Anal. Chem. 1997, 69, 3140-3147
Automated In-Tube Solid-Phase Microextraction Coupled to High-Performance Liquid Chromatography Ralf Eisert and Janusz Pawliszyn*
Department of Chemistry, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada
Recently, solid-phase microextraction (SPME) was successfully coupled to high-performance liquid chromatography. However, the efficiency of this analytical method, in terms of manpower, still suffers from its manual operation technique. Furthermore, the selectivity obtained for the analysis of very polar compounds is still poor because of a limited selection of commercially available fiber coatings that can withstand the aggressive HPLC conditions (solvents). This paper describes the first approach to developing an automated SPME-HPLC system. A mixture of polar thermally labile analytes, phenylurea pesticides, was selected for microextraction directly from an aqueous sample. A piece of a ordinary capillary GC column with its coating (Omegawax 250) was used for the absorption of analytes from the aqueous sample (in-tube solid-phase microextraction). A needle hosts the capillary when it is pierced through the septum of the vial containing the spiked aqueous sample. The aqueous samples were stored in 2 mL vials on the tray of a commercial autosampler. A sample of 25 µL was aspirated and dispensed several times from the sample into the capillary using a syringe. After the extraction the absorbed analytes were released from the coating by aspiring methanol into the column and then dispensing the methanol into the HPLC injector loop. The absorption-time profiles, the amounts absorbed by different coatings, linearity, and precision were studied under different sampling conditions using spiked aqueous samples. SPME selectivity for polar compounds, which represent an important compound class for water analysis, can be improved by using more polar column coatings such as Carbowax instead of poly(dimethylsiloxane)coated columns. Compared to the manual version this automated SPME-HPLC system could increase productivity and reproducibility. Furthermore, the desorption step is quantitative; i.e., no carryover could be detected. This entire method for automated SPME sample preparation is simple and controlled by a commercial autosampler from LC Packings which was modified to operate in-tube SPME. The automated SPME-HPLC device obtains RSD for all investigated compounds below 6%. A simple mathematical model was used to calculate the concentrations vs length profiles in the column for any time. The model was in good agreement with experimental data which was obtained for benzene as a model compound. Thus, the main parameters affecting the partitioning
process were determined and the amount absorbed by the coating could be predicted.
3140 Analytical Chemistry, Vol. 69, No. 16, August 15, 1997
S0003-2700(97)00319-3 CCC: $14.00
Optimization of solid-phase microextraction (SPME) methods has concentrated mostly on the effects of matrix conditions such as the influence of pH, salt content of an aqueous sample, the presence of polar, low molecular weight solvents (e.g., methanol),1 or humic material; competition between main and trace compounds; and the use of agitation techniques.2 Automation is necessary for many applications. Sample preparation techniques that cannot be automated are less often used for routine analysis even if they offer other attractive features, like high selectivity or sensitivity. However, no suitable automation of the entire SPME method especially for SPME-HPLC is the most often cited disadvantage of SPME. To date, the applications of SPME-HPLC are all based on the manual device and interface.3-6 So far SPME has been automated only for the GC sample preparation with static absorption, fiber vibration, and direct aqueous or headspace sampling using a commercial GC autosampler.7,8 Direct extraction from the aqueous matrix, with the SPME fiber directly exposed to the aqueous matrix, is usually used for semivolatile compounds, including polar pesticides such as triazines.1,2 The SPME methods developed so far have had limited effectiveness for thermally labile compounds, such as phenylurea and carbamate pesticides. These compounds could be extracted from an aqueous matrix only with a very polar coating like Carbowax. Besides having high polarity, the SPME fibers have to be stable against the solvents used in HPLC, for example, methanol or acetonitrile. The capacity (total volume times partition coefficient of the stationary phase) of the commercially available fibers is low. Thus, the SPME-HPLC method suffers from poor sensitivity. Sensitivity can be increased instrumentally by using hyphenated techniques, in particular LC-MS. This paper describes the first demonstration a new variation of SPME that uses GC capillary columns and that can be coupled to a commercial HPLC autosampler. The automation of the entire SPME-HPLC system for handling samples was the main goal of (1) (2) (3) (4) (5)
Eisert, R.; Levsen, K. Fresenius J. Anal. Chem. 1995, 351, 555-562. Eisert, R.; Levsen, K. J. Am. Soc. Mass Spectrom. 1995, 6, 1119-1130. Chen, J.; Pawliszyn, J. B. Anal. Chem. 1995, 67, 2530-2533. Boyd-Boland, A. A.; Pawliszyn, J. B. Anal. Chem. 1996, 68, 1521-1529. Liao, J. L.; Zeng, C. M.; Hjerte´n, S.; Pawliszyn, J. J. Microcolumn Sep. 1996, 8, 1-4. (6) Jinno, K.; Muramatsu, T.; Saito, Y.; Kiso, Y.; Magdic, S.; Pawliszyn, J. J. Chromatogr., A 1996, 754, 137-144. (7) Arthur, C. L.; Killam, L. M.; Buchholz, K. D.; Pawliszyn, J.; Berg, J. R. Anal. Chem. 1992, 64, 1960-1966. (8) Eisert, R.; Pawliszyn, J. J. Chromatogr., A, in press. © 1997 American Chemical Society
the study. Furthermore, this concept extends the application range of SPME-HPLC to very polar compounds, which are more difficult to extract from water than relatively apolar compounds such as PAHs.3 Even coated fiber SPME would be capable of the extraction step when more polar coatings are used, but it still suffers from carryover and a lack of automation. Therefore, more polar coatings were investigated to increase the sensitivity of the method for polar compounds. To date, a larger range of coatings is available for GC capillaries than for SPME fibers. Eventually, each coating used for GC capillaries is expected to be offered for SPME fiber coatings. The newly developed automated SPME-HPLC method uses a flow-through process which is expected to reduce the total extraction time per sample and increase the precision of the entire method. The open tubular column used for the direct absorption of the target analytes from the aqueous sample is enclosed in a needle device and can automatically be exposed to a vial containing the sample. The device combines features of earlier developed SPME devices where the inner surface of a syringe was coated with a polymer and microcolumn LC. The absorption equilibrium will be achieved faster because the extraction process involves agitation by sample flow in and out of a column. Theory of In-Tube SPME. The in-tube SPME consists of a piece of fused-silica capillary, internally coated with a thin film of extraction phase (a piece of open tubular capillary GC column), or a capillary packed with extracting phase dispersed on an inert supporting material (a piece of LC microcolumn). In these geometric arrangements, the concentration profile along the axis, x, of the tubing containing the extracting phase as a function of time, t, can be described by adopting the expression for dispersion of a concentration front:9-11
[
]
x - ut/(1 + k) 1 C(x,t) ) C0 1 - erf 2 σx2
(1)
where u is linear velocity of the fluid through the tube and k is the partition ratio defined as
Vf ds k ) Kfs ) 4Kfs VV dc
(2)
where Kfs is a coating/sample distribution constant, Vf is the volume of extracting phase, VV is a void volume of the tubing containing the extracting phase, ds is the stationary phase film thickness, and dc is the diameter of the column bore. σ is the mean square root dispersion of the front defined as
σ)
x
u Ht 1+k
These contributions are dependent on the particular geometry of the extracting system.10 The value for H can be derived from Golay’s equation for capillary open tubular column systems where H is defined as
H)
[
] [
]
2 2Dm 2kds2u 1 + 6k + 11k2 udc + + m 96(1 + k)2 Dm 3(1 + k)2Ds
(4)
where u is the linear velocity of the solvent, Dm is the diffusion coefficient of the solute in the mobile phase, and Ds is the diffusion coefficient of the solute in the stationary phase. The last term in eq 4 represents slow equilibrium in the stationary phase. To a first approximation it can be ignored in water extraction for open tubular capillaries, which leads to the following equation:
H)
[
]
2 2Dm 1 + 6k + 11k2 udc + u 96(1 + k)2 Dm
(5)
Equation 1 indicates that the front of analyte migrates through the capillary with a speed proportional to the linear velocity of the sample and inversely related to the partition ratio. For short capillaries with small dispersion, the extraction time can be assumed to be similar to the time required for the center of the band to reach the end of the capillary:
te )
L(1 + k) u
(6)
where L is the length of the capillary holding the extraction phase. As expected, the extraction time is proportional to the length of the capillary and inversely proportional to the laminar flow rate of the fluid. Extraction time also increases with an increase in the coating/sample distribution constant and with the volume of the extracting phase but decreases with an increase in the void volume of the capillary. It should be emphasized that the above equation can be used only for direct extraction when the sample matrix passes through the capillary. This approach is limited to particulate-free gas and clean water samples. The headspace SPME approach can broaden the application of in-tube SPME. In that case, careful consideration of the mass transfer between the sample and headspace should be given in order to describe the process properly.12 Also, if the flow rate is very rapid, producing turbulent behavior, and the coating/sample distribution constant is not very high, then the perfect agitation conditions are met and the eq 7 can be used to estimate equilibration times.
te ) t95% ) ds2/2Ds
(7)
(3)
where H is equivalent to the HETP (height equivalent to a theoretical plate) in chromatographic systemssthis can be calculated as a sum of individual contributions to the front dispersion. (9) Crank, J. Mathematics and Diffusion; Clarendon Press: Oxford, UK, 1989; p 14. (10) Pawliszyn, J. J. Chromatogr. Sci. 1993, 31, 31-37. (11) Pawliszyn, J. Solid-Phase Microextraction: Theory and Practice; WileyVCH: New York, 1997.
Using this equation, one can estimate the shortest equilibration time possible for the practical system by substituting appropriate data for the diffusion coefficient of an analyte in the coating (Ds) and the coating thickness (ds). Removal of analytes from a tube is an elution problem analogous to frontal chromatography which has been discussed in detail in ref 10. In general, if the desorption temperature of a GC is high and thin coatings are used, then all the analytes are (12) Zhang, Z.; Pawliszyn, J. Anal. Chem. 1993, 65, 1843-1852.
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in the gas phase as soon as the coating is placed in the injector, and the desorption time corresponds to the elution of two void volumes of the capillary. For liquid desorption (for example, LC systems as shown in this paper), the desorption volume can be even smaller since the analytes can be focused at the front of the desorption solvent. The mass present in the capillary tubing at any given moment (t), can be calculated by
M(t) )
∫ C(x,t) dx L
0
(8)
This mass is equal to the amount of analyte absorbed by the polymeric coating at any given moment. Thus, the absorption time profile can be derived from eq 8. EXPERIMENTAL SECTION Reagents. The pesticide standards of diuron, fluometuron, linuron, monuron, neburon, and siduron were purchased from Chem Service (West Chester, PA). In addition eight carbamates (carbaryl, chlorpropham, s-ethyldipropylthiocarbamate, methiocarb, promecarb, propham, propoxur, and venolate) from Chem Service were used. They were of g98% quality and used as received. Acetonitrile and methanol (HPLC grade quality) were from EM Science (Gibbstown, NJ). Water was obtained from a Barnstead/Thermodyne NANO-pure ultrapure water system (Dubuque, IA). Single standards with a concentration of 1000 ng/µL were prepared for each compound using methanol as a solvent. A standard mixture using methanol was prepared containing the organic compounds at a concentration of 100 ng/µL each. The aqueous samples were distilled water spiked with this or diluted standard mixtures reducing the concentration to 10, 100, 1000, or 10 000 µg/L. Liquid Chromatography. High-performance liquid chromatographic investigations were carried out using a TSK-6010 HPLC pump and a TSK-6041 UV detector (TosoHaas, Philadelphia, PA). A Nova-Pak C18 8 mm × 100 mm (4 µm) HPLC column was operated at 1.0 mL/min and isocratic conditions with an acetonitrile/water 60:40 mixture. The HPLC cartridge resides in a waters RCM 8 × 100 cartridge holder (Waters, Milford, MA). The optimum wavelength for detection of phenylureas was found to be 245 nm. Data acquisition and processing was provided by a PC interfaced to the detector using Star 4.5 software (Varian, Palo Alto, CA). In-Tube Solid-Phase Microextraction. GC capillary columns with different coatings were selected for the absorption of the six phenylureas and were compared according to the selectivity obtained. All capillary tubings were 60 cm long and 0.25 mm i.d. Thus, the total internal volume of each capillary was 29.5 µL. Due to the different coating thicknesses, the volumes of the stationary phases differed by a factor of 5. The volume of the stationary phase for the Omegawax 250 (Supelco, Bellefonte, PA) capillary (0.25 µm film thickness) was 59 nL. The SPB-5 (Supelco) capillary (1 µm film thickness) had a volume of 235 nL for the stationary phase. The SPB-1 (Supelco) capillary (0.25 µm film thickness) had a volume of 59 nL for the stationary phase. This column represents the poly(dimethylsiloxane)-coated column, a liquid coating widely used in classical SPME. In addition, a retention gap capillary (no coating) was tested for comparison. The in-tube SPME device is based on a open tubular capillary column that hosts in a needle device. The design has some major 3142
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advantages compared to older devices used for SPME. The direct coating of the inner surface of a syringe makes the device less flexible. The in-tube SPME device allows easy changes of the coated capillary modifying the selectivity. Furthermore, the device fits perfect to the LC Packings autosampler, which uses a capillary for aspirating the sample into the HPLC loop. Commercial columns can be used in this setup which leads to unique application range based on a large variety of GC columns available. Autosampler Operation and Interface Design. A 60 cm long piece of one of the GC capillaries mentioned above was mounted in the Famos autosampler (LC Packings, Amsterdam, The Netherlands) replacing the noncoated capillary which is usually installed there. The total internal volume of this tubing was 29 µL. The autosampler software was manually programmed to control the SPME absorption and desorption technique. For desorption, pure methanol from a second vial was flushed through the SPME unit and directly transferred into the injection loop. The HPLC injection loop was built of a 56 cm long PEEK tubing (300 µm i.d.) which had a total volume of 40 µL. The instrumental setup is illustrated in Figure 1. The 2 mL vials were filled with 1.4 mL of sample for the absorption of the compounds from the aqueous sample. The spiked aqueous samples were sonicated before use. For absorption-time experiments, water from a NANOpure water purification system was spiked with standard solutions containing 10 000 µg/L of each compound in methanolic solution. Therefore, an uncoated piece of capillary (2.4 µL) was installed instead of the coated SPME column. The first step in the method was to rinse the GC capillary with methanol, and it still contained methanol before the first absorption step. A sample volume of 25 µL (total volume of the syringe used in this study) was aspirated from the sample vial at a flow rate 63 µL/min. Then the same sample volume was dispensed back into the vial. Usually, these two steps were repeated 10 times. After the absorption step, the six-port valve was switched to the LOAD position. A 38 µL aliquot of methanol was aspirated from a solvent vial and transferred to the injection loop for the desorption of the extracted analytes from the capillary coating. The six-port valve was switched to the INJECT position, and a trigger was sent to the PC for starting the data acquisition. The sample was transferred from the loop to the analytical column by the isocratic eluent mixture. During the analysis of the first sample, a subsequent sample could be extracted. HPLC calibration was performed by injecting liquid standards (5 µL loop) containing the organic compounds in an organic solvent such as methanol. RESULTS AND DISCUSSION In-Tube SPME Calculations. Results of this model are presented in Figure 2 for the extraction of benzene from water by a 0.25 µm PDMS-coated GC capillary having the following characteristics: Kfs ) 126, Dm ) 1.08 × 10-5 cm2/s, and Ds ) 2.8 × 10-6 cm2/s (benzene).12-14 The series of curves in this figure represent normalized concentration profiles for benzene obtained for different extraction times over the length of the capillary column L. For this compound, extensive physicochemical data are available to describe the in-tube SPME processes in more (13) Wendt, J. O. L.; Franzier, G. C. Ind. Eng. Chem. Fundam. 1973, 12, 239243. (14) News, A. C.; Park, G. S. J. Polym. Sci.: Part C 1969, 22, 927-937.
Figure 1. Instrumental setup of the new on-line SPME-HPLC interface based on an in-tube SPME capillary technique. A piece of GC column (in-tube SPME) hosts in the position of the former needle capillary. The aqueous sample is frequently aspirated from the sample vial through the GC column and dispensed back to the vial (INJECT position) by movement of the syringe. After the extraction step, the six-port valve is switched to the LOAD position for the desorption of the analytes from the in-tube SPME by flushing 100% methanol from another vial through the SPME capillary. The volume is transferred to the loop. After switching the Valco valve to the INJECT position, an isocratic separation using a mixture of 60:40 acetonitrile/water was performed. A detailed view of the in-tube SPME capillary is included at the left side of the figure.
Figure 2. Normalized concentration profiles for in-tube SPME calculated for the equations presented in the theory. The graphs show curves for different times after the in-tube SPME start for t ) 1, 5, 10, 20, 27.5, and 50 s for benzene (K ) 126).
detail. The initial concentration of benzene in the aqueous solution is reached at the end of the capillary tubing within 1.5 void volumes aspirated into the capillary column. This time represents the equilibration of the compound in the column. As shown before, the number of void volumes necessary to reach equilibrium increases with K-values of the analytes. The integration of the C(x,t) over the length of the capillary column L leads to the amount absorbed by the coating as stated in eq 8. Validation of the Model. For benzene, the equilibration time for PDMS coatings is very short. It takes only 1-1.5 void volumes aspirated into the capillary column to reach equilibrium conditions, as shown in Figure 3. Taking a flow rate of 63 µL/min into account, this is equal to an extraction time of te ) 50 s when continuous-flow conditions are used. The experimental data are in good agreement with the model developed. Some discrepancy between the data and the model, seen in Figure 3, can be explained by instrumental conditions. The absorption equilibrium
Figure 3. Fraction of the analyte n/no (normalized by the total amount absorbed at equilibrium, no) extracted by the coating during the in-tube SPME absorption vs the number of void volumes, to, passing through the column. The line represents the calculated and the marker the experimentally determined absorption curve for benzene at flow rate of 63 µL/min using a poly(dimethylsiloxane)coated capillary (50.9 cm length, 0.25 mm i.d., and 0.25 µm film thickness).
was reached faster in the experiment then in theory. The extracted amounts are higher for flow volumes below the void volume of the column. The model predicts the absorbed amount from the concentration profiles at different extraction times. Experimentally, these values are difficult to obtain. For example, when 10 µL is aspirated into the column the absorbed amount cannot be determined without removing the aqueous volume from the column. Thus, the volume was dispensed back to the vial, which leads to additional absorption of analyte onto the coating. The absorbed amounts are higher than expected from the model. However, a good correlation was achieved, which means the model is valid for the calculation of optimized operation conditions, such as flow rate. Practical limits can prevent increasing the efficiency further. Very high flow rates affect the precision. Under Analytical Chemistry, Vol. 69, No. 16, August 15, 1997
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Figure 4. Normalized concentration profiles for in-tube SPME calculated for the equations presented in the theory. The graphs show curves for different times after the in-tube SPME start for t ) 1, 5, 10, 30, and 60 s for three compounds with different K-values (solid lines, K ) 100; dashed lines, K ) 1000; dotted lines, K ) 10 000), assuming diffusion coefficients similar to benzene which was used for this calculation.
these conditions, air bubbles are formed at the edges of the capillary dipping into the sample. The aspiration of air reduces the accuracy of the volumes transferred into the column, which affects the RSD of the extraction. The fraction of analyte absorbed by the coating normalized by the total amount that can be extracted at equilibrium, no, within a defined time decreases with increasing K-value of the compound. The model for in-tube SPME is based on liquid polymer coatings, which usually show a lower affinity toward more polar analytes, like phenylureas. Thus, the model does not fit perfectly for mixed polymer coating materials such as Carbowax used in this study. However, the model explains the basic principles to understand and optimize the intube SPME parameters. Figure 4 shows the normalized concentration curves for three different K-values (100, 1000, 10 000). The number of void volumes necessary to reach equilibrium significantly increases with increasing K-values. The absorption-time profiles, which can be calculated, are in good correlation with results from experimental data, as explained above. In our final experimental design, the sample is aspirated and dispensed instead of using a one-way flow. The contamination of the buffer tubing makes the flow-through approach less efficient compared to the repeated aspirate/dispense mode. The increase of the flow rate of the sample is a significant factor which determines the equilibration time. However, the increase of this parameter is limited by practical factors. Air bubbles are formed at high flow rates at the edge of the capillary which are aspirated into the column; difficult sample handling (uncontrolled backflush) in the capillaries when air is aspirated and contamination of the upper buffer tubing significant affect the precision. Furthermore, the total volume that can be aspirated in one step is limited by the syringe volume. A continuous one-way aspiration system shows no significant advantages and further improvement of the extraction efficiency. The aspiration concept, however, could improve the automated in-tube SPME handling substantially by obtaining a higher precision. More rugged extraction conditions are demonstrated in the validation part below. The newly developed automated SPME system based on a coated capillary that is usually used for GC separations shows very good results. The number of aspirate/dispense steps was varied from 0 to 50 steps to monitor the absorption-time profiles. 3144 Analytical Chemistry, Vol. 69, No. 16, August 15, 1997
Figure 5. Absorption-time profiles for six phenylurea pesticides using a 60 cm long piece of a Omegawax 250 GC column tubing (0.25 mm i.d., 0.25 µm film thickness) and up to 50 aspirate/dispense steps at a sample flow rate of 63 µL/min from a 1.4 mL aqueous sample with a concentration of 1000 µg/L. Peak assignment: (O) neburon, (b) linuron, (4) diuron, (2) monuron, (0) siduron, and (9) fluometuron.
Even after 50 aspirate/dispense steps, no equilibrium conditions were obtained for the extraction of all six compounds, as demonstrated in Figure 5. However, the extraction is very efficient and in good agreement with the octanol-water partitioning coefficient, KOW (for values of the compounds, see Table 1). For a first approximation, the compounds with lower KOW values show a faster equilibration time. However, the KOW value is directly related to the Kfs partitioning coefficient when liquid coatings are used. Every time the sample was dispensed back to the vial, the amount previously absorbed by the coating was at least partly desorbed by the methanol which follows the sample inside the capillary. This complex three-phase system can no longer be quantitatively described by the model. A chromatogram obtained by automated SPME-HPLC was compared to a chromatogram obtained by injection of a methanolic standard to determine the selectivity and sensitivity achieved for the six compounds, as shown in Figure 6. The “less” polar compounds, later eluting pesticides, show a higher affinity to the Omegawax 250 coating. The peak shape is no longer affected by the composition of the eluent solvent mixture which was observed for the manual SPMEHPLC interface. The extracted compounds are first desorbed and then transferred into an HPLC loop. The injection into the analytical column is similar to the injection of methanolic standards. The separation of the desorption step from the injection to HPLC step is a big advantage compared to the manual SPME-HPLC interface. Using this procedure, a different solvent mixture can be used for the desorption, which shows a higher eluting power compared to the initial solvent composition of the HPLC eluent. Precision. Ten aspirate/dispense steps were used for all precision experiments. The precision obtained using an Omegawax 250 capillary varies between 1.6 and 5.6% RSD (n ) 10), depending on the compound and concentration studied. For 10 000 µg/L concentrations the precision was
