Anal. Chem. 2001, 73, 1382-1386
On-Chip Integration of Neutral Ionophore-Based Ion Pair Extraction Reaction Hideaki Hisamoto,† Takayuki Horiuchi,† Manabu Tokeshi,‡ Akihide Hibara,† and Takehiko Kitamori*,†,‡
Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, and Integrated Chemistry Project, Kanagawa Academy of Science and Technology, 3-2-1 Sakado, Takatsu-ku, Kawasaki, Kanagawa 213-0012, Japan
Integration of a neutral ionophore-based ion pair extraction reaction onto a glass microchip was performed. Since the liquid microspace provides a short molecular diffusion distance and a large specific interfacial area of the liquidliquid interface, novel attractive analytical features arise such as extremely fast ion sensing and ultrasmall reagent solution volume. In contrast to the slow response time of a standard ion optode, in which the response time is basically governed by slow diffusion of ionic species in the viscous polymer membrane, that of the on-chip ionsensing system is clearly faster due to the short molecular diffusion distance and low viscosity of organic solution. In this case, the organic solution containing a neutral ionophore and a lipophilic pH indicator dye and the aqueous solution containing sample ion were independently introduced into microchannel to form an organicaqueous interface. Then determination of the ion was performed by thermal lens microscopy at the downstream of the organic phase under continuous-flow conditions. The response time and minimum required reagent solution volume of the on-chip ion-sensing system are about 8 s and 125 nL, respectively, indicating the advantages of using the liquid microspace. Other advantages of the on-chip ion pair extraction system arising from using the liquid microspace and microfluidic system are also discussed in detail. Recent chemical sensor technology provides widespread applications in analytical chemistry. In particular, numerous investigations on neutral ionophore-based ion-selective electrodes (ISEs) or optodes have demonstrated that these sensors are applicable in various fields such as medicine and environmental science.1,2 Generally, ion sensor technology is based on ion-ionophore complexation chemistry and membrane technology involving immobilization of the ion-sensing components such as an ionophore or a dye molecule. In this case, sensor membranes are utilized because they allow continuous measurements or easy †
The University of Tokyo. Kanagawa Academy of Science and Technology. * Corresponding author. (1) Bakker, E.; Buhlmann, P.; Pretsch, E. Chem. Rev. 1997, 97, 3083-3132. (2) Buhlmann, P.; Pretsch, E.; Bakker, E. Chem. Rev. 1998, 98, 1593-1687 ‡
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disposal after use. However, continuous measurements require good membrane durability and a disposable sensor requires large quantities of expensive ion-sensing components. Furthermore, to apply this technology to the sensing of different ions sequentially, single device-based sensing is basically limited, and a parallel configuration of plural sensor devices is required. Thus, more sophisticated ion-sensing technology is necessary to realize multiion-sensing devices soon. On the other hand, “downsizing” chemical systems onto glass or silicon microchips has become one of the largest active research field in recent years.3,4 Among them, micro total analysis systems (µ-TAS) have attracted much attention for their attractive analytical performance features such as fast response time and small sample volume. Most research papers dealing with µ-TAS have involved electroosmotic pumping of liquids and laser-induced fluorescence (LIF) as a detection technique, both of which were developed and verified for capillary electrophoretic analysis. However, the applicability of electroosmotic pumping is limited to only ionic liquids (and most work has used water), and that of LIF is limited to only detecting fluorescent molecules or using fluorescence labeling of the analyte. In contrast to this, we have focused on the characteristics provided by the liquid microspace, such as short molecular diffusion distance and large specific interfacial area of the liquidliquid interface or solid-liquid interface, to demonstrate highly effective chemical reactions in microchips. We have proposed the integration of general chemistry principles on a glass microchip rather than electrophoretic analysis techniques. Generally, our system is based on pressure-driven liquid manipulation in a glass microchip and photothermal microscopy detection. This system allows (1) introduction of all liquids including water and organic solvents, (2) demonstration of general chemical reactions, and (3) ultrasensitive detection of nonfluorescent general chemical species with high spatial resolution.5-7 We developed the thermal (3) Freemantle, M. Chem. Eng. News 1999, (Feb 22), 27-36. (4) Proceedings of the µTAS′2000 Symposium; van den Berg, A., Olthuis, W., Bergveld, P., Eds., 2000. (5) Sato, K.; Tokeshi, M.; Kitamori, T.; Sawada, T. Anal. Sci. 1999, 15, 641645. (6) Tokeshi, M.; Minagawa, T.; Kitamori, T. Anal. Chem. 2000, 72, 17111714. (7) Sato, K.; Tokeshi, M.; Odake, T.; Kimura, H.; Ooi, T.; Nakao, M.; Kitamori, T. Anal. Chem. 2000, 72, 1144-1147. 10.1021/ac001271v CCC: $20.00
© 2001 American Chemical Society Published on Web 02/10/2001
lens microscope (TLM) and demonstrated that it has a detection limit on the single-molecule level.8-10 By using these techniques, we have demonstrated ultrasensitive detecting of dye molecules,8-10 monitoring of basic chemical reactions such as a chelating reaction under flow-analytical conditions,5 carrying out a highly effective immunoreaction,7 and solvent extraction6,11-13 all in glass microchips. In the present study, we investigated integration of a neutral ionophore-based ion pair extraction reaction in a microchip. Although, the utilized microchip is the simplest Y-shaped microchip, we can investigate widely applicable ion-sensing technology by exploiting characteristics of the liquid microspace and microfluidic system. We used a highly lipophilic pH indicator dye and lipophilic neutral ionophore as the ion-sensing components in an organic phase, and we detected the selective extraction process of analyte ions from the water phase by the TLM. Selective extraction of the analyte cation causes the lipophilic pH indicator dye to be deprotonated to maintain the electroneutrality in the organic phase. Therefore, detection of the proton-releasing process across a large liquid-liquid interface allows highly efficient and selective ion determination. The ion pair extraction scheme is an established methodology employed for ion-selective optodes which provides highly selective optical ion determination of various kinds of ions by using a single lipophilic pH indicator dye and highly selective neutral ionophores.1,2,14,15 The ion pair extraction scheme is already in use for various kinds of optical chemical-sensing devices such as flow analytical devices, sensing plate devices, waveguide devices, and fiber-optic devices.16-21 However, exploitation of an ion pair extraction reaction and microchip technology provide attractive advantages which would not be achieved by conventional ion sensors. The advantages provided by this method are as follows. (1) Reaction time (response time) can be extremely reduced by the fast molecular transport in a microspace. (2) Since the required reagent solution volume is extremely small (∼100 nL), fresh organic phase can be used in every measurement. Subsequently, response degradation caused by the leaching of ion-sensing (8) Tokeshi, M.; Uchida, M.; Uchiyama, K.; Sawada, T.; Kitamori, T J. Lumin. 1999, 83-84, 261-264. (9) Sato, K.; Kawanishi, H.; Tokeshi, M.; Kitamori, T.; Sawada, T. Anal. Sci. 1999, 15, 525-529. (10) Uchida, M.; Tokeshi, M.; Sawada, T.; Kitamori, T. Anal. Chem., submitted. (11) Tokeshi, M.; Minagawa, T.; Kitamori, T. 13th International Symposium on High Performance Capillary Electrophoresis and Related Microscale Techniques (HPCE2000), Book of Abstracts; 2000; p 110. (12) Sato, K.; Tokeshi, M.; Sawada, T.; Kitamori, T. Anal. Sci. 2000, 16, 455456. (13) Tokeshi, M.; Minagawa, T.; Kitamori, T. J. Chromatogr., A 2000, 894, 1923. (14) Suzuki, K.; Ohzora, H.; Tohda, K.; Miyazaki, K.; Watanabe, K.; Inoue, H.; Shirai, T. Anal. Chim. Acta 1990, 237, 155-164. (15) Morf, W. E.; Seiler, K.; Rusterholz, B.; Simon, W. Anal. Chem. 1990, 62, 738-742. (16) Hisamoto, H.; Suzuki, K. Trends Anal. Chem. 1999, 18, 513-524. (17) Hisamoto, H.; Satoh, S.; Satoh, K.; Tsubuku, M.; Siswanta, D.; Shichi, Y.; Koike, Y.; Suzuki, K. Anal. Chim. Acta 1999, 396, 131-141. (18) Hisamoto, H.; Miyashita, N.; Watanabe, K.; Nakagawa, E.; Suzuki, K. Sens. Actuators, B 1995, 29, 378-385. (19) Hisamoto, H.; Sato, S.; Sato, K.; Siswanta, D.; Suzuki, K. Anal. Sci. 1998, 14, 127-131. (20) Hisamoto, H.; Kim, K.-H.; Manabe, Y.; Sasaki, K.; Minamitani, H.; Suzuki, K. Anal. Chim. Acta 1997, 342, 31-39. (21) Kurihara, K.; Ohtsu, M.; Yoshida, T.; Abe, T.; Hisamoto, H.; Suzuki, K. Anal. Chem. 1999, 71, 3558-3566.
components, which is a typical problem of ion-selective optodes,21-23 would not need to be taken into consideration. This merit directly reflects excellent reproducibility of the response during continuous measurements and effective reduction of amounts of expensive reagents consumed in one measurement. (3) Essentially, ion determination can be carried out by detection of the protonation/ deprotonation process of a single lipophilic anionic dye in the organic phase. Therefore no special color-changeable chelating reagents are required for measurement of another kind of analyte ion. We only need to alter the neutral ionophore for different ionselective ionophores. From the viewpoint of optical instrumentation, this merit is quite important; there is no need to change the excitation laser to match the excitation wavelength of different chelating reagents. (4) Highly selective ionophores or carriers for various kinds of ions developed for application to ion-selective electrodes are now commercially available and can be used without any chemical modification. In the present report, we evaluated analytical features of onchip ion pair extraction experiments under flow analytical conditions using KD-A3 as a lipophilic pH indicator dye and dibenzo 18-crown-6 (DB18C6) and double-decalino 16-crown-5 (DD16C5) as lipophilic neutral ionophores. The many advantages of the onchip ion-sensing system compared to those of ion sensors are discussed. EXPERIMENTAL SECTION Reagents. Reagents of the highest grade commercially available were used for the preparation of the aqueous test electrolytes. A lipophilic pH indicator dye (N-2,4-dinitro-6-octadecyloxyphenyl2′,4′-dinitro-6′-trifluoromethylphenylamine, KD-A3), and a highly selective sodium ionophore (2,6,13,16,19-pentaoxapentacyclo[18.4.4.4.7,120.1,2007,12]dotriacontane, DD16C5) were kindly supplied by Prof. Koji Suzuki, Department of Applied Chemistry, Keio University. Synthesis of these molecules was reported elsewhere.24,25 Dibenzo 18-crown-6 (DB18C6) and butyl acetate were purchased from Merck AG (Darmstadt, Germany) and Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), respectively, and used as-received. Distilled and deionized water had resistivity values of more than 1.7 × 107 Ω cm at 25 °C. Microfabrication of the Quartz Glass Chip. The microfabrication procedure for the quartz glass chip was described in our previous report.5 Briefly, the microchannel of the glass chip was fabricated by thermally laminating three quartz glass plates (3 × 6 cm rectangles): a cover plate possessing three holes for two liquid inlets and an outlet, a middle plate possessing a Y-shaped channel pattern fabricated by a highly focused and intensified excimer laser irradiation, and a bottom plate. The microchannel prepared in the glass chip was 250 µm wide and 100 µm deep. Preparation of Organic and Aqueous Solutions, and Measurement Procedure. The chemical structures of the ionophores and the lipophilic pH indicator dye used for the (22) Dinten, O.; Spichiger, U. E.; Chaniotakis, N.; Gehrig, P.; Rusterholz, B.; Morf, W. E.; Simon, W. Anal. Chem. 1991, 63, 596-603. (23) Hisamoto, H.; Nakagawa, E.; Nagatsuka, K.; Abe, Y.; Sato, S.; Siswanta, D.; Suzuki, K. Anal. Chem. 1995, 67, 1315-1321. (24) Watanabe, K.; Nakagawa, E.; Yamada, H.; Hisamoto, H.; Suzuki, K. Anal. Chem. 1993, 65, 2704-2710. (25) Suzuki, K.; Satoh, K.; Hisamoto, H.; Siswanta, D.; Hayashi, K.; Kasahara, N.; Watanabe, K.; Yamamoto, N.; Sasakura, H. Anal. Chem. 1996, 68, 208215.
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Figure 1. Chemical structures of ion-sensing components.
Figure 2. Schematic illustration of experimental setup and ion pair extraction model (for details, see Experimental Section).
sensing components are shown in Figure 1. The ion-sensing organic phase was prepared by dissolving DB18C6 or DD16C5 as neutral ionophore and KD-A3 as lipophilic pH indicator dye in butyl acetate (5 ×10-6 M each). Aqueous sample cation solutions were prepared by dissolving respective the chloride salts in 0.05 M Tris-HCl buffer solutions of pH 10.0. Figure 2 shows the experimental setup of this work. The organic and aqueous solutions were introduced into the microchannel by using a microsyringe pump (Kd Scientific, model 200, Japan) in order to control the flow rate. Electroosmotic pumping is a common technique to introduce a liquid into a microchannel. However, it is limited for aqueous solutions or strongly polarized solvents and not applicable to most organic solvents. In our case, pressure-driven flow using a syringe pump enabled multilayer flow of organic phase and water phase. Pressure-driven fluid control is indispensable for demonstrating multilayer flow and ion pair 1384
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extraction. For the ionophore-free ion pair extraction experiment, flow rate was varied from 1 to 100 µL/min. An ionophore-based ion pair extraction experiment was performed under a fixed flow rate of 1 µL/min. In each case, flow rates of two syringe pumps were fixed at the same flow rate. The volume flow rates indicated in the figure are shown as set flow rates of the syringe pumps. Thus, total volume flow rate of organic and water phases was twice as much as the indicated value. TLM detection was carried out in the organic phase, 10 mm downstream from the confluence point of organic and water phases and the central position perpendicular to the organic-phase flow direction (∼60 µm from the channel wall). For TLM, a experimental apparatus similar to that reported in our previous studies was used.5-7 In the present work, the protonation/deprotonation process of the lipophilic pH indicator dye was monitored. Therefore, according to the absorption maximum wavelength of KD-A3 in the deprotonated form in butyl acetate (510 nm), the 514.5-nm argon ion laser beam and 633-nm helium-neon laser beam were used as excitation beam and probe beam, respectively. The chopping frequency of the excitation beam was set to 1.06 kHz. RESULTS AND DISCUSSION Response Time of Ionophore-Free Ion Pair Extraction Experiments. Here we demonstrated integration of an ion pair extraction reaction on a glass microchip capable of fast ion determination under flow-analytical conditions. In our previous report, introduction of an organic solution and water into the Y-shaped microchannel by pressure-driven flow formed an organicwater interface at the center position of the microchannel along the channel depth as illustrated in Figure 2.6 Thus, direct observation of the extraction process with TLM is possible by adjusting the focal point at the organic phase. To evaluate response time of the simplest on-chip ion extraction system, we investigated flow rate dependence of the TLM signal under ionophore-free ion pair extraction conditions by introducing organic solution containing protonated KD-A3 and sodium hydroxide aqueous solution, prior to application of this system to neutral ionophore-based ion sensing. Since the detection point was fixed at 1 cm from the confluence point of organic and water phases, flow rate dependence of the TLM signal directly indicates required reaction time to complete the ion pair extraction reaction occurring at the organic-water interface. Figure 3 shows the flow rate dependence of the TLM signal generated from the organic phase containing KD-A3. Since the TLM signal is slightly influenced by flow rate, Figure 3 was obtained by subtracting fundamental flow rate dependence of the TLM signal. Thus, it clearly shows flow rate dependence of the ion pair extraction reaction in the microchannel. In this case, the ion pair extraction reaction occurring in the microchannel can be expressed as the following equation.
AHorg + Na+aq ) A-Na+org + H+ aq where AH, Na+, A-Na+, and H+ indicate the protonated lipophilic anionic dye, a sodium ion, the ion pair produced by deprotonated lipophilic anionic dye and sodium ion, and a proton, respectively. Subscripts org and aq indicate organic phase and water phase. A
Figure 3. Flow rate dependence of thermal lens signal intensity upon introduction of butyl acetate containing protonated KD-A3 and 0.1 M sodium hydroxide solution.
sodium ion can be extracted into the organic phase, and subsequently, the proton of AH is released to the water phase to maintain electroneutrality in the organic phase. In Figure 3, the TLM signal is saturated at the flow rate of 10 µL/min (∼13.4 mm/s as a linear flow rate), indicating that the sodium ion extraction by KD-A3 is completed within 1 s. In the present extraction system, the lipophilicity of KD-A3 is large (log Po/w ) 7.8; see ref 24); thus the ion pair extraction reaction can occur at the organic-water interface. Since the protonation/deprotonation reaction at the organic-water interface is fast, the reaction time to reach equilibrium can be estimated as a simple diffusion of the formed ion pair species (the deprotonated KD-A3 and sodium ion), from the organic-water interface to the bulk organic phase. In this case, Fick’s law of diffusion can be simply expressed as L ) (Dt)0.5, where L, D, and t are diffusion distance, diffusion coefficient, and diffusion time, respectively.26 According to the flow rate dependence of the TLM signal indicated in Figure 3, the order of the diffusion coefficient is estimated as 10-5-10-4 (cm2/s). This range corresponds to that of an ionic species, indicating that the reaction time is basically governed by diffusion of the ion pair species. In such a case, employing the liquid microspace, which provides a short molecular diffusion distance and a large specific interfacial area of the liquid-liquid interface, is quite advantageous to achieve fast response time. In the case of polymeric membrane-based ion sensors, diffusion of chemical species is generally slow due to high viscosity of the polymer support (usually the diffusion coefficient of an ionophore in a polymer membrane is ∼10-8 cm2/s).1,27 By contrast, for the microfluidic device, a low-viscosity organic liquid can be utilized to “immobilize” functional molecules such as lipophilic dyes or ionophores. Thus, utilization of low viscous liquid in the present study also helps to achieve fast response time, and it is another merit gained by employing the microfluidic device. Required Reagent Solution Volume and Quantity of Reagents. Concerning the reagent solution volume, we know the minimum reagent solution volume required for ion determination, estimated as a channel volume from the confluence point to the detection point, is ∼125 nL, indicating the possibility of demonstrating fast ion determination with an extremely small reagent solution volume. In the case of previously reported ion sensors, (26) CRC Handbook of Chemistry and Physics, 78th ed.; Lide, D. R., Ed.; CRC Press: Boca Raton, 1997. (27) Oesch, U.; Simon, W. Anal. Chem. 1980, 52, 692-700.
leaching of ion-sensing components from the organic phase to the water phase in sequential measurements was a serious problem, and it usually limited long-term stability of the sensor response.21-23 This problem was basically caused by continuous use of a single organic phase (usually a polymer membrane) in every measurement. In contrast, since the consumption of reagent solutions is extremely small in the integrated ion-sensing system, fresh organic phases can be used for every measurement, so that leaching of ion-sensing components from the organic phase to the water phase need not be taken into consideration. This advantage cannot be obtained by standard ion sensor technology. In actual experiments, relative standard deviation of thermal lens signals for sequential measurements performed by on-off switching of the syringe pump was 1.1% (n ) 10), indicating excellent reproducibility. Concerning required amount of reagents such as lipophilic dyes or neutral ionophores, the following simple calculation makes it easier to understand the advantage of the microchip system quantitatively. Usually, 2-3 mg (depending on the molecular weight) of dyes or ionophores is required to prepare several (usually three to five membranes) optical sensor membranes when the spin-coating technique is used.18 In contrast, under the present experimental conditions, the same amount of reagents provides ∼1 L of reagent solution. Since the required reagent solution volume for one measurement is ∼100 nL, 107 sequential measurements are possible. The same sequential measurements cannot be demonstrated by optical sensor membranes without response degradation, even if three to five membranes are used. At the present stage of development, introduction of reagent solution was performed by manual switching of the syringe pump; thus several hundred nanoliters of reagent solution were introduced in each measurement. However, since the integrated ion pair extraction system proposed here involves the liquid introduction in a thin and long space along the channel, segmentation of the organic phase at the 100-nL level is possible by alternate injection of organic phase containing ionsensing components and that containing no reagents. Recently we carried out a successful preliminary investigation concerning the segmentation described above.28 Thus, exploitation of the segmentation technique would provide us a further advantage in the required reagent solution volume. From the viewpoint of channel size, we used a microchip possessing a channel size of 250-µm width and 100-µm depth. However, further reduction of channel size is technically easy and this would also reduce the required volume for one measurement. Thus, the advantage concerning reagent quantity would become more dominant. A Neutral Ionophore-Based Ion Pair Extraction Experiments under Flow-Analytical Conditions on a Glass Microchip. On the basis of the fundamental experiments shown above, a typical neutral ionophore for potassium ion of DB18C6 was applied to demonstrate ionophore-based ion sensing on a glass microchip. Employing a neutral ionophore gives selective extraction of the analyte ion to be determined. Figure 4 shows typical response curves obtained by using the organic phase containing DB18C6 and KD-A3. Selective determination of potassium ion is realized. The selectivity coefficients defined by ion pair extraction theory,1,14,15,24 log KoptK,Na, is -1.0. (28) Horiuchi, T.; Hisamoto, H.; Tokeshi, M.; Kitamori, T. 61th Jpn. Soc. Anal. Chem. Annu. Meet. Book of Abstracts; 2000; p 54.
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Figure 4. Response curves obtained by using DB18C6 as an ionophore.
This value agrees well with that of previously reported ion sensors based on DB18C6,14 indicating that the selective determination of potassium ion is demonstrated without any loss of ion selectivity of the ionophore itself. In addition, since the flow rate was fixed at 1 µL/min, response time is calculated to be ∼8 s. This value is much faster than that previously reported for an ion-selective optode membrane using a similar dye, which exhibited a response time of more than 10 min.18 The concentration detection limit in Figure 4 was around 10-4 M. In the case of the ion pair extraction principle, concentration detection limit basically depends on the complexation constant of the ionophore itself and the polarity of the organic phase, when the same lipophilic dye is used. Thus, exploitation of the ionophore molecule possessing strong complexation ability or organic phase of high dielectric constant would make it possible to lower the concentration detection limit. Concerning the number of dye molecules probed by the probe beam, that calculated from detection volume and molar absorptivity is estimated to be 4.5 zmol for the plot obtained at 10-4 M in our experimental conditions. Therefore, in consideration of the detection limit of the TLM measurement,8 detection of a lower concentration of ion can be principally possible. Altering the neutral ionophore makes it possible to determine other kind of ions. Figure 5 shows the response curves obtained with an organic phase containing the sodium ionophore of DD16C5 rather than the potassium ionophore of DB18C6 as a neutral ionophore. As expected, we obtain the sodium-selective response. This suggests that any analyte ions can be determined by simply altering the neutral ionophore without changing the operating parameters of the optical instrument. In addition, the
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Figure 5. Response curves obtained by using DD16C5 as an ionophore.
selectivity coefficient against potassium ion is -2.1, indicating that ion selectivity of the ionophore itself can also be demonstrated for any ionophores in the microchip extraction system. Numerous highly selective neutral ionophores have been developed for applications in ion sensors,2 and many of them are commercially available. Thus, the integrated system can be widely applied to many analyte ions of interest. Here we have demonstrated successful operation of a fast ion analysis system with ultrasmall reagent solution volume by using characteristics of the liquid microspace and microfluidic system. Recent research concerning microchip technology has mostly focused on microfabrication of chips or integration of the capillary electrophoretic technique. In contrast, we have focused on the characteristics provided by the liquid microspace and microfluidic system in our application example. Further work focusing on these two points should provide promising ways to realize novel analytical devices using microchips. ACKNOWLEDGMENT We gratefully acknowledge Prof. Koji Suzuki, Department of Applied Chemistry, Keio University, for supplying the lipophilic pH indicator dye molecule (KD-A3) and highly sodium-selective neutral ionophore (DD16C5). This work was partially supported by Grants for Scientific Research from the Ministry of Education, Science, and Culture, Japan, Showa Shell Sekiyu Foundation for Promotion of Environmental Research, Sasakawa Scientific Research Grant, and The Kao Foundation for Arts and Sciences. Received for review October 23, 2000. Accepted January 7, 2001. AC001271V
