Anal. Chem. 1996, 68, 2559-2563
Sample Preparation Using a Miniaturized Supported Liquid Membrane Device Connected On-Line to Packed Capillary Liquid Chromatography Eddie Thordarson, Sveinbjo 1 rg Pa´lmarsdo´ttir, Lennart Mathiasson,* and Jan A ° ke Jo 1 nsson
Department of Analytical Chemistry, University of Lund, P.O. Box 124, 221 00 Lund, Sweden
A miniaturized supported liquid membrane device has been developed for sample preparation and connected online to a packed capillary liquid chromatograph. The device consists of hydrophobic polypropylene hollow fiber, inserted and fastened in a cylindrical channel in a Kel-F piece. The pores of the fiber are filled with an organic solvent, in this study 6-undecanone, thus forming a liquid membrane. The sample is pumped on the outside of the hollow fiber (donor), and the analytes are selectively enriched and trapped in the fiber lumen (acceptor). With this approach, the volume of the acceptor solution can be kept as low as 1-2 µL. This stagnant acceptor solution is then transferred through capillaries attached to the fiber ends to the LC system. The system was tested with a secondary amine (bambuterol), as a model substance in aqueous standard solutions as well as in plasma. The best extraction efficiency in aqueous solution, with an acceptor volume of 1.9 µL, was 32.5% at a donor flow rate of 2.5 µL/min. At flow rates above 20 µL/min, the concentration enrichment per time unit was approximately constant, at 0.9 times/min, i.e., 9 times enrichment in about 10 min. The overall repeatability (RSD) for spiked plasma samples was ∼4% (n ) 12). Linear calibration curves of peak area versus bambuterol concentration were obtained for both aqueous standard solutions and spiked plasma samples. The detection limit for bambuterol in plasma, after 10 min of extraction at a flow rate of 24 µL/min, was 80 nM. Liquid membranes, comprising an organic liquid in contact with two separated aqueous phases, have many technical applications.1,2 One of the configurations used is the supported liquid membrane (SLM), where the organic liquid is entrapped in a porous membrane, separating the two aqueous phases. This configuration can be used in a flow system, connected to analytical instruments for various sample handling operations.3,4 The technique is useful for enrichment of ionizable and charged species and gives an efficient sample cleanup of complex matrices. (1) Noble, R. D., Way, J. D., Eds. Liquid Membranes. Theory and Applications; ACS Symposium Series 347; American Chemical Society: Washington, DC, 1987. (2) Araki, T., Tsukube, H., Eds. Liquid Membranes, Chemical Applications; CRC Press: Boca Raton, FL, 1990. (3) Audunsson, G. Anal. Chem. 1986, 58, 2714-2723. (4) Jo¨nsson, J. Å.; Mathiasson, L. Trends Anal. Chem. 1992, 11, 106-114. S0003-2700(95)00929-2 CCC: $12.00
© 1996 American Chemical Society
As such, it is an interesting alternative to established techniques for the extraction of analytes from biomedical samples.5 The SLM device is mounted in a flow system, where the sample is pumped through the donor channel and sample components are trapped in the stagnant acceptor solution. The selectivity of the method is tuned for analytes of interest by choosing a proper composition of the three phases. The conditions in the sample at the donor side are chosen so that the analytes, as uncharged species, can diffuse across the membrane. Normally, the trapping process involves ionization of an ionizable analyte by proper choice of pH. Macromolecules will not be enriched, nor will constantly charged species unless special operations are undertaken, e.g., ion pairing. The basic theory has been described in a number of papers,3,4,6 while others describe applications of the technique, such as trace enrichment of herbicides in natural waters as recently reviewed,7 carboxylic acids in soil liquids,8 amines and basic drugs in urine9 and blood plasma,10,11 and metals in water.12 The SLM technique has been used connected on-line to GC,9,10 and to LC.7,11,13 There are a number of important reasons for miniaturizing the SLM technique. One of these reasons is related to the maximal concentration enrichment obtainable in sample workup. This is limited to the volume ratio between the original sample and the extract. It is thus important that the volume of the enriched sample is as small as possible when the access of sample is limited, as for plasma samples. Another reason is to achieve low detection limits in systems with low sample volume capacity by injecting the total extracted volume after enrichment. Examples of systems with low sample capacity are packed capillary LC and capillary electrophoresis (CE). The combination of a miniaturized SLM and either of these two techniques will take full advantage of a high concentration enrichment of the analytes during the SLM (5) Metha, A. C. Talanta 1986, 33, 67-73. (6) Jo ¨nsson, J. Å.; Lo ¨vkvist, P.; Audunsson, G.; Nilve´, G. Anal. Chim. Acta 1993, 227, 9-24. (7) Knutsson, M.; Nilve´, G.; Mathiasson, L.; Jo ¨nsson, J. Å. J. Chromatogr. A, in press. (8) Shen, Y.; Obuseng, V.; Gro ¨nberg, L.; Jo ¨nsson, J. Å. J. Chromatogr. A 1996, 725, 189-198. (9) Audunsson, G. Anal. Chem. 1988, 60, 1340-1347. (10) Lindegård, B.; Jo¨nsson, J. Å.; Mathiasson, L. J. Chromatogr. 1992, 573, 191-200. (11) Lindegård, B.; Bjo ¨rk, H.; Jo ¨nsson, J. Å.; Mathiasson, L.; Olsson, A. M. Anal. Chem. 1994, 66, 4490-4497. (12) Papantoni, M.; Djane, N.-K.; Ndung’u, K.; Jo ¨nsson, J. Å.; Mathiasson, L. Analyst 1995, 120, 1471-1477. (13) Jo ¨nsson, J. Å.; Mathiasson, L.; Lindegård, B.; Trocewicz, J.; Olsson, A. M. J. Chromatogr. 1994, 665, 259-268.
Analytical Chemistry, Vol. 68, No. 15, August 1, 1996 2559
Figure 1. Membrane extraction unit. A, hollow fiber (reaching through a hole drilled through the whole block); B, fused silica capillaries inserted in the ends of the fiber; C, O-rings for fixing the fiber and capillaries; D, connectors for donor channel.
process and a high signal-to-noise ratio in the final detection system due the narrow peaks obtained in both packed capillary LC and CE. Furthermore, the consumption of organic solvent is kept at an absolute minimum in the pretreatment step as well as in the HPLC analysis. We have previously shown that it is possible to construct supported liquid membrane devices based on flat, porous membranes, yielding acceptable extraction efficiencies and enriched sample volumes down to 10-15 µL. These devices were connected on-line to LC with 4.6 mm columns11 and off-line to CE,14 but they are too large for on-line connection to packed capillary LC or CE. To further decrease the volume while preserving the enrichment efficiency requires shallower channels. This may lead to increased pressure drop and possible clogging problems, especially when dealing with biological samples. The use of porous hollow fibers (i.e., bundles of many fibers) as the support for a liquid membrane is common in technical applications,1,2,15 where high throughput is essential. Here we outline the possibilities of decreasing the enriched sample volume by utilizing a single porous hollow fiber. The cylindrical geometry gives a high surface area-to-internal volume ratio and low pressure drops, in principle allowing for acceptable extraction efficiency with a comparatively low acceptor volume. EXPERIMENTAL SECTION Hollow Fiber Membrane Device. Two types of hollow fibers were used interchangeably, depending on the sample matrix. When enriching aqueous samples, the hollow fiber used was a Celgard X-20 (Hoechst Celanese, Charlotte, NC), made of polypropylene with an inner diameter of 400 µm and a wall thickness of 30 µm. It had an average pore size of 0.03 µm and an overall porosity of 40%. The fiber used for plasma analyses was Plasmaphan (Akzo Nobel, Wuppertal, Germany), also made of polypropylene. It had an inner diameter of 330 µm, a wall thickness of 170 µm, and an average pore size of 0.2 µm. The hollow fiber membrane module is shown in Figure 1. It was manufactured from Kel-F and made in one piece with a 15 mm long cylindrical channel at the center. For use with the Celgard X-20 fiber, the channel was 0.50 mm in diameter. Thus, an annular volume of ∼0.45 µL was created when the fiber was (14) Pa´lmarsdo´ttir, S.; Lindegård, B.; Deininger, P.; Edholm, L.-E.; Mathiasson, L.; Jo ¨nsson, J. Å. J. Cap. Electrophor. 1995, 2, 185-189. (15) Garea, A.; Urtiaga, A. M.; Ortiz, M. I.; Alonso, A. I.; Irabien, J. A. Chem. Eng. Commun. 1993, 120, 85-97.
2560 Analytical Chemistry, Vol. 68, No. 15, August 1, 1996
Figure 2. Structural formulas of bambuterol and its metabolite terbutaline.
mounted in the channel. This annular volume served as the donor channel, while the the inner volume in the fiber lumen was used as acceptor with a volume of approximately 1.9 µL. When the Plasmaphan fiber was used, the center channel was made 0.7 mm in diameter. This fiber had an inner (acceptor) volume of ∼1.3 µL, and the annular donor volume was ∼0.50 µL. For mounting of the fiber, two identical holes, 2.4 mm in diameter, were drilled at both ends of the channel, and two O-rings (2.5 mm o.d.) were fixed in position. The fiber (A), with a 350 µm o.d. fused silica capillary (B) and an O-ring (C) in each end, was inserted into the channel. Upon tightening, a large frictional force between the O-rings prevents them from rotating, and their inner diameters are simultaneously reduced, leaving the arrangement stable. Fixing the inner O-rings in position also minimizes their possible movement along the capillary axis, which reduces the risk of the fiber being crumpled. The fiber was impregnated in situ by introducing 6-undecanone into both the donor and acceptor channels and keeping it stagnant for 30 min. Before beginning the first enrichment, the fiber was washed with water for 30 min to remove excess solvent. After the membrane has been used for a number of extractions, the liquid is partly lost from the support, leading to decreased extraction efficiency. The regeneration of such an exhausted membrane was conducted in situ with the following procedure: The fiber was washed with ethanol (4 + 4 mL) on the donor and acceptor side to remove the aqueous buffer and then dried by an air-flow on both sides for 10 min and finally impregnated with organic solvent and washed as above. Packed Capillary LC. The LC system consisted of a HPLC pump (Model 2150, LKB, Bromma, Sweden), a splitter device (Acurate, LC Packings, Zu¨rich, Switzerland), a six-port external volume injection valve (Valco Vici, Valco Instruments Co. Inc., Houston, TX), a C18 packed capillary column either with 3 µm Hypersil BDS particles, 180 µm i.d. and 10 cm long (column A), or 5 µm Zorbax SB particles, 100 µm i.d. and 15 cm long (column B) (LC Packings, Zu¨rich, Switzerland), a variable wavelength detector (Linear Uvis 200, Linear Instruments, Reno, NV), and a recorder (BD 41, Kipp & Zonen, Delft, Holland). Chemicals. Bambuterol hydrochloride was obtained from Astra Draco AB (Lund, Sweden). Bambuterol itself has no pharmacological effect but is slowly metabolized to terbutaline, which is a long-acting bronchodilator used in the treatment of asthma (Bricanyl). The structures of bambuterol and terbutaline are shown in Figure 2. 6-Undecanone was obtained from Janssen Chimica (Geel, Belgium), triethylamine (TEA) from Fluka Chemie
Figure 3. Enrichment and separation equipment setup. For details, refer to the text.
(Buchs, Switzerland), physostigmine from Sigma (St. Louis, MO) and other chemicals from Merck (Darmstadt, Germany). All chemicals beside 6-undecanone (97%) were of analytical grade or better. All water used was purified with a Milli-Q-RO 4 system (Millipore, Bedford, MA). Plasma. Blood plasma is a very complex matrix consisting of numerous organic and inorganic substances. The main constituents in plasma (besides water) are proteins such as globulins, albumins, and fibrinogens. For a complete listing of plasma constituents and their concentrations, see ref 16. Plasma was obtained from Astra Draco AB (Lund, Sweden). The plasma samples were centrifuged, spiked with bambuterol, and diluted to twice their initial volume with buffer solution (pH 11). This serves for converting the analyte into an extractable form by neutralization, and it decreases sample viscosity and subsequently diminishes problems associated with the latter. Furthermore, 10 µM physostigmine was added as an inhibitor to each sample to suppress the enzymatically catalyzed biodegradation of bambuterol in plasma. After the plasma samples are spiked, they should be analyzed on the same day because the inhibitor itself, when added in these small amounts, is degraded fully within 3 days. The values of bambuterol concentration in plasma samples are reported as before dilution. Experimental Setup of the SLM Packed Capillary LC System. Figure 3 shows the system schematically. The sample, mixed with 0.3 mM phosphate buffer (pH 11), was pumped with a peristaltic pump (A) (Minipuls 3, Gilson Medical Electronics, Villiers-le-Bel, France) and introduced into the membrane device (B). The donor flow rate was usually kept between 10 and 20 µL/min, and the acceptor solution used was 1 mM H3PO4 (pH 3). After enrichment, the donor side was washed with 10 volumes of water by manually connecting the washing line (D) to the inlet of the membrane device at point C. Simultaneously, the microinjection pump (E) (CMA/100, CMA/Microdialysis AB, Stockholm, Sweden) was started (flow rate 10 µL/min), and the content of the hollow fiber (the acceptor) was transported into the loop (F). A low-pressure filter from a microconcentrator (Centricon, Danvers, MA) was punched and inserted between the column (G) and the injector to prevent plasma from entering the column if the hollow fiber was accidentally damaged. After the loop was filled, 10 volumes of 1 mM H3PO4 was passed through the fiber for washing before starting a new extraction. By removal of ∼0.5 cm of the capillary coating (done with a flame), a detection “window” was created for on-capillary detection (H). (16) Vander, A. J.; Sherman, J. H.; Luciano, D. S. Human physiology: the mechanisms of body function, 5th ed.; McGraw-Hill: New York, 1990; p 352.
Figure 4. Dependency of the extraction efficiency (E, 9) and the concentration enrichment per time unit (Et, [) on donor flow rate. Concentration of bambuterol in water solution was 250 nM, and the enrichment time was 10 min for each run.
The splitter unit (I), connected to the high-pressure pump (J), gives a split ratio of 1:70 when connected with original connections and before the column is installed. As the column acts as a restrictor itself, it increases the split ratio. A set flow rate of 300 µL/min resulted in a flow of approximately 2 µL/min through the column A (i.e., a split ratio of 1:150) and 0.8 µL/min through the narrower column B (split ratio 1:375). The mobile phase was, in most experiments, 45% methanol in water with 0.1 mM TEA and 1 mM H3PO4. TEA was used to deactivate free silanol groups in the column packing. RESULTS AND DISCUSSION Studying the performance of a supported liquid membrane system, the extraction efficiency (E) and the concentration enrichment per time unit (Et) are important parameters to consider. With a stagnant acceptor phase, these values can be calculated as E ) CAVA/CDVD and Et ) tCA/CD, where CA and CD are analyte concentrations in acceptor and donor, respectively. VA is the acceptor volume, and VD is the volume of sample having passed the donor channel during the time t. These relations are demonstrated in Figure 4 for the extraction of bambuterol. The curves in Figure 4 show that the enrichment process is essentially performed under so-called membrane-controlled conditions.4,6 This means that the extraction is governed mainly by the diffusion rate through the membrane. Under such conditions, Et tends to level out at relatively low flow rates, and the gain in E is large only at very low flow rates. This behavior is expected for analytes with relatively low partition coefficients (
