higher amount of SDS admicelles formed on the alumina surface at pH 2 compared to pH 6, the breakthrough volume for quats was higher at the lower pH. It was found to be
75 mL for CQ and MQ, 100 mL for DQ and PQ, and 125 mL for FQ at pH 2. The breakthrough volume decreased to 60 mL for CQ and MQ, 75 mL for DQ and PQ, and 100 mL for FQ at pH 6. So, sample volumes lower than 75 mL should be used under the experimental conditions proposed. The preconcentration factors achieved under the experimental conditions recommended were about 250 by using 50 mL of sample. Analytical Performance. Calibration curves were run by injecting 2-400 ng (absolute amount) of each quat under fullscan conditions (m/z scan range 105-255). No gains in sensitivity were achieved by reducing the m/z scan range to 10 (termed SIM mode in the operation of the Agilent system), which is logical considering how an ion trap works.18 On the other hand, quats quantification by MS/MS by using the m/z of the most abundant fragment ions (Table 1) also caused losses of sensitivity by a factor between 100 and 1000 compared to the measurement of parents ions. The sensitivity obtained for quats under full-scan conditions, expressed as the slope of the calibration curves in the concentration ranges specified under Experimental Section, were 0.021 ( 0.003, 0.045 ( 0.005, 0.011 ( 0.002, 0.010 ( 0.001, and 0.14 ( 0.01, for CQ, MQ, DQ, PQ, and FQ, respectively. The practical detection limits19 were estimated from six independent complete determinations of analyte concentrations in a typical matrix low-level material. They were calculated by using a signal-to-noise ratio of 3 (the ratio between the peak areas for each quat and internal standard and peak area of noise). The detection limits ranged between 20 and 30 ng/L for CQ, MQ, PQ, and DQ, and it was about 10 ng/L for FQ. These values were similar in distilled, tap, and mineral water. Selectivity studies were carried out to investigate the influence of surfactants and salts on the efficiency of the admicelles-based SPE for quats since these matrix components cause serious negative interference on the recoveries of most of the SPE methods described for quats.20-22 With regard to surfactants, special attention was paid to alkylbenzenesulfonates (LAS) and nonionic surfactants (nonylphenol ethoxylate, NPE, was selected as a model) since they are the main surfactants present in environmental waters. The effect of electrolytes was investigated by using sodium chloride. No interference was found from LAS and NPE up to surfactant concentrations of about 500 mg/L, that is the maximum level generally found in sewage effluents.23 We checked that both the LAS adsorption isotherm and the adsolubilization of quats onto admicelles of LAS were similar to those obtained for SDS. Therefore, if competition between SDS and LAS by the adsorptive sites of the alumina surface or by binding to the quats was established, the SPE efficiency obtained was constant. On the other hand, sodium chloride concentrations below 0.04 M did not affect the adsorption of quats. Higher electrolyte concentrations, however, decreased their recoveries. Thus, recoveries of 48% CQ, 49% MQ, 88% PQ, 85% DQ, and 94% FQ and 18% CQ, 19% MQ, (18) Yates, N. A.; Booth, M. M.; Stepheson, J. L.; Yost, R. A. In Practical Aspects of Ion Trap Mass Spectrometry; Raymon, E. M., Todd, J. F. J., Eds; CRC Press: New York, 1995; Chapter 4. (19) Thompson, M.; Ellison, S. L. R. Pure Appl. Chem. 2002, 74, 835-855. (20) Agudo, M.; Rı´os, A.; Valca´rcel, M. Anal. Chim. Acta 1993, 281, 103-109. (21) Iba´n ˜ez, M.; Pico´, Y.; Man ˜es, J. J. Chromatogr. A 1998, 823, 137-146. (22) Cserha´ti, T. Fresenius J. Anal. Chem. 1993, 345, 541-548. (23) Matthew, J. S.; Jones, M. N. Biochim. Biophys. Acta 2000, 1508, 235-251.
Table 4. Mean Recovery (%) and (Relative Standard Deviation (%)) of Quats in Fortified (n ) 5) Drinking Water Samplesa fortified tap water (µg/L)
CQ MQ DQ PQ FQ a
fortified mineral water (µg/L)
0.1
0.6
1.8
0.1
0.6
1.8
90 (11) 99 (13) 107 (8) 96 (10) 104 (9)
98 (10) 100 (8) 102 (8) 93 (9) 97 (7)
97 (6) 98 (5) 98 (6) 103 (4) 101 (3)
90 (12) 104 (11) 108 (9) 100 (7) 103 (10)
101 (9) 96 (9) 104 (7) 101 (8) 97 (8)
98 (6) 97 (7) 99 (5) 104 (6) 100 (4)
Analyzed by on-line admicelle-based SPE-LC/MS.
29% PQ, 30% DQ, and 45% FQ were found in the presence of 0.08 and 0.2 M NaCl, respectively. The possible interference of matrix components that could elute with quats causing ion suppression or space-charge effects on the ion trap was assessed by comparison of the calibration curves obtained from standards and those obtained from tap and mineral water samples fortified with known amounts of quats. As internal standard ethyl viologen was used. Since the analytical characteristics of both types of calibration curves were similar, we recommend external calibration for determination of quats in environmental water. However, it is advisable to investigate the influence of matrix components when the sample to be analyzed is expected to have a composition very different than those investigated here. The identification of quats was carried out through MS/MS spectrometry by using the ion trap instrument. Table 1 shows the fragmentation efficiencies obtained from the precursor ion specified for each quat, under the proposed conditions of fragmentation. Assignation of structures to the product ions was based in a detailed MS/MS and MSn study of quats, previously reported.24 Since the ion trap instrument offers the possibility of switching between full-scan MS and a collision induced dissociation (CID) product scan in the presence of helium collision gas, with no loss in signal or CID efficiency, we used this strategy to quantify and identify quats in environmental water samples. Analysis of Quats in Drinking Water Samples. The developed method was applied to the analysis of drinking water samples in order to check its suitability. Tap and mineral water samples free of quats were fortified at different levels (0.1, 0.6, and 1.8 µg/L) and processed as described in the Experimental Section. Table 4 shows the mean of the percentages of recoveries obtained and the corresponding relative standard deviation. These results indicate very good accuracy and precision for the analysis of quats at the maximum admissible level established by the European Community directive (0.1 µg/L). Panels A and B of Figure 5 show, respectively, the MS extracted ion chromatograms from a standard solution and a fortified tap water sample at a concentration level for quats of 0.6 µg/L. According to the results obtained, the admicelle-based method surpasses the EPA method14 when applied to the determination of quats in drinking water. Thus, the detection limits achieved are lower in the admicelle-based method (10-30 ng/L) compared to the EPA one (680-720 ng/L), permitting the identification and (24) Evans, C. S.; Startin, J. R.; Goodall, D. M.; Keely, B. J. Rapid Commun. Mass Spectrom. 2001, 15, 699-707.
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Figure 5. LC/MS extracted ion chromatograms obtained from (A) a standard solution and (B) a fortified tap water sample that were treated using the whole procedure. [quat] ) 0.6 µg/L.
quantitation of quats at the levels required by the European Community directives for human consumption (100 ng/L for individual pesticides). The relative standard deviation values obtained with the proposed method in the quat concentration range 0.1-1.8 µg/L (3-13%) are very similar to those obtained with the EPA method in the concentration range 2.5-52.5 µg/L (2.5-8.4%). On the other hand, the admicelle-based method has proved suitability for the determination of CQ, MQ, and FQ in addition to DQ and PQ. CONCLUSIONS The results obtained in this research constitutes a new step toward the objective of proving the suitability of surfactant-coated mineral oxides as sorbents in the SPE of organic compounds.
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Their versatility for this application arises from the fact that different mechanisms of retention of analytes (e.g. ion exchange, hydrophobic interactions, ion-pair formation, etc.) can be exploited by changing surfactant concentration or their nature (e.g. cationic, anionic, mixed nonionic-ionic, etc.). In this article, the suitability of admicelles to concentrate highly water soluble organic ions (quats) from drinking water has been proved. Previous investigations have also proved their applicability to concentrate apolar8 and amphiphilic9 compounds. Rank ordering for incorporation of analytes to adsorbed surfactant aggregates is similar to that known for aqueous surfactant aggregates.5-7 Partition of analytes to the surfactant aggregate phase increases for the more hydrophobic and oppositely charged compounds. For this reason, extraction of amphiphilic compounds9 is extremely favorable. It is worth noting that an on-line setup coupling of hemimicelles/admicelles-based SPE to LC can be easily performed in any laboratory, and regeneration of the sorbent becomes easy by removing the micellar aggregates with organic solvents or changing the pH. So, one can have a “new” sorbent in each run in a simple way. Because of the dynamic nature of the micellar aggregates, special attention must be paid to method development in order to achieve an accurate optimization and interpretation of the data obtained. In this context, some guidelines can be established, namely as follows: (a) The effect of parameters affecting SPE (e.g. pH) on the surfactant adsorption isotherms must be known, and optimization experiments must be designed based on this knowledge. (b) Partition constants of analytes will probably change with the type and/or amount of sorbent, so, the initial conditions selected should be kept constant throughout operation. For this purpose, a fixed amount of surfactant, that can be inferred from the adsorption isotherm, must be supplied with the sample. (c) For a fixed amount of mineral oxide, the amount of sorbent available for adsolubilization increases at determined pH values since this parameter affects the charge density of the mineral oxide surface. This fact should be not very important for off-line SPE applications, but in on-line applications higher breakthrough volumes can be achieved working under conditions of maximal charge density. ACKNOWLEDGMENT The authors gratefully acknowledge financial support from MCyT (Project BQU2002-01017). Received for review February 16, 2004. Accepted April 22, 2004. AC049736V
