Anal. Chem. 1995, 67,2530-2533
Solid Phase Microextraction Coupled to HighlPerformance Liquid Chromatography Jian Chen and Janusz B. Pawliszyn*
Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G 1, Canada
A new interface has been developed to couple the solid phase microextraction (SPME) sampling technique with narrow-bore high-performance liquid chromatography (HPLC). This interface maintains the advantages of SPME as a fast, solvent-free, portable, and inexpensive sampling technique. In addition, it provides possibilities for the analysis of semi- and nonvolatile organic comto achieve pounds in water, an analysis that is " I t with gas chromatography. Polycyclic aromatic hydrocarbons (PAHs) were selected as test analytes for the system's evaluation. The interface was shown to provide a successful way of coupling SPME with HPLC for the analysis of PAHs. As a universal, convenient, and solvent-free sampling method, the solid phase microextraction (SPME) technique has been investigated for the analysis of volatile organic compounds by gas chromatography (GC).l-l0 However, many classes of organic compounds widely used today are semi- or nonvolatile, such as pharmaceutical products, drugs, peptides and proteins, some pesticides, and polycyclic aromatic hydrocarbons, and are best separated by high-performance liquid chromatography (HPLC) methods. Thus, it would be advantageous to design an interface to couple the SPME sampling device with the HPLC system, providing easy sample preparation as well as high resolving power for semi- and nonvolatile analytes. The principle of SPME is based on the partitioning of analytes between a sample matrix and the stationary phase coated on a fiber. The amounts of analytes absorbed by the liquid polymer coating can be described by Nemst's partitioning law.2 The larger the partition coefficient of an analyte between the coating and the matrix, the greater the amount of analyte extracted. Different types of coatings provide different absorption properties for different kinds of analytes. The SPME method has two steps. The first is to extract analytes from a sample matrix. The second is to desorb those analytes directly into a GC or HPLC column for further analysis. (1) Belardi, R. G.; Pawliszyn, J. J. Water Pollut. Res. Can. 1989,24, 179. (2) Arthur, C. L.; Pawliszyn, J. Anal. Chem. 1990,62, 2145. (3) Arthur, C. L.; Killam, L. M.; Buchholz, K D.; Pawliszyn, J. Anal. Chem. 1992.64, 1960. (4) Louch, D. S.; Motlagh, S.; Pawliszyn, J. Anal. Chem. 1992,64, 1187. (5) Potter, D. W.; Pawliszyn J. J. Chromatogr. 1992,625, 247. (6) Arthur, C. L.; Potter, D. W.; Buchholz, K D.; Motlagh, S.; Pawliszyn, J. LC-
GC 1992,10,656 (7) Potter, D. W.; Pawliszyn, J. Environ. Sei. Technol. 1994,28, 298. (8) Zhang. Z.; Pawliszyn, J. Anal. Chem. 1993,65, 1843. (9) Zhang, 2.; Pawliszyn, J. J. High Resolut. Chromatogr. 1993,16, 689. (10) Chai, M.;Arthur, C. L.; Pawliszyn, J.; Belardi, R P.; Pratt, K F. Analyst 1993,118, 1501.
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For SPME-HPLC coupling, the extraction procedure is similar to that used for GC analysis. The SPME device is a modified syringe which is portable and easy to handle. The plunger moves the fiber in and out of the needle to protect the coating. The fiber is coated with a polymeric phase, e.g., poly(dimethylsi1oxane), poly(acrylate), etc. During sampling, the fiber is exposed to the sample matrix air or aqueous phase. Analytes partition from the sample matrix into the fiber coating until equilibrium is reached. The main difference between SPME-HPLC and SPME-GC is in the second step, the desorption procedure. For GC analysis, the fiber is introduced into the injector port where the analytes are thermally desorbed from the coating. However, for HPLC analysis, thermal desorption at high temperature creates practical problems such as degradation of the polymer, and in addition, many nonvolatile compounds cannot be completely desorbed from a fiber. Solvent desorption is thus proposed as an alternative way for SPME-HPLC coupling. The challenge in this application is to desorb analytes completely with a minimum amount of solvent to avoid signifkant extracolumn dispersion contributed by a large sample volume. In this paper, an interface design for SPME-HPLC analysis is reported. The interface maintains the advantages of SPME in sample preparation and is compatible with the requirements of HPLC analysis. Thirteen semi- and nonvolatile polycyclic aromatic hydrocarbons were extracted from an aqueous matrix by SPME with a poly(dimethylsi1oxane) coating and then separated on a narrow-bore (2.1 mm id.) ODS column. EXPERIMENTAL SECTION Apparatus and Reagents. The SPME-HPLC system developed for this investigation is depicted in Figure 1. The system consists of the following three parts: (1) SPME device, (2) interface, (3) HPLC system. The interface is enlarged to show its construction. The assembly of the SPME device is similar to that described previo~sly.~,~ The 1.0 cm long fiber is coated with a 7 pm thick stationary phase of poly(dimethylsi1oxane) and was obtained from Supelco Canada (Missisauga, ON). A new fiber should be conditioned before use as specified in the literature accompanying the commercial SPME products. A fiber is conditioned by inserting it into a GC injection port set to 250 "C, under a helium stream for 8 h. After removal from the injector, the fiber is cooled to room temperature. The fiber is then conditioned again using the same GC conditions for 0.5 h two to three times, until a very stable baseline is obtained. The interface includes a custom-designed desorption chamber and a six-port Rheodyne 7161 injection valve. The construction of the desorption chamber is shown in detail in Figure 1. It 0003-270019510367-2530$9.00/0 0 1995 American Chemical Society
Procedm. A conditioned fiber coated with 7 pm poly(dimethylsiloxane) was exposed to a stirred water sample spiked with PAH standard for 30 min. Before transferring the fiber into the desorption chamber, the injection valve was placed in the "load" position. The fiber was then introduced into the desorption chamber by lowering the syringe plunger. The twwpiece PEEK union was closed tightly. The valve was then switched to the "injection" position, and the desorption procedure started. Solvents from the HPLC pump passed through the desorption chamber in an upstream direction to avoid air bubbles being introduced to the column and disturbing detection. Analytes that were absorbed by the fiber desorbed into the solvent and were canied to the LC column. Column separation was then initiated and a solvent program applied when necessaty to achieve good separation.
uv-VIS Da,ec,or
Figure 1. Consiruciiin of the SPME-HPLC system. Componenis
are explained in the text. consists of the following: (a) a stainless steel (SS) '/I6 in. tee joint (Valco, Houston, TX); 6)a piece of '/I6 in. o.d., 0.02 in. i.d. SS tubing (Supelco, Bellefonte, PA); (c) a piece of '/I6 in. o.d., 0.02 in. i.d. poly(ether ether ketone) (PEEK) tubing (0.02 in. i.d.); (d) a two-piece finger-tight PEEK union; (e) a piece of 0.005 in. i.d. PEEK tubing with a onepiece PEEK union. All PEEK products were obtained from S i a A l d r i c h , Milwaukee,WI. The upper portion of PEEK tubing in part c was enlarged to fit the needle of the syringe. The SS rod in the SPME device can be tightly sealed by the PEEK tubing and union, withstandingsolvent pressures as high as 4500 psi. The desorption chamber is placed in the position where the injection loop normally resides on the Rheodyne 7161 valve. When the injection valve is at the "load" position. the desorption chamber is at ambient pressure so that the fiber can be placed into the chamber. The HPLC system used in this study includes an Eldex 9Mx) pump (Eldex Laboratories,Inc., San Carlos, CA), a TosohaasTSK6041 W detector (distributed by Supelco Canada), or a Varian 9Mx) fluorescence detector (Varian Associates, Inc., Walnut,CA). All separations were carried out on a narrow-bore HPLC column (2.1 mm id,) packed with 5 pm ODS phase (Alltech Associates, Inc.. Deerlield, IL). Data were collected by a PC computer using Varian Star sofhmre. All reagents used were HPLC grade and purchased from Fisher Scientific (Fair Lawn, NJ). The polycyclic aromatic hydrocarbon (PAH) samples were obtained from Aldrich. The 1 mg/mL PAH standard solutions were prepared in methanol/ methylene chloride (l/l, v/v) and M e r diluted to 1 pg/mL with methanol. PAH mixture 525, which contains the 13 PAHs at a cmcentratjon of 0.5 mg/mL per compound in methylene chloride, was obtained from Supelco Canada. All water samples were prepared by spiking the PAH standard solution into highpurity water produced by an ultrapure water system (Barnstead/ Thermolyne, Dubuque, IA).
RESULTS AND DISCUSSION To evaluate the SPMEHPLC interface, PAHs and poly(dimethylsiloxane) (PDMS) were selected as the test sample and extraction coating. The determination of PAHs in water using SPME with GC/MS has already been investigated by Potter and Pawliszyn.7 The SPME extraction procedure was optimized using 15pm thick PDMS and Carbopack B coatings. For large PAHs such as benz[alanthracene and benzo[alpyrene, the distribution constants are about 10s between the poly(dimethylsi1oxane) coating and water. Large PAHs also require a much longer equilibration time with the 15pm PDMS coating, e.g., 60 min for benz[olanthracene, than for smaller PAHs, e.g., 6 min for naphthalene. The extraction procedure for SPMEHPLC was the same as for SPMEGC reported by Potter and Pawliszyn.7 The desorption procedure is totally different from that used for GC. Solvent desorption is used instead of thermal desorption: The d& contact of crosslinked polymers with organic solvent often results in swelling of polymers. A 7 pm PDMS coating was thus used to avoid blockage of flow inside the desorption chamber due to swelling of PDMS. The desorption kinetics of analytes from the fiber coating into the gas phase bas already been discussed by Louch et aL' For desorption in SPMEHPLC, the gas phase is replaced by an organic solvent phase. The desorption process will be 90% complete at time f = L2/2D, where Lis the coating thickness and D is the diffusion coefficient of an analyte. In this case, the thickness of the poly(dimethylsi1oxane) coating is 7 pm, and the dfision coefficientof analytes in a liquid layer is about m2/ s. The desorption time required is thus less than 1 s. assuming continuously flowingmobile phase and good solubility of analyte in the mobile phase. The performance of solvent desorption for SPME was inves tigated by connecting the interface directly to the detector cell. Solvent mixtures of different compositions of acetonitrile/water were passed through the desorption chamber. The &e, be[olpyrene, desorbed from the fiber was detected by the W detector, and the elution curves were recorded. A very small desorption volume, less than 0.2 pL, was found when 99/10 of CH&N/H20 was used as solvent. However, the desorption volume increased drasticaliy when a solvent mixlure with a higher concentration of water was used, due to the lower solubility of benzo[olpyrene. The desorption procedure is extremely important in HPLC and needs to be optimized for each application with Analytical Chemistv, Vol. 67, No. 15, August 1, 1995
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Figure 2. lsocratic separation of a four PAH mixture by (a) 1 pL loop injection and (b) fiber injection, 7 pm PDMS extraction for 30 min from 100 ppb of each compound spiked into water. Chromatographic conditions: column, 25 cm long x 2.1 mm i.d., 5 pm ODs; detection, UV 254 nm; solvent, acetonitrile/water (90/10, v/v); flow rate, 0.2 mUmin. Peak identification: (1) fluorathene, (2) pyrene, (3) benz[a]anthracene, and (4) benzo[a]pyrene.
different solvent compositions adjusted for solubility of target analytes in the mobile phase. Figure 2 compares the chromatograms obtained for the separation of a four PAH mixture using a 1 p L loop injection (Figure 2a) and SPME sample introduction (Figure 2b) under isocratic conditions. The peaks of PAHs desorbed from the fiber in the desorption chamber are all very sharp, with no differences from the peak shape produced by loop injection. The evidence agrees well with the discussion above that the desorption volume in the interface is small enough for direct injection. In addition, the chromatograms also show identical retention times for loop injection and SPME fiber injection. Although the retention of analytes is only controlled by the properties of the stationary phase and the mobile phase, a slow desorption procedure could affect the retention of analytes. The identical retention times indicate a very fast desorption of analytes from the fiber coating and very little contribution of desorption chamber geometry to the dead volume of the whole system. During earlier investigationsof SPME-GC, cany-over was often observed with the thermal desorption method. It has been reported that, when a 15 pm PDMScoated fiber is used for extraction of semivolatile compounds, carry-over is still significant for benz[a]anthracene and benzo[a]pyrene even after four fiber blanks7 This drawback can be overcome by applying higher desorption temperatures using a highly thermally stable coating as mentioned in the same paper. In SPME-HPLC analysis, however, no carry-over was found with solvent desorption. No peaks appeared in the chromatogram of the second blank. Solvent gradients are widely used in HPLC to analyze complex samples. This investigation illustrated that fiber injection does not affect the retention of the PAH compounds under solvent gradient conditions. As shown in Figure 3, a separation of a 13 2532 Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
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0 5 10 15 20 min Figure 3. Separation of PAH mix 525 with solvent gradient by (a) 1 pL loop injection and (b) fiber injection, 7 pm PDMS extraction for 30 min from 100 ppb of each compound spiked into water. Chromatographic conditions: column, 25 cm long x 2.1 mm i.d., 5 pm ODs; flow rate, 0.2 mumin; detection, UV 254 nm; solvent program, CH&N/H?O (80/20, v/v) linear gradient to 100% CHGN in 15 min. Peak identification: (1) acenaphthylene, (2) fluorene, (3) phenanthrene, (4) anthracene, (5) pyrene, (6) benz[a]anthracene, (7) chrysene, (8) benzo[b]fluorathene, (9) benzo[k]fluorathene, (10) benzo[alpyrene, (1l ) dibenzo[ah]anthracene, (12) indeno[l,2,3-cd]pyrene, and (13) benzo[gh/]perylene.
PAH mixture was performed by both loop injection (Figure 3a) and fiber injection via the SPMEHPLC interface F i r e 3b). The retention times of PAHs by fiber injection agree well with the loop injection under linear solvent gradient mode. The benz[alanthracene and chrysene (peaks 6 and 7) cannot be separated on a monomeric ODS phase, as reported earlier, because monomeric phases lack shape selectivity.11J2 The improved separation can be obtained on polymeric ODS phases specially designed for PAH analysis. A chromatographic separation aims to characterize or quantify compounds of interest or do both. In turn, the good reproducibility of peak retention time and peak area is of great importance in the evaluation of a chromatographic system. As the SPMEHPLC interface was installed onto an ordinary HPLC system, the reproducibility of retention times and peak areas of the whole system was evaluated with the PAH compounds. The results of five replicate SPME-HPLC injections are listed in Table 1. The peak retention times and areas both have very good reproducibility with the SPMEHPLC interface. Therefore this instrumentation setup can be used successfully for analysis of semivolatile or nonvolatile compounds. Some new applications based on this interface setup are in progress and will be reported later. CONCLUSIONS
The SPME sampling technique has been coupled to the HPLC via the interface reported. The solvent desorption does not result (11)Wise, S. A; Sander, L.C. J. High Resolut. Chromatogr. Chromatop. Commun. 1985,8, 248. (12) Chen, J.; Steenackers, D.; Sandra, P. J. High Resolut. Chromatogr. 1993, 16,608.
Table 1. Reproduclblllty of Retentlon Times and Peak Areas
fluoranthene pyrene benz [a]anthracene benzo [ajpyrene
t~ (min)
RSD (%)
6.86 7.70 8.84 13.63
5.00 4.70 3.60 4.00
peak area (counts)
RSD (%)
43 568 56874 127 352 88028
6.80 6.90 6.90 5.80
reproducibility in both retention and peak area, the SPME-HPLC can be used for both qualitative and quantitative analyses. ACKNOWLEDGMENT The financial support of Supelco, Varian, and the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged. Received for review November 9,1994. Accepted May 10,
in a large injection volume and thus avoids large extracolumn band broadening contributed by injection volume. No carry-over was found by the solvent desorption procedure. A solvent gradient can also be applied to enhance the separation. With its good
1995.B AC9410950 @
Abstract published in Advance ACS Abstracts, June 15, 1995.
Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
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