On-Line Solid-Phase Extraction of Triazine Herbicides Using a

Anal. Chem. 1999, 71, 2152-2156

On-Line Solid-Phase Extraction of Triazine Herbicides Using a Molecularly Imprinted Polymer for Selective Sample Enrichment Bjarni Bjarnason,*,† Luke Chimuka,† and Olof Ramstro 1 m‡

Department of Analytical Chemistry and Department of Pure and Applied Biochemistry, Lund University, Box 124, S-221 00 Lund, Sweden

A coupled-column system, consisting of a combination of a molecularly imprinted polymer (MIP) and a C18-silica column, was used for selective triazine detection in the HPLC mode. Complex aqueous samples, spiked with triazines, were selectively extracted by the MIP followed by subsequent identification by analytical reversed-phase chromatography. The MIP showed good performance for selectively discriminating triazines from humic acid, whereas urine and apple extract had some tendency to be retained by the MIP. Enrichment was observed in all cases, and triazine-enrichment factors of up to 100 could be recorded, with good extraction efficiency (74-77%). The results indicate that the high selectivity of MIPs can be favorably used for selective sample enrichment in conjunction with reversed-phase HPLC. Solid-phase extraction (SPE) using C18-silica sorbents and other similar matrixes is today often used in environmental analysis for sample enrichment of triazines.1,2 These sorbents retain the analytes primarily by hydrophobic interactions and are thus fairly nonspecific in nature. By use of SPE, the detectability of diluted analytes can be greatly enhanced by applying large sample volumes, but as coextracted interfering substances are also retained by the matrixes, an increase in sensitivity may not be obtained. The identification of the compounds is also a problem since confirmation by retention times using UV detection alone is not sufficient for determination. Efforts have therefore been made to increase the selectivity in the extraction of the analytes from large sample volumes. Supported liquid membrane (SLM) techniques,3 immunosorbents,4-6 or ion exchangers7 have been applied for this purpose. * Corresponding author. Fax: +46 46 222 45 44. E-mail: Bjarni.Bjarnason@ analykem.lu.se. † Department of Analytical Chemistry. ‡ Department of Pure and Applied Biochemistry. (1) Pichon, V.; Hennion, M. C. J. Chromatographia 1994, 665, 269-281. (2) Pichon, V.; Charpak, M.; Hennion, M.-C. J. Chromatogr. A 1998, 795, 8392. (3) Knutsson, M.; Nilve´, G.; Mathiasson, L.; Jo¨nsson, J. A° . J. Chromatogr., A, 1996, 754, 197-205. (4) Lawrence, J. F.; Me´nard, C.; Hennion, M.-C.; Pichon, V.; LeGoffic, F.; Durand, N. J. Chromatogr., A 1996, 752, 147-154. (5) Pichon, V.; Chen, L.; Durand, N.; Le Goffic, F.; Hennion, M.-C. J. Chromatogr., A 1996, 725, 107-119. (6) Pichon, V.; Chen, L.; Hennion, M.-C.; Daniel, R.; Martel, A.; Le Goffic, F.; Abian, J.; Barcelo, D. Anal. Chem. 1995, 67, 2451-2460.

2152 Analytical Chemistry, Vol. 71, No. 11, June 1, 1999

Molecular imprinting is a technique by which highly selective binding matrixes can be prepared using a chosen target molecule as a template in a casting procedure.8-11 Imprinted matrixes used as stationary phases in molecular imprinting chromatography (MIC) have been an extensively studied application area, and several intriguing separations resulting in high separations and resolutions have been performed.12-14 Molecularly imprinted materials have been found to exert antibody-like affinities toward the target substances and can therefore, in likeness to immunosorbents, be used for selective extraction.15-19 The present paper involves the use of molecularly imprinted polymers (MIP) as recognition matrixes in a coupled SPE system in order to selectively enrich triazine analytes in dilute complex solutions. Recently presented results indicate that highly selective MIPs can be prepared against triazines.20-22 However, these polymers were only used in off-line experiments. By coupling a C18-SPE column on-line in combination with a MIP column, the high extraction efficiency of the SPE and the high selectivity of the MIP could be used for matrix discrimination prior to detection. Even though we could have enriched the water samples on the top of the MIP column and then later eluted it by changing the mobile phase, we chose to separate the enrichment from the purification procedure. This is for practical reasons as it gives us the possibility in later developments to enrich one sample while another is being analyzed. Another factor is that it is possible to (7) Land, C. C. J.; LC-GC Int. 1994, 7, 215-218. (8) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812-1832. (9) Mosbach, K.; Ramstro¨m, O. Bio/Technology 1996, 14, 163-170. (10) Whitcombe, M. J.; Alexander, C.; Vulfson, E. N. Trends Food Sci. Technol. 1997, 8, 140-145. (11) Ramstro¨m, O.; Ansell, R. J. Chirality 1998, 10, 195-209. (12) Ramstro¨m, O.; Nicholls, I. A.; Mosbach, K. Tetrahedron: Asymmetry 1994, 5, 649-656. (13) Kempe, M.; Mosbach, K. J. Chromatogr., A 1995, 694, 3-13. (14) Matsui, J.; Nicholls, I. A.; Takeuchi, T. Tetrahedron: Asymmetry 1996, 7, 1357-1361. (15) Sellergren, B. Anal. Chem. 1994, 66, 1578-1582. (16) Andersson, L. I.; Paprica, A.; Arvidsson, T. Chromatographia 1997, 46, 5762. (17) Martin, P.; Wilson, I. D.; Morgan, D. E.; Jones, G. R.; Jones, K. Anal. Commun. 1997, 34, 45-47. (18) Matsui, J.; Okada, M.; Tsuroka, M.; Takeuchi, T. Anal. Commun. 1997, 34, 85-87. (19) Muldoon, M. T.; Stanker, L. H. Anal. Chem. 1997, 69, 803-808. (20) Siemann, M.; Andersson, L. I.; Mosbach, K. J. Agric. Food Chem. 1996, 44, 141-145. (21) Muldoon, M.; Stanker, L. J. Agric. Food Chem. 1995, 43, 1424-1427. (22) Matsui, J.; Doblhoff-Dier, O.; Takeuchi, T. Chem. Lett. 1995, 489. 10.1021/ac9810314 CCC: $18.00

© 1999 American Chemical Society Published on Web 04/21/1999

Figure 1. System configuration for on-line sample purification using MIP prior to detection in a HPLC system. For details see text.

have higher flow rate through a short SPE column than through the longer MIP column. EXPERIMENTAL SECTION Materials. Simazine, atrazine, propazine, and terbuthylazine were from Larodan Fine Chemicals AB (Malmo¨, Sweden). Humic acid, Mw 600-1000, was from Fluka (Buchs, Switzerland). Methacrylic acid (MAA, dried over CaCl2, CaH2, distilled), ethylene glycol dimethacrylate (EDMA, dried over CaH2, CaCl2, distilled), and azobisisobutyronitrile (AIBN, used as delivered) were from Merck (Darmstadt, Germany). Dichloromethane (DCM, anhydrous) used in the imprinting protocol was from Lab-Scan (Stillorgan, Ireland). All other solvents were of HPLC-grade and used as delivered. Instrumentation. The system setup is presented in Figure 1. It consisted of two LKB 2150 (LKB, Bromma, Sweden) pumps (A, B) and one Kontron Instruments HPLC pump 422 (C). The detector was a Lambda-Max model 480 LC spectrophotometer from Waters. Injector I was a Rheodyne model 9125 (Rheodyne, Cotati, CA) and the six-port injection valves (H and J) were from Valco (Houston, TX). For SPE we used C18 silica (E) (LiChrosorb, RP-18 from Merck) 55 × 2 mm packed in a stainless-steel HPLC column. For selective sample discrimination we used a MIP packed in column F which was a 150 × 4.6 mm stainless steel column. The analytical column G was a C18 Hichrome 5 µm × 250 mm × 4.6 mm. The detector was connected to a Shimadzu C-R3A Chromatopac integrator (L) and to a 16-bit A/D converter coupled to a 286 Ericsson computer (N) for data sampling. The data were integrated using an integration program written in MATLAB (Math Works Inc., Natick, MA). Preparation of MIPs. Polymers were prepared using simazine as the print molecule and methacrylic acid as the functional monomer. The print molecule (1.68 mmol), the functional monomer (MAA, 6.7 mmol), the cross-linker (EDMA, 34 mmol), and the initiating agent (AIBN, 0.4 mmol) were mixed and dissolved in the porogen (dry dichloromethane, 10 mL). The solutions were subsequently purged with nitrogen for 10 min and left to polymerize in a Rayonet photochemical reactor (Southern New England Ultraviolet Co., Bradford, CT) at 350 nm and 4 °C for 16 h. Each polymer was ground with a mechanical mortar (Retsch, Haan, Germany) and sieved through a 0.025-mm sieve (Retsch).

Following repeated sedimentation in acetone, polymer particles ranging from approximately 0.01 to 0.025 mm were collected. Control polymers were prepared, using the same protocol, in the absence of any print molecule. Sample Preparation. Humic acid was ground in a mortar and mixed with water to give a concentration of 20 ppm. The mixture was stirred for a week and then filtered with 0.22 µm filters (Duopore Membrane Filters, GVWPO4700, Millipore AB, Sundbyberg, Sweden) and spiked with triazines to 0.5 ng/mL. An apple extract was prepared by homogenizing an apple in a homogenizer. The homogenate (40 g) was then mixed with 160 mL of methanol and extracted overnight. The methanol was evaporated at reduced pressure at room temperature until a thick, oily liquid appeared, which was subsequently dissolved in 200 mL of 250 mM phosphate buffer pH 7.0. The solution was finally filtered and spiked with triazines to 100 ng/g (20 ng/mL). The urine sample was spiked with 20 ng/mL of each triazine and analyzed without further treatment. The operation of the System. The SPE column (E) was conditioned with 2 mL of distilled water from pump A prior to the extraction. Using the same pump, the sample was introduced to the SPE column at a flow rate of 2 mL/min. The effluent, in both cases, was passed to waste through valve K. When sufficient sample volume had been extracted, a switch was made to the second pump, B, by turning the switching valve H. Simultaneously, the valve K was switched and the SPE column thus connected to the MIP column. Pump B pumped the mobile phase (acetonitrile) through the SPE column (at 1 mL/min), eluting all extracted species to the MIP column. The triazines were retained on the MIP column (F) allowing contaminants to pass the column, resulting in the relief of the matrix and improvement of the later chromatogram (acquired in the on-line analytical HPLC system) for the retained species. After a certain time (7.4 min) a 200-µL water plug was injected into the MIP column through injection valve I. When the front of the plug reached injection valve J, after 10 min the compounds captured in the 100-µL injection loop of the valve were injected into the analytical column (G) for separation. The mobile phase (acetonitrile/50 mM NaOAc pH 7.0 in proportion 50:50) for the analytical column was pumped by pump C at 1 mL/min. Detection was made spectrophotometrically at 235 nm, and the peaks were recorded on a computer (N) and a recorder (L). Extraction Efficiency. The extraction efficiency (E) for the triazines was in this case taken as the amount of each triazine in the eluted peak from the MIP column (nA) divided by the total amount of the extracted triazine (nI) on the SPE column:

E ) nA/nI

RESULTS AND DISCUSSION Retention Behavior of Triazines by MIPs. The structures and retention factors (k) of the triazines used in this work are presented in Table 1. The retention factor is defined as k ) (tr to)/to, were to is the column dead time (determined by acetone injection) and tr the retention time. Since we wanted to use the MIPs for selectively retaining the analyte triazines and removing the nonretained matrixes in the process, we were interested in fairly long retention times for the analytes. This would allow time Analytical Chemistry, Vol. 71, No. 11, June 1, 1999

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Table 1. Triazine Structure, Different Substituents Making the Herbicides, and The Corresponding Retention Factors for Direct Injection Using Mobile-Phase Compositions of 2% Acetic Acid in Dichloromethane (k1), 1% Water in Acetonitrile (k2), and Acetonitrile (k3)a

substituent group

simazine atrazine propazine terbuthylazine

Table 2. Extracted Sample Volume, Concentration of the Injected Plug, and Enrichment Factor (Ee) for Humic Acid, Apple Extract and Urine humic acid (200 mL of 20 ppm)

retention factor

R1

R2

R3

k1

k2

k3

Cl Cl Cl Cl

NHCH2CH3 NHCH2CH3 NHCH(CH3)2 NHCH2CH3

NHCH2CH3 NHCH(CH3)2 NHCH(CH3)2 NHCH(CH3)3

3.6 2.8 2.1 2.5

2.1 2.3 2.5 2.8

3.9 4.2 4.4 5.1

triazines

conc (ng/mL)

Ee


50.6 50.1 50.0 50.4

101 100 100 100

apple extract (5 mL)

urine (10 mL)

conc conc (ng/mL) Ee (ng/mL) Ee 39.2 41.1 39.0 45.1

2 2.1 2.0 2.3

58.1 57.2 60.5 102.3

2.9 2.9 3.0 5.1

Table 3. Extraction Efficiency (E) and Relative Standard Deviation for the SPE Combined with MIP as Analyzed from the Eluted Peak from the MIP Column


E

RSD (%)

0.77 0.77 0.74 0.76

4 3.5 5 3.2

a For K the triazines were eluted from an SPE column instead of 3 directly injected.

Figure 2. Detector signal as the analytes are eluted from the SPE column through the MIP column to the detector.

for the mobile phase to remove the matrix from the MIP columns. Acetonitrile was chosen for elution as it is water-miscible and has moderately low polarity and was therefore expected to result in selective binding to the polymer. For polymers printed in dichloromethane, acetonitrile has been demonstrated earlier to give selective retention in HPLC experiments.20 Generally, better recognition effects are achieved when less polar solvent is used for elution. This is the result of reduced influence on the retention mechanism, which primarily is considered to be hydrogen bindings, by low-polarity solvents.23 Figure 2. shows the detector signal when the triazines were eluted from the SPE through the MIP column to the detector. The figure shows the retention of the four triazines that each had been extracted (3 mL of 100 ng/ mL) on the SPE column. Reference polymers were also tested by extracting 1 mL of a 100 ng/mL mixture of each triazine in reagent water. The results indicate clearly that the molecularly imprinted polymers selectively retain the triazines. As the triazines are retained to a different extent on the polymer, we needed to release them from the column simulta(23) Matsui, J.; Miyoshi, Y.; Doblhoff-Dier, O.; Takeuchi, O. Anal. Chem. 1995, 67, 4404.


neously so as to be able to detect them all in one run. For this purpose we used a 200 µL injection of water from injection valve I. Water interferes with the noncovalent interactions (hydrogen bindings) of the triazines to the polymer, resulting in a release from the column. But 5% water in acetonitrile is sufficient to cancel all retention capability of the polymer.23 Acetic acid, sometimes used for this purpose, is not suitable because of its UV absorption. Another effect resulting from the water injection was a significant enrichment effect in the water plug. The highest concentration was found in the front of the plug, giving 3.1 times enrichment as compared to captured peak eluting from the MIP without injection of the water plug. Therefore, a 200 µL water plug was injected in the following experiments. By varying the capturing times for the plug, its concentration profile could be established. It turned out to contain 44% of the initially extracted triazine (either extracting 1 mL of 100 ng/mL in reagent water or 200 mL of water containing 20 ppm humic acid spiked to 0.5 ng/mL of each triazine). Additionally 11.5% of the triazine was in the front of the plug that was captured and injected in the 100-µL injection loop of injection valve J. Extraction Efficiency. For calculating the extraction efficiency (E), the MIP column was connected directly to the detector. The extracted triazines on the SPE column are thus passed from the extraction column to the detector via the MIP column. This extraction efficiency thus gives us the amount that could have been obtained purified in a vial if the work had been done off line. The losses are mainly due to severe band broadening caused by inhomogeneity of the binding site on the MIP. This was done also because the whole peak itself could not be captured in the 100-µL loop of injection valve J as discussed earlier. The peak detected was integrated and compared to a calibration curve for a directly injected triazine. The extraction efficiencies were 74-77% for the extraction of a 3 mL sample containing 100 ng/mL of the triazine in reagent water (Table 3). Sample Analysis. To demonstrate the suitability of the MIP for on-line sample pretreatment, samples containing humic acid,

Figure 4. The figure shows the difference between C18-SPE/MIP (a) and SPE only (b) for extraction of a 10-mL urine sample spiked with 20 ng/mL of each triazine. Chromatogram peak identification as in Figure 3.

Figure 3. Peaks 1, 2, 3, and 4 are the signals from simazine, atrazine, propazine, and terbutylazine, respectively. (a) Selective extraction of triazines from the highly contaminated sample containing 20 ppm humic acid. (b) The chromatogram when the MIP column is omitted. The extraction was made on a 200-mL solution containing 20 ppm humic acid spiked with 0.5 ng/mL of each triazine.

urine, and apple extract were investigated. Humic acid is commonly encountered in natural water samples, influencing the detectability of the analytes if found in high concentration.24,25 These complex matrixes make it difficult to determine low concentrations of triazines, using chromatography and ELISA assays without extensive sample pretreatment26 because of a large amount of interfering compounds. Therefore, we wanted to study the potential of MIPs to remove the negative influence of these matrixes prior to a chromatographic separation. The difference between C18-SPE, and C18-SPE in combination with antitriazine MIP, is shown in the chromatograms produced by the respective procedures in Figure 3. Peaks 1, 2, 3, and 4 are the signals from simazine, atrazine, propazine, and terbuthylazine, respectively. As can be seen in Figure 3a, the triazines are selectively extracted from the highly contaminated sample containing 20 ppm humic acid. Figure 3b shows the chromatogram from the eluate directly from the C18-SPE column, bypassing the MIP column. Large amounts of interfering compounds are detected in the chromatogram, making the determination process difficult, if not impossible, as the simazine and propazine peaks are completely hidden by the matrix. Thus, by use of the MIPs, a 100-fold selective sample enrichment could be recorded. The detection limits for extraction of 200 mL 20 ppm humic acid were determined to be 0.07 ng/mL, when defined as 3 times the noise. (24) Barcelo´, D.; Hennion, M. C. Anal. Chim. Acta 1995, 318, 1-14. (25) Berg, M.; Muller, R. S.; Schwazenback, R. P. Anal. Chem. 1995, 67, 18601865. (26) Baum, E. J.; Robertshaw, V. L. In New Frontiers in Agrochemical Immunoassay; Kurtz, D. A., Skerritt, J. H., Stacker, L., Eds.; AOAC International, 1995, 17-29.

Figure 5. The figure shows the C18-SPE/MIP extraction for 5 mL of apple extract spiked with 20 ng/mL of each triazine. Chromatogram peak identification as in Figure 3.

The degree of enrichment of the urine samples was lower, and a tendency to give nonspecific interactions with the MIPs was observed. The chromatograms in Figure 4 show a comparison between the C18-SPE/MIP and the SPE only. The chromatograms show a reduction of the interfering matrix signal, using the MIP; but, as the analytical signal is reduced as well, a less pronounced improvement was obtained. Nevertheless, up to a 5-fold selective sample enrichment could be recorded. Samples of the apple extracts showed a similar behavior (Figure 5). These results are indicative of the presence of compounds in the urine and appleextract samples (e.g., amino compounds) that exert strong, nonspecific binding to the carboxylate groups of the MIP. However, it can be envisaged that further optimization of the MIP, with respect to, e.g., choice of functional monomer, may lead to more efficient matrixes for such samples. We are currently in the process of extending the technique to other polymer systems. In Table 2, we present a summary of the results for the three sample matrixes. The typical coefficient of variation for three separate injections was 3.5%. In all cases, selective sample enrichment was acquired, demonstrating that the MIPs could be used to selectively improve an SPE process. The enrichment factor was calculated by dividing the obtained concentration in the captured fraction by the initial concentration in the spiked sample. As a result of the highly efficient discrimination of the humic acid Analytical Chemistry, Vol. 71, No. 11, June 1, 1999

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using MIP, large volumes of sample could be extracted and low detection limits acquired. CONCLUSIONS It has been shown that MIPs can be used for on-line selective trace enrichment of water samples containing 20 ppm humic acid. As a result of the MIP selectivity, large volumes (at least 200 mL) of water samples containing humic acid (20 ppm) can be extracted with little influence of this matrix on the resulting chromatogram. High sample-enrichment factors could therefore be obtained. Sample matrixes such as apple extract or urine were retained on the MIP to some extent, reducing the sample volume that could


be extracted, but still resulted in sample enrichment. However, further optimization of the MIPs may lead to more efficient matrix discrimination even for such samples. ACKNOWLEDGMENT The authors want to thank Professor Gillis Johansson and Dr. Jan A° ke Jo¨nsson for valuable comments throughout this work.

Received for review September 15, 1998. Accepted February 23, 1999. AC9810314

On-Line Solid-Phase Extraction of Triazine Herbicides Using a

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