Anal. Chem. 2005, 77, 7472-7477
ATR-FT-IR Membrane-Based Sensor for Integrated Microliquid-Liquid Extraction and Detection Rafael Lucena, Soledad Ca´rdenas, Mercedes Gallego, and Miguel Valca´rcel*
Department of Analytical Chemistry, Marie Curie Building (Annex), Campus de Rabanales, University of Co´ rdoba, E-14071 Co´ rdoba, Spain
A novel straightforward membrane-based sensor, which uses attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopy has been developed. The flow cell designed permits the on-line microliquid-liquid extraction of the target analyte into a organic solvent layer (OSL), which was deposited on the ATR surface using a sequential injection manifold. The aqueous and organic phases are separated via a commercial hydrophobic membrane placed on the PTFE piece of the cell. The main advantage of the proposed device is that the OSL can be created and regenerated in a continuous manner using the automatic manifold without opening the cell. The analytes are enriched into the OSL after diffusion through the membrane, which excludes the typical absorption bands of water. In addition, the behavior of different organic solvents was evaluated in order to increase the applicability and versatility of the proposed system. Finally, the analytical performance of the design was established for the detection and quantitation of Triton X100 in water. Attenuated total reflection (ATR)1,2 is a useful technique for measuring in the infrared (IR) region, due to its versatility and simplicity. In this technique, an IR beam is focused into a high refractive index medium (Ge, ZnS, diamond) in which multiple reflections happen. In the ATR surface, an electromagnetic disturbance occurs, called an evanescent wave, which is the fundamental of the measurement. ATR-FT-IR has been used in different application fields. For example, in food analysis,3 some methods for food quality control4,5 based on this technique have been developed. It is also remarkable for its applicability for industrial fermentation process control.6,7 In some applications, a chemometrics data treatment is essential, owing to the large amount of information provided by IR spectroscopy. * Corresponding author: (tel/fax) +34-957-218-616; (e-mail)
[email protected]. (1) Goormaghtigh, E.; Raussens, V.; Ruysschaert, J. M. Biochim. Biophys. Acta 1999, 1422, 105. (2) Hind, A. R.; Bhargava, S. K.; McKinnon, A. Adv. Colloid Interface Sci. 2001, 93, 91. (3) Wilson, R. H.; Tappl, H. S. Trends Anal. Chem. 1999, 18, 85. (4) Che Man, Y. B.; Syahariza, Z. A.; Mirghani, M. E. S.; Jinap, S.; Bakar, J. Food Chem. 2005, 90, 815. (5) Ozen, B. F.; Mauer, L. J. J. Agric. Food Chem. 2002, 50, 3898. (6) Kansiz, M.; Gapes, J. R.; McNaughton, D.; Lendl, B.; Schuster, K. C. Anal. Chim. Acta 2001, 438, 175. (7) Mazarevica, G.; Diewok, J.; Baena, J. R.; Rosenberg, E.; Lendl, B. Appl. Spectrosc. 2004, 58, 804.
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Due to its nature, the ATR has a potential applicability in membrane process studies, mainly for membrane characterization8 but also for the study of diffusion processes. Several studies of drug diffusion across epithelial or synthetic membranes have been developed,9,10 being useful for topical applied drugs. The behavior of the membrane ATR-FT-IR combination has also been proposed for in-batch study of the permselective ion extraction process at the surface of an ionophore incorporated solvent polymeric membrane.11 Recently, different sensors based on ATR measurements have been developed.12 The ATR element can be chemically modified to allow specific interactions13 or be coated either with a hydrophobic polymeric membrane14,15 or a thin silica sol-gel film for analyte retention.16 In some cases, a mid-IR transparent fiber optic can be used to develop the so-called fiber-optic evanescent wave sensing technology.17 The membrane protects the ATR from the presence of water, which is an interference in multibounce methods although this is not so important for single bounce ones,18,19 and at the same time permits analyte preconcentration following solid-phase extraction principles. Some sensors, based on this foundation, have been developed to determine analytes such as aromatic compounds,20 pesticides,21 or chlorinated hydrocarbons.22 The main problem with these sensors is that they require a long regeneration time. (8) Kyotani, T.; Sato, K.; Mizuno, T.; Kakui, S.; Aizawa, M.; Saito, J.; Ikeda, S.; Ichikawa, S.; Nakane, T. Anal. Sci. 2005, 21, 321. (9) Hartmann, M.; Hanh, B. D.; Podhaisky, H.; Wensch, J.; Bodzenta, J.; Wartewig, S.; Neubert, R. H. H. Analyst 2004, 129, 902. (10) Tantishaiyakul, V.; Phadoongsombut, N.; Wongpuwarak, W.; Thungtiwachgul, J.; Faroongsarng, D.; Wiwattanawongsa, K. Rojanasakul, Y. Int. J. Pharm. 2004, 283, 111. (11) Umezawa, K.; Lin, X. M.; Nishizawa, S.; Sugarawa, M.; Umezawa, Y. Anal. Chim. Acta 1993, 282, 247. (12) Vigano, C.; Ruysschaert, J. M.; Goormaghtigh, E. Talanta 2005, 65, 1132. (13) Se´vin-Landais, A.; Rigler, P.; Tzartos, S.; Hucho, F.; Hovius, R.; Vogel, H. Biophys. Chem. 2000, 85, 141. (14) Murphy, B.; Kirwan, P.; McLoughlin, P. Anal. Bional. Chem. 2003, 377, 195. (15) Phillips, C.; Jakusch, M.; Steiner, H.; Mizaikoff, B.; Fedorov, A. G. Anal. Chem. 2003, 75, 1106. (16) Rivera, D.; Poston, P. E.; Uibel, R. H.; Harris, J. M. Anal. Chem. 2000, 72, 1543. (17) Mizaikoff, B. Anal. Chem. 2003, 75, 258A. (18) Patterson, B. M.; Danielson, N. D.; Sommer, A. J. Anal. Chem. 2003, 75, 1418. (19) Patterson, B. M.; Danielson, N. D.; Sommer, A. J. Anal. Chem. 2004, 76, 3826. (20) Karlowatz, M.; Kraft, M.; Mizaikoff, B. Anal. Chem. 2004, 76, 2643. (21) Regan, F.; Meaney, M.; Vos, J. G.; MacCraith, B. D.; Walsh, J. E. Anal. Chim. Acta 1996, 334, 85. 10.1021/ac0507643 CCC: $30.25
© 2005 American Chemical Society Published on Web 10/11/2005
Continuous flow systems constitute a versatile, inexpensive, well-established alternative for automation of the preliminary operation of the analytical process. Flow injection analysis and, more recently, sequential injection analysis (SIA) manifolds have been coupled to a wide variety of detectors (optical, electrochemical, piezoelectric). However, FT-IR spectroscopic detection has been scarcely used as it has been recently pointed out23 because of the likely result of the potential interference of common solvent (viz. water) and the poor sensitivity of the technique. Moreover, there are only few references dealing with the on-line coupling of a SIA manifold to a FT-IR spectrometer using a transmission flow cell. Recently Kansiz et al. proposed the use of a SIA-ATR-FT-IR configuration for on-line bioprocess monitoring.6 In this work, a fully automated alternative to these sensors is proposed based on the on-line coupling of a SIA system and an ATR-FT-IR spectrometer. A commercial hydrophobic PTFE membrane (1-µm pore size) is placed in the ATR cell, being the gap between the membrane and the internal reflection element (IRE) filled with an organic solvent, being the organic solvent layer (OSL) monitored against the time in a continuous way. Analyte can diffuse from the aqueous sample through the membrane to the OSL providing the analytical signal according to its characteristic absorption bands. The OSL is renewable using the proposed SIA system, with no need to open the cell. The presence of the membrane guarantees the OSL stability and increases the selectivity of the measurement process. The features of the system have been studied using Triton X-100 as model analyte. EXPERIMENTAL SECTION Reagents. All reagents were of analytical grade or better. n-Hexane, n-heptane, carbon disulfide, and ethyl acetate were supplied by Scharlau (Barcelona, Spain). Carbon tetrachloride (Acros Organics, Geel, Belgium), 1,1,2trichlorotrifluoroethane (Sigma Aldrich, Madrid, Spain), chloroform, and dichloromethane (Merck, Darmstad, Germany) were also employed. Fluoropore membranes (1-µm pore size, 175-µm thickness, and 25-mm diameter) were purchased from Millipore (Madrid, Spain). These hydrophobic PTFE-based membranes, bonded to a highdensity polyethylene support, provide broad chemical compatibility. A stock standard solution of Triton X-100 (10 g/L) was prepared by dissolving the appropriate amount of this compound in Milli-Q water (Millipore Corp., Madrid, Spain). Working solutions were prepared on a daily basis by rigorous dilution of the stock in Milli-Q water. Safety Considerations. The organic solvents used in this work are relatively volatile and slightly toxic if exposed to lungs and skin, and they should be handled using protective gloves and face mask. The wastes of the automated system (aqueous and organic) were collected in glass bottles, and polychlorinated organic solvents (specially carbon tetrachloride) were recovered by distillation and reused to minimize the cost derived from the use of the proposed configurations in terms of reagent and proper waste management and to reduce environmental damage. Apparatus. The automated configuration used in this work is schematically shown in Figure 1. It consists of a SIA manifold (22) Jakusch, M.; Mizaikoff, B.; Kellner, R.; Katzir, A. Sens. Actuators, B 1997, 38-39, 83. (23) Galignani, M.; Brunetto, M. R. Talanta 2004, 64, 1127.
Figure 1. Schematic diagram of the SIA configuration on-line coupled to the ATR-FT-IR spectrometer. W, waste.
on-line connected to a ATR-FT-IR spectrometer through an inhome-built flow-through accessory (obtained from the working group on Chemical Analysis and Vibrational Spectroscopy, Vienna University of Technology, Vienna, Austria), which enables pumping of the solutions over the ATR diamond surface with minimal dead volume (∼3 µL). The SIA configuration comprises a Cavro XP 3000 syringe pump (Sunnyvale, CA) equipped with a 1-mL syringe, a Cavro six-port selection valve equipped with a microactuator, and a 4-mL holding coil. PTFE tubing of 0.5-mm i.d. and standard connectors are also employed. The setup is computer controlled by a Sagittarius 3.0 software package (obtained from the working group on Chemical Analysis and Vibrational Spectroscopy, Vienna University of Technology, Vienna, Austria). This software also allows triggering the FT-IR spectrometer for exact timing of the collection of spectra. The selection valve was connected via 15-cm-long PTFE tubing to a Bruker Tensor37 FT-IR spectrometer, equipped with a diamond ATR cell with a circular surface of 3-mm diameter and three internal reflections. A liquid nitrogen-cooled mercurycadmium-telluride detector was used for spectra acquisition. Spectra are collected between 4000 and 700 cm-1 at a 4-cm-1 resolution with 128 coadded scans each. The main spectral information interval varies depending on the proposed application. For Triton X-100 monitoring, two regions were selected, 18001000 and 3000-2800 cm-1. Data collection and processing was made using OPUS software (Bruker, Ettligen, Germany). A spectrum of the dry system was used as background. Flow Cell Description. The on-line connection of the SIA manifold to the ATR unit of the FT-IR spectrometer was done by using a custom-built PTFE accessory, which is depicted in Figure 2. The inlet and outlet are connected via a microchannel (1.5 cm long) made in the lower part of the piece, which is in contact with the ATR surface, and where the commercial hydrophobic membrane was deposited. This accessory is then screwed on a stainless steel piece (which is not depicted in Figure 2 for simplicity) and thus tightly sealed to avoid any leakage. This unit acts as a phase separator as only organic solvents, immiscible with water, can pass through the membrane, reach the evanescent field, and settle onto the ATR surface generating a stable OSL. The thickness of the organic layer beneath the membrane can be estimated in ∼25 µm, which resulted in a sensing volume of ∼2 µL. Taking into account the reduced volume of the organic phase Analytical Chemistry, Vol. 77, No. 22, November 15, 2005
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Figure 2. Scheme of the SIA diffusion cell used for membrane location and organic solvent layer formation over the IRE of the ATR module.
situated in the diffusion cell, high preconcentration factors can be achieved for the analytes that can diffuse through the membrane from the aqueous to the organic phase, which dramatically increases the sensitivity of this technique. The continuous nature of the ATR diffusion cell developed offers the inherent advantages of the automated systems as all the operations involved in the whole process, including regeneration of the OSL, can be done without opening the cell. In addition, as the system is computer controlled, it can work unattended. Experimental Procedure. The proposed configuration requires the following sequential steps: background acquisition, organic solvent layer situation, analyte monitoring, and system cleaning/OSL regeneration. Background was acquired at the beginning of the working session after the system was dried with air. Next, a variable volume of organic solvent (between 2 and 5 mL) was passed through the continuous diffusion cell at the optimum flow rate (which also depends on the organic solvent used) to generate the OSL onto the ATR surface. For analyte monitoring, a volume of 20 mL of aqueous solution containing the target analyte was passed through the cell, the analyte diffused (from the aqueous phase to the organic medium) through the hydrophobic membrane, and the spectrum recorded under the above-described instrumental conditions. The flow was halted for ∼2 min to complete the measurement cycle. In the final step, the OSL was regenerated by flushing a fresh volume of the organic solvent at the optimum flow rate and time values. The whole process is carried out without opening the cell, and every cycle took ∼30 min to be completed for 20 mL of sample. RESULTS AND DISCUSSION IR determinations present problems when measuring in strongly IR-absorbing matrixes such as water. This problem can be overcome by using hydrophobic polymeric membranes coated on the IRE of the ATR. These membranes avoid the water interference and enhance the sensitivity of the determinations of hydrophobic analytes, preconcentrating them following the principles of solid-phase extraction. In some cases, these so-called 7474
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sensors require a long equilibration regeneration time, which is the main shortcoming of the configurations proposed in the literature together with the chemical modification of the analytical instrument. The proposed configuration avoids the water interference in the infrared spectra without the limitation inherent to the chemical modification of the IRE. By way of example, Figure 3A shows the ATR surface monitoring, when plugs of 20 µL of water and an organic solvent (carbon tetrachloride) are pumped in a sequential way through the flow system at a flow rate of 1 mL/min for 30 min. In this case, the typical water IR bands (3500 and 1640 cm-1) can be observed. As can be seen (Figure 3B), when the PTFE membrane is used, the carbon tetrachloride spectrum is the record without water interference. A similar behavior is expected for other organic solvents. Solvent selection depends on the characteristic absorption bands of the target analyte (viz. for mineral oil determination, a solvent without bands at 3000 cm-1 is required). Solvent Layer Formation. The organic solvent layer is located between the ATR surface and the PTFE membrane using the above-described SIA manifold. Solvent polarity plays a key role in this process as the passage through the membrane is favored for those solvents of lower polarity index. The flow rate is a critical variable for OSL formation, as it provides the pressure needed, and it is closely related to the polarity of the organic solvents. Therefore, it was systematically studied for different organic solvents, immiscible with water, covering a wide polarity interval (see Table 1). In all cases, discrete volumes of 1 mL of the organic solvents were aspirated sequentially into the SIA manifold and driven to the detector at increasing flow rates (between 0.2 and 2.5 mL/min). After each milliliter, the IR absorption spectrum was recorded on the ATR surface. For the seven organic solvents assayed (n-heptane, n-hexane, carbon tetrachloride, chloroform, dichloromethane, ethyl acetate, 1,1,2-trichlorotrifluoroethane), the monitored signal increases with increasing flow rate, reaching a steady state at an average flow rate of 2.5 mL/min. A double-
Figure 3. Variation of IR absorption with time after sequential injection of water and carbon tetrachloride plugs without (A) and with (B) hydrophobic membrane within the ATR diffusion cell over a period of 30 min. For details, see text. Table 1. Influence of the Flow Rate in the Organic Solvent Layer Formation
solvent
polarity index
viscosity at 20 °C (cP)
critical flow ratea (mL/min)
n-heptane n- hexane 1,1,2-trichlorotrifluoroethane carbon tetrachloride dichloromethane chloroform ethyl acetate
0 0 0 1.6 3.1 4.1 4.4
0.42 0.31 0.71 0.61 0.44 0.57 0.45
1.56 1.41 1.28 1.22 1.98 2.45 2.96
a
Minimum flow rate required for organic solvent layer formation.
Table 2. Volume of Aqueous Solution That Can Be Tolerated by Different Organic Solvent Layers
solvent n-heptane n- hexane 1,1,2trichlorotrifluoroethane carbon tetrachloride carbon disulfide chloroform dichloromethane ethyl acetate
density (kg/L)
water solubility (% w/w)
organic solvent layer stabilitya
0.68 0.67 1.58
0.0003 0.001 0.02
>20 (0.8) >20 (1.0) >20 (0.6)
1.59 1.26 1.49 1.32 0.90
0.08 0.2 0.8 1.8 8.7
>20 (0.8) >20 (1.0) 10 (0.6) 3 (0.8) 1 (0.8)
a Volume of aqueous solution (mL) at the optimum sample flow rate (mL/min).
logarithmic plot of signal against flow rate permits us to calculate the critical flow rate for each organic solvent (inflection point of the curve) considered as the flow rate above which pressure prevails over diffusion through the membrane. The critical values are listed in Table 1 and correspond well with the polarity index as the lower the polarity, the lower the critical flow rate. Solvent Layer Stability Studies. The stability of the solvent layer against the aqueous stream is a critical feature of the proposed device as it markedly affects the robustness of the analytical application. The experiments were carried out using different organic solvents with adequate properties for future applications. Immiscibility with water and solvent density appeared to be crucial properties to ensure OSL stability. Therefore, waterimmiscible organic solvents with densities higher than water are expected to provide the best results. To determine the maximum volumes of aqueous sample that is tolerated, for each organic solvent, the OSL was generated as previously described, then a water stream (20 mL) was passed through the ATR diffusion cell at variable flow rate (between 0.2 an 2.5 mL/min), and the IR absorption spectrum was recorded after each milliliter. Table 2 summarized the maximum water volume that can be passed through the system without altering the OSL and the optimum flow rate. As can be seen, the organic solvents showing lower water solubility tolerated the highest volume of water. By way of example Figure 4 shows the absorption spectra of the carbon tetrachloride layer within the whole interval
and in the maximum absorption region (850-650 cm-1). The spectra were recorded after each milliliter of water. As can be seen, no significative difference in the absorbance was obtained after passing 20 mL of water, which clearly testifies to the stability of the OSL with the aqueous stream. After each run, the continuous regeneration of the OSL was accomplished by passing 4 mL of fresh organic solvent through the system at the critical flow rate. Therefore, the system is ready for new measurement. Analyte Diffusion Characterization. Once the instrumental parameters were optimized, the variables affecting the difussion of the analyte through the membrane were studied. For this purpose, Triton X-100 was selected as model analyte. Its amphiphilic character permits an interaction with both organic and aqueous phases, and its surface properties can influence the diffusion through the membrane also transporting molecules of water than can interfer in the absorption spectrum recorded. It also provided an estimation of the capability of the proposed configuration to determine the presence of nonionic surfactant in environmental water samples. Taking into account the IR absorption spectrum of the analyte, with absorption bands in the 30002800- and 1600-1000-cm-1 regions, carbon tetrachloride was selected to form the OSL on the ATR surface. Analytical Chemistry, Vol. 77, No. 22, November 15, 2005
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Figure 4. ATR-FT-IR absorption spectra (n ) 20) of the carbon tetrachloride layer generated on the ATR surface. Each spectrum was recorded after each milliliter of water. For details, see text.
The optimization of the chemical and hydrodynamic variables was carried out using an aqueous standard solution of the model analyte at a concentration of 100 µg/mL and the configuration depicted in Figure 1. The flow rate of the aqueous stream was studied within the interval 0.2-5.0 mL/min. In all cases, 20 mL of sample was pumped through the system and the IR absorption spectrum (128 coadded scans) was recorded after each milliliter. To study the influence of this variable on the analytical signal, the area of the band between 3000 and 2800 cm-1 was calculated for the 20-mL sample. The area increased when the flow rate increased, reaching a steady state (maximum analyte diffusion) within the interval 0.8-1.0 mL/min and then decreasing after this value. This maximum diffusion is a compromise between the pressure of the aqueous flow and the analyte-membrane interaction time. Longer contact times were achieved at lower flow rate while pressure prevailed at higher values. To decrease the aqueous solubility of the analyte in the sample and shift the partition ratio in favor of the organic solvent, the effect of ionic strength was studied by adding variable ammounts (0-20 g/L) of Na2SO4. It was observed that saturation of the sample was necessary to help the kinetic transfer of small amounts of Triton X-100 through the membrane, increasing the sensitivity and robustness of the method. Thus, 20 g/L was selected for further experiments. Figure 5 shows the IR absorption spectra of Triton X-100 obtained using the proposed configuration and passing 12 mL of a standard solution of the analyte. The spectra were recorder after each milliliter of sample. As can be seen in the zoom (1400-900 cm-1), the analytical signal increases with the sample volume as the likely result of the increasing amount of Triton X-100 passing 7476 Analytical Chemistry, Vol. 77, No. 22, November 15, 2005
through the membrane and being then preconcentrated on the OSL. To evaluate the potential application of the optimized methodology, the analytical figures of merit were established by using the proposed configuration working under the optimum chemical and flow conditions. The calibration graph for Triton X-100 was constructed by using 30 standard solutions analyzed by triplicate. Data evaluation was performed by peak area integration in the 3021-2810- and 1207-997-cm-1 regions, and the integrated areas were plotted versus the amount of analyte (mg) resulting in linear fit functions as indicated in Table 3. The precision of the method (repeatability), expressed as relative standard deviation was checked using 11 individual standards within the lower part of the linear interval. CONCLUSIONS In the proposed configuration, a commercial PTFE membrane is used to achieve the separation between the monitored organic phase and the aqueous one. This membrane, which is not in contact with the IRE, permits analyte transference between the two phases, enhancing the selectivity of the determinations. In addition, the favorable organic/aqueous volume ratio allows analyte preconcentration, increasing the sensitivity of the instrumental technique. The analyte should have affinity with the organic solvents and favorable diffusion coefficient through the membrane. On the other hand, the organic solvent has to be chosen, for each application, taking into account three main factors, namely: chemical affinity with the analyte; spectral compatibility to avoid overlapping with the characteristic absorption bands of the target analyte; and the OSL stability. The system
Figure 5. ATR-FT-IR absorption spectra (n ) 12) of Triton X-100 diffusion on a carbon tetrachloride OSL. Each spectrum was recorded after each milliliter of aqueous sample.
Table 3. Analytical Figures of Merit of the Automated System Developed Using Triton X-100 as Model Analyte IR region (cm-1)
regression equationa
R
linear range (mg)
RSD (%)
3021-2810 1207-997
Y ) 26.22X - 0.06 Y ) 10.63X - 0.05
0.999 0.999
0.6-20 0.6-20
3.0 3.2
a
Y, peak area, X, concentration (mg).
is easy and automatically regenerated using a few milliliters of the fresh organic solvent. The optimized system can also be used as phase separator in liquid-liquid and solid-phase extractions to avoid the interference of the potential microliter of water remaining in the system. In addition, by selecting the appropriate membrane, diffusion studies can also be implemented using this simple and reproducible setup.
It is also a powerful alternative to existing available portable instrumentation for environmental monitoring. Current investigation is focused on the direct introduction of raw aqueous samples into a simple SIA manifold for the multiparametric analysis (either qualitative or quantitative). Moreover, the sensitivity of the proposed sensor is also a clear objective in order to reach the values established by legislation for the determination of pollutants and toxic compounds in different application fields such as food analysis, industrial fermentation, and environmental monitoring. ACKNOWLEDGMENT This work was supported by Grant CTQ2004-01220 of the DGI of the Spanish Ministry of Science and Technology. Received for review May 4, 2005. Accepted September 12, 2005. AC0507643
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