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Synthesis of pH-sensitive modified cellulose ether halfesters and their use in pH detecting systems based on fiber optics
journal of
ELSEVIER
controlled release Journal of Controlled Release 35 (1995) 155-163
Synthesis of pH-sensitive modified cellulose ether halfesters and their use in pH detecting systems based on fiber optics Tim F. Liebert a, David R. Walt b,, a Fritz-Kalisch-Strasse 3, 07743 Jena, Germany b Max TishlerLaboratoryfor Organic Chemistry, Department of Chemistry, Tufts Universi~, Medford, MA 02155, USA
Received 3 October 1994; accepted 24 February 1995
Abstract The syntheses of several derivatized cellulose ether halfesters with dissolution points above pH 7 are described. Fluorescent dyes were incorporated into these polymers, and the rate of fluorescent dye release as a function of pH was determined. Dye containing polymers were employed to create a new fiber optic sensor system for detecting pH changes over long time periods. In this study, sensors were prepared using polymers with high sensitivity in the pH region between 9.2 and 12. Sensors responded within 30 s after being exposed to basic solutions. Keywords: Modified cellulose ether halfester; Synthesis; Fiber optics; pH detection
1. Introduction
The detection of important chemical parameters such as CO2, 02, and pH in aqueous media is important for medical, environmental and process control applications. Optical sensors are a particularly promising approach because they can measure these parameters in real time, they are insensitive to electromagnetic interference, they can be internally calibrated and they can be operated remotely over long distances [ 1-3]. Such sensors operate on the principle of total internal reflection. Light is introduced into a fiber composed of two materials of differing refractive index - a core in which the light travels and a clad that provides a reflective surface for light traveling down the core. Light travels to the distal (far) tip on which is placed a sensitive indicating dye. The dye interacts with light in * Corresponding author. Tel.: (617)627-3470; Fax: (617)6273443; e-mail: [email protected] 0168-3659/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDIO 1 6 8 - 3 6 5 9 ( 9 5 ) 0 0 0 3 2 - 1
such a way as to give a change in the light proportional to the presence of a particular analyte species. For example, a fluorescent pH indicator could be disposed on the distal tip that emits a fluorescent signal proportional to the pH of the solution. To date, all optical pH sensors have been based on measuring absorbance or fluorescence changes between the ionized and the nonionized form of an indicator dye as a function of pH. Two configurations have been used previously to prepare optical sensors. In the first configuration, a reversible analyte-sensitive indicating layer is applied directly to the fiber. For example, the pH-sensitive indicator fluorescein has been attached covalently to a polymer coated on the fiber tip [4]. In the second configuration, reagents are released continuously from controlled release polymers [ 5,6 ]. In this approach, the indicators are released into the sample medium, where they react with solution analyte and are detected through an optical fiber. In this paper we describe a
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T.F. Liebert, D.R. Walt~Journal of Controlled Release 35 (1995) 155-163
third approach in which pH-sensitive polymers are used to provide the requisite sensitivity. Fluorescent dyes are incorporated into the pH sensitive polymers. When exposed to solutions that cause the polymer to dissolve, fluorescent dye is released in a pH-dependent manner. In this way, polymer release is coupled to fluorescence signal generation enabling precise pH measurements to be made via an optical fiber. A number of pH-sensitive polymers have been developed over the course of the last three decades for use as coating materials. These materials are used mainly to target drug release to specific organs [7-11 ]. The majority of these polymers undergo a fast irreversible response and their lifetimes vary from a few hours to up to two months. Three major dissolution mechanisms for pH-sensitive polymers have been established and include: ( 1) cleavage of the polymer backbone to smaller soluble units; (2) reaction of pH sensitive side groups and cleavage of the backbone; (3) simple reaction of pH sensitive units (such as ionization) [ 12]. pH-sensitive polymers combined with fiber optics for pH detection could solve major problems limiting the lifetime of existing sensors. Photobleaching processes resulting from the constant excitation of the dye can be diminished by isolating the unreleased dye away from the light source. In the present sensor, the chemistry is designed such that reaction occurs only if the solution being monitored deviates from a defined pH value. This kind of an 'alarm or threshold'-type system is a desirable tool in environmental analysis because it allows pH changes to be detected without giving constant background signals for the normal state of the system. If the release rate under neutral conditions is almost zero, a long lasting pH sensor becomes accessible. Ideally, if diffusional processes are ignored, polymer dissolution should be reversible so that the rate of polymer erosion is equal to the dye release. In such a system, it would be possible to calibrate the release rate versus pH. In principle such a system would enable the use of pH insensitive dyes and thereby extend the range of suitable fluorescent dyes. We report an optical pH sensor for measuring basic pH values. Our goal was to develop a reversible sensor based on the sensitivity of the polymer rather than the indicator. These requirements suggest the use of polyacids as ionizable polymers that dissolve under basic conditions. For example, studies on maleic anhydride
copolymers have shown that it is possible to tailor the dissolution behavior of the substance by partial esterification. In addition, the use of cellulose ether halfesters [13-15] as the polyfunctional backbone offers the potential for other derivatizations in order to prepare polymers sensitive to other analytes or other pH ranges. 2. Materials and methods
2.1. Materials Hydroxypropylmethylcellulose (HPMC), methylcellulose, fluorescein and the esterification reagents were from Aldrich Chemical Co. (Milwaukee, WI). HPMC phthalate (HPMCP, 23.2% phthalyl, 22.3% methoxy and 7.2% hydroxypropyl) was obtained from Eastman Kodak Chemical Co. (Rochester, NY). Sulforhodamine 640 (SR 640) was purchased from Exciton (Dayton, OH).
2.2. Syntheses Preparation of HPMC halfesters 4 g HPMC and 2 g sodium acetate were mixed with 0.45 ml acetic anhydride and 1.2 ml acetic acid. The mass was stirred for 5 min in a waterbath without heating. The system was agitated and heated to 80°C after addition of 2.6 g maleic anhydride and 3 g sodium acetate. Within 30 min it became a brown mixture of high viscosity. Isolation was carried out by addition of 100 ml water, 20 ml 10% HC1, filtration and washing with water. The HPMC pyromellitate, methylcellulose phthalate and polyvinylalcohol phthalate were prepared in the same manner.
Crosslinking of HPMC 0.5 g HPMC was dissolved in 25 ml dry pyridine and 0.39 g pyromellitic dianhydride was added. Gelation occurred within 20 min. The product was isolated after 2 h by precipitation in 200 ml water and addition of 30 ml conc. HC1. In the case of dye-containing samples, the dye (ca. 5 mg) was added before addition of dianhydride. The formed gel was sliced and purified by addition and washing with dilute HC1.
Crosslinking of HPMCP 100 mg HPMCP was dissolved in 9 ml dry pyridine and 61.3 mg tosyl chloride (4 mol/mol free acid group) was added. Gelation occurred within 20 min.
157
T.F. Liebert, D.R. Walt/Journal of Controlled Release 35 (1995) 155-163 fiber
The product was isolated by addition of 70 ml water and 10 ml conc. HC1. Crosslinking of methylcellulose phthalate and polyvinylalcohol phthalate were carried out in the same way. Crosslinking o f H P M C P with resorcinol
100 mg HPMCP was dissolved in 9 ml pyridine. 26.6 mg resorcinol (3 mol/mol free acid group) was added and after 5 min tosyl chloride ( 184 rag, 12 mol/ mol free acid group). Gelation occurred within 30 min. Isolation was carried out in the same way as in the previous case. The dye-containing material was prepared by adding the dye (approx. 1 mg) before addition of resorcinol. Isolation was carried out by slicing the gel and adding dilute HC1. The same procedure was applied for the crosslinking of polyvinylalcohol phthalate with resorcinol. 2.3. UV-VIS examinations
A Perkin Elmer Lambda 6 spectrophotometer was used for all spectroscopic measurements. Determination o f the dissolution rate:
The samples were ground and sieved to obtain a particle size <0.225 mm. Polymer (1 mg) was suspended in 3 ml 0.1 N NaOH. The absorption was measured at 288 nm and plotted versus time. Sensor configuration
The cross section of the sensor (configuration 1 ) is shown in Fig. 1. A polished fiber (core diameter 200 /~m, outer diameter 240/~m) was placed in the tube system after cleaving, washing with conc. sulfuric acid and rinsing with distilled water. The tube system consisted of a piece of polyethylene tubing (length approx. 1.5 cm, outside diameter 1.6 mm), a piece of siloxane tubing (length approx. 1 cm, outside diameter 5 mm), and the insulation material of the fiber. All pieces fit together tightly, thus no further fixation was necessary but the formed chamber was adjustable to the size of the polymer by simply shifting the position of the tubing. Two slices of polymer (approximately 4 mm X 0.5 m m X 0 . 5 mm) were inserted between the Siloxane tubing and the fiber and were fixed by swelling in 0.1 N NaOH for 10 rain. Polymer that had swelled over the tip of the fiber was removed mechanically.
tubing
polymer
Fig. 1. Cross-sectionof the fiber optic sensor (Configuration 1). 2.4. Measurements
The measurement system has been described previously [4,5 ] and consisted of an argon ion laser (Spex. air cooled), a dichroic filter for separating excitation and emission signals and a monochromater with photomultiplier tube as the detection unit. The excitation wavelength was 488 nm and the emitted light was detected at 525 nm for fluorescein and at 620 nm for sulforhodamine. 0.2 M phosphate buffer, 0.4 M phosphate buffer, 0.2 M carbonate buffer and aqueous solutions of NaOH and HC1 were used to vary the pH. Qualitative release experiments could be conducted without calibration by simply moving the sensor from one buffer solution to another with a brief water rinse between measurements. In the case of calibration experiments with sensor configuration 1, the fiber with the sensor tip was kept in the medium and the pH was adjusted by addition of aqueous solutions of NaOH and HC1. pH values were simultaneously detected by a glass pH electrode. The baseline (background signal) was obtained by stirring the medium between the measurements to distribute accumulated dye. 2.5. Flow cell experiments
A cross section of the flow cell (configuration 2) is shown in Fig. 2. The lower part was separated from the
158
T.F. Liebert, D.R. Walt~Journal of Controlled Release 35 (1995) 155-163
upper part by a layer of filter glass and black filter paper to avoid direct irradiation of the dye-containing polymer. The lower part was filled with a homogeneous mixture of dye-containing polymer (particle size 0.225-0.45 mm) and controlled pore glass (pores 500 Angstrom). The mixture was poured in the tube and was treated with distilled water, stirred and sealed with filter paper. The lower part was connected to a peristaltic pump that delivered a constant flow of 14 ml per min. The fiber and the glass electrode were inserted in the upper part. The buffer solutions and wavelengths were the same as in the sensor experiments with configuration 1.
1 water out pH electrod(
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3. Results and discussion
Several halfesters of hydroxypropylmethylcellulose 1 (HPMC) were synthesized (Fig. 3) and examined to determine if they could be used to prepare dyecontaining polymers. The maleate 2 was obtained from a heterogeneous esterification reaction of HPMC with maleic anhydride. This polymer began to dissolve when exposed to solutions at pH 3.12. The pKa of the polymer was determined to be 3.9 by titration [ 11 ]. Because of this low value and the possibility of undefined side reactions on the double bond, no further experiments were carried out with this polymer. In order to increase the polymer pKa value, the HPMC pyromellitate 3 was prepared using partially hydrolyzed pyromellitic anhydride. The resulting polymer was insoluble in common organic solvents and soluble in basic media only by irreversible saponification. The pKa value of the polymer was determined to be approximately 6.3. Pyromellitate-modified polymers could also be synthesized by a homogeneous reaction of partially hydrolyzed pyromellitic anhydride with HPMC in pyridine but the resulting products show the same insolubility. The insolubility probably results from competitive network formation via intermolecular crosslinking processes by unhydrolyzed dianhydride. It is not possible to dissolve these networks without saponification. Although the polymer is not reversibly soluble in basic media it shows pH sensitivity by swelling and shrinking processes. Comparable observations were made for polymers obtained from the reaction of unhydrolyzed dianhydride with HPMC. For the first release experiments the dye Sulforhodamine was chosen because of its solubility both in
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water and pyridine and its pH insensitivity. Therefore, changes in fluorescence intensity should reflect the degree of polymer hydrolysis. Dye-containing samples of the polymer were prepared by dissolving the dye and HPMC in pyridine and adding pyromellitic anhydride. The resulting gel was sliced and then purified by precipitation with dilute HCI. After drying, the polymer was cut in strips. Strips were placed with a needle in the container around the fiber and fixed by swelling in dilute NaOH as shown in Fig. 1. The optical system for the release studies consisted of a laser based fiber fluorimeter reported previously [ 4,5 ]. Excitation was at 488 nm with emission at 620 nm. Measurements were carried out by changing the medium (phosphate and car-
T.F. Liebert, D.R. Walt~Journal of Controlled Release 35 (1995) 155-163
backbone
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R" = H, HPMCP-units Fig. 3. Structures of the polymers and their derivatives. bonate buffers with defined pH values). The sensor responded to pH values above 9 within 30 seconds. Unexpected signals were obtained in acidic media and in solutions with pH values around 7. These results will be discussed below. The prepared sensor was applicable for at least 20 measurements (pH > 9) but released the dye within 2 weeks during storage in pH 7 phosphate buffer. Similar results were obtained with fluorescein-containing polymers.
The limitations of the maleate and pyromellitate, including their low pKas and rapid release prompted us to examine commercially available HPMC phthalate 4. This polymer has a pKa of 3.82 and starts to dissolve at pH 4.23. Sensors were prepared from HPMCP and Sulforhodamine by dissolving both substances in pyridine and evaporating the solvent. Such sensors respond to pH values above 5, however, they can be used only for one measurement because of their high dissolution
T.F. Liebert, D.R. Walt~Journal of Controlled Release 35 (1995) 155-163
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rate and their constant release even in strong acidic solutions. Consequently, the rate of dissolution and the structure of the polymer matrix had to be changed. Attempts to vary the dissolution behavior of HPMCP by simple esterification of the acid residues were unsatisfactory. Crosslinking the polymer was considered to be a promising method, however, the formation of a network leads to a dramatic increase in the dissolution time. Crosslinking was carried out by intermolecular esterification of the free phthalic acid residues of the polymer with free hydroxyl groups on the cellulose backbone. Esterification was effected with tosyl chloride in pyridine. Reactions were carried out at room temperature in the absence of moisture resulting in formation of a gel. The resulting polymer 4a was isolated by precipitation with water and dilute HC1. The dissolution time was determined with a UV-Vis spectrometer. A plot of the absorbance at 288 nm versus time provides information about the dissolution rate as both phthalate esters and acids absorb at this wavelength. Four crosslinked samples were prepared using different amounts of tosyl chloride and the dissolution times in 0.1 N NaOH were determined (Fig. 4). The resulting polymers exhibited bimodal dissolution behavior characteristic of the starting HPMC [ 16]. Because of an observed stronger influence of the dye sulforhodamine on the release behavior caused by its free solubility in water and its ampholytic character, the less soluble and acidic dye fluorescein was used for
the release studies. The samples utilized were obtained from the reaction of tosyl chloride with HPMCP in pyridine in the presence of fluorescein. Measurements were carded out in the above-mentioned manner by using 488 nm light for excitation and determining the emission signal at 525 nm. The prepared sensor responded within 30 s to pH values above 9. The signals responded rapidly when the pH was changed to values below 7.5 however, within two min another signal appeared. Depending on the pH of the initially applied basic solution and the time of contact with basic solution, up to three additional signals appeared. This behavior can be explained by assuming that the basic medium is entrapped by the polymer matrix via fast formation of a thin stiff layer of protonated polymer around a highly swollen particle. This process is followed by the dissolution of this thin layer by the base from the inside out. An equilibrium can be established and a new layer of insoluble polymer is formed. The result is the occurrence of a new smaller signal. This conceptual model would explain the periodicity of the observed subsequent signals. One possible way to avoid this process would be to introduce spacer groups into the polymer structure to increase its accessibility and consequently slow down the formation of the insoluble layer. Resorcinol was used as a spacer unit to provide polymer 4b. Different amounts of crosslinking reagent were applied to the polymer to determine the dependence of the dissolution time on the degree of crosslinking. The reactions were carded out by dissolving the polymer, resorcinol and tosyl chloride at room temperature in pyridine. The molar ratio of tosyl chloride to the resorcinol spacer and crosslinker was 4" 1. The amount of free acid groups of the polymer per dehydrating reagent in the reaction mixtures was varied from 1:2 up to l:12. Dissolution experiments were carried out by placing the polymer in 0.1 N NaOH and monitoring dissolution at 288 nm. These experiments showed that an increase of the molar ratio of free acid to dehydrating reagent beyond one to eight does not result in a significant increase in the dissolution time. The maximum value achieved for the dissolution of 0.05 gram of polymer in l0 ml 0.1 N NaOH was 165 rain. A bimodal dissolution behavior is observed once again for these derivatives. Fluorescein- and sulforhodamine-containing polymers were then prepared. Fluorescein sensors responded within 30 s to pH values above 9 and the
T.F. Liebert, D.R. Walt~Journal of Controlled Release 35 (1995) 155-163
signal decay after changing the pH below 7.5 was almost as fast as in the previously discussed case but the subsequent signals disappeared almost entirely. Sulforhodamine containing samples were prepared to examine how this highly water-soluble, ampholytic dye is released over the entire pH range and whether it is possible to generalize the observations. The same crosslinking reaction was carried out with resorcinol. Measurements were performed in the above-mentioned manner with an excitation wavelength of 488 nm and emission at 620 nm. The response of this sensor was as fast as in the fluorescein case and the base line was equally stable. Usually the signal decay took longer (up to 2 min). When the sensor was exposed to basic medium, followed quickly by pH 7 and then with acid solution, a strong signal was obtained that disappeared almost entirely within a few min. We had already determined that dissolution of the polymer does not occur even in strong acidic solutions (UV-Vis experiments). This result can be explained by a 'squeezing effect'. When the pH is changed from slightly basic to acidic conditions the swollen polymer shrinks and the entrapped dye-saturated medium is squeezed out. A second possible reason for an unexpected release pattern could be the ionic strength of the medium. It was found that by changing the concentration of pH 7 buffer solutions from 0.4 M to 0.2 M and back, a signal is obtained for the sulforhodamine-containing polymer. The signal is due to osmotic effects. This influence on the release behavior occurs only in the region around pH 7-8. At higher pH values, it is not detectable because of a preferred erosion mechanism due to hydrolysis. The influence of temperature on the release was not investigated. The amount of dye has a strong influence on the release pattern. Dyes are generally hydrophilic and increasing the content of the water soluble dye increases the dissolution rate. Dissolution and release times for samples with different amounts of dye in 0.5 N NaOH were determined by UV-Vis analysis (Fig. 5). As can be seen, the lower loadings provided for greater release times. Since fluorescence detection is not limiting, we used samples with 1% dye loadings for our experiments. We next assembled a working demonstration of how such polymers could be used for continuous monitoring. A Sulforhodamine-containing resorcinol-crosslinked HPMCP sample was dried at room temperature.
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Amountof Dye(%) Fig. 5. Hydrophilizingeffect of the increasing amountof Sulforhodamine on resorcinolcrosslinked HPMCPillustrated by decreasing dissolution and release times. After three weeks' storage, it showed exactly the same release behavior as the freshly prepared polymer. A sensor was prepared with this material (Fig. 1) and kept in phosphate buffer at pH 7. After four months' storage, the sensor was exposed to base. A signal appeared after about three min of base exposure. The lag in response was probably due to a dye-depleted layer at the polymer surface as a consequence of slow leakage upon storage. Our attempts to calibrate the sulforhodamine-containing derivatized HPMCP were unsuccessful because no clear correlation was observed between the applied pH values and the signal intensities. The reasons could be the free water solubility of the dye or the capability of the ampholytic dye to buffer the interface layer to its own isoelectric point [ 17]. We next turned to the fluorescein-containing polymer in a sensor configuration (Fig. 1). Measurements were carried out by using aqueous solutions of 0.1 N NaOH and HC1 to adjust the pH of the medium. The baseline was obtained by constant stirring between measurements to distribute accumulated dye. As can be seen in Fig. 6, signals returned to baseline upon stirring, but rapidly went back to the steady state values when stirring was stopped. Although the sensor showed good pH sensitivity the signal levels were low, resulting in low precision. To obtain stronger and more stable signals and to avoid several irreproducible variables caused by the small interface between polymer and medium, it was
T.F. Liebert, D.R. Walt~Journal of Controlled Release 35 (1995) 155-163
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necessary to increase the reactive surface of the polymer. For this purpose a flow cell was constructed as shown in Fig. 2. It consisted of two chambers that were separated by a layer of filter glass. The upper chamber contained the fiber to detect the released dye, a glass electrode to measure the pH, and a black filter on the bottom to avoid photobleaching of the dye entrapped in the polymer. The lower chamber contained the polymer and was covered by a filter. A blend of polymer 100
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and controlled pore glass must be used, otherwise the polymer swells and occludes the flow cell thereby preventing flow through the system. Dilute solutions of NaOH or buffers were used as media and were pumped with a constant flow of 14 ml/min from the lower chamber through the upper chamber over periods of five rain. Usually the system responded at the same time a change in pH was detected by the pH electrode. Two measurements per pH value were carried out and the obtained signal intensities were plotted versus the pH values. The calibration curve obtained by this procedure for fluorescein-containing resorcinol-crosslinked HPMCP shows an excellent sensitivity to basic pH values (Fig. 7). The fluorescein intensity changes exhibited by the system are not due to the pH sensitivity of fluorescein because it is fully ionized at pH values above 8 [ 18]. Above this value no further ionization is possible and consequently no further increase of the fluorescence intensity is possible. We conclude that the dependence of the intensity on pH is due to a controlled dye release. Measurements performed over the course of three days still fit this curve. The system can be used to detect pH values above 10.5 in aqueous solution. The sensitivity of simple crosslinked HPMCP, was similar to the resorcinol-crosslinked polymer (Fig. 8). This polymer exhibits a small but measurable release at pH 9.2. In
T.F. Liebert, D.R. Walt/Journal of Controlled Release 35 (1995) 155-163
m e r matrix and not e x p o s e d continuously to solution, there is little chance that dye degradation or reaction with interfering substances will occur. Finally, it should be noted that p o l y m e r s with different pH sensitivity ranges as well as sensitivity to other analytes (e.g., organic solvents) can be prepared to broaden the applicability o f such a transduction m e c h a n i s m .
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4. Conclusion W e h a v e d e m o n s t r a t e d the use o f resorcinol crosslinked H P M C P in c o m b i n a t i o n with the dye fluorescein for construction o f an ' a l a r m / t h r e s h o l d ' - t y p e sensor to detect p H values in the basic region ( a b o v e p H 9) with a 30 s response time. O p t i m i z a t i o n of the sensor configuration will further i m p r o v e its p e r f o r m ance with respect to reduced response t i m e and i m p r o v e d signal levels. T h e overall principle o f this sensor differs significantly f r o m other p H sensors in that the p H sensitivity is a function o f the p o l y m e r , not the indicator dye. Seitz and c o - w o r k e r s h a v e reported p H sensors based on p o l y m e r s w e l l i n g [ 19 ] and changes in p o l y m e r opacity [ 2 0 ] . T h e present sensor can be d e s i g n e d so that it is always in an o f f state, unless a threshold p H is e x c e e d e d . Such sensors should be o f v a l u e in process control and e n v i r o n m e n t a l m o n i t o r i n g in w h i c h only o c c a s i o n a l deviations f r o m nominal p H values are expected. B e c a u s e the dye is trapped within the poly-
[ 1] M.A. Arnold, Fiber-optic chemical sensors, Anal. Chem. 64 (1992) 1015-1025. [2] C. Camera, M.C. Moreno, G. Orellana, For a comprehensive review on pH fiber-optic sensors, in: D.L. Wise and L.B. Wingard (Eds.), Biosensors with Fiber Optics, Humana Press, Clifton, NJ, 1991, pp. 37--43. [3] O. Wolfbeis (Ed.), Fiber Optic Chemical Sensors and Biosensors, CRC Press, Boca Raton, FL, 1991, Vols. 1 and 2. [4] C. Munkholm, D.R. Walt, F.P. Milanovich and S.M. Klainer, Anal. Chem. 58 (1986) 1427. [5] S. Luo and D.R. Walt, Anal. Chem. 61 (1989) 174. [6] S.B. Barnard and D.R. Walt, Science 251 ( 1991 ) 927-929. [7] R.M. Ottenbrite, in: L.G. Donaruma, R.M. Ottenbrite, and O. Vogel (Eds.), Anionic Polymeric Drugs, Wiley, New York, 1980, p. 21. [8] S. Matsumara and S. Yoshikama, in: J.E. Glass, G. Swift, (Eds.), Agricultural and Synthetic Polymers, American Chemical Society, Washington DC, 1990, p. 124. [9] C. Brand, C. Bunel, and M. Vert, Polym. Bull. 13 (1985) 293. [10] M.E.K. Krealing and W.A. Ritschel, Methods Find. Exp. Clin. Pharmacol. 14(3), (1992), 199. [11] J.H. Kou, D. Fleisher, and G.L. Amidon, Cosmet. Pharm. Appl. Polym. 1990 (Pub 1991), 201. [12] J. Heller, R.W. Baker, R.M. Gale, and J.O. Rodin, J. Appl. Polym. Sci. 22 (1978) 1991. [13] M. Schnabelrauch, T. Heinze, and D. Klemm, Acta Polymerica 41(2), (1990), 112. [14] P.C. Schmidt and F. Niemann, Drug. Dev. Ind. Pharm. 18(18) (1992) 1962. [151 P. Giunchedi, U. Conte, and L. Maggie, Int. J. Pharm. 85( 13) (1992) 141. [ 16] A.C. Shah, N.J. Britten, L.S. Olanoff, and J.N. Badalamenti, J. Control. Release 9 (1989) 169. [ 17] J.C. Shah and M. Maniar, J. Control. Release 23 (1993) 261. [ 18] C.-S. Chen, H. Nakumura, and Z. Tamura, Chem. Pharm. Bull. 27(2) (1979) 475. [ 19] S. Pan, V. Conway, Z.M. Shakhsher, S. Emerson, M. Bai, W.R. Seitz, K.D. Legg, Anal. Chim. Acta 1993, 279, pp. 195-202. [20] Z. Shakhsher, W.R. Seitz and K.D. Legg, Single fiber-optic pH sensor based on changes in reflection accompanying polymer swelling, Anal. Chem., 66, 1994, pp. 1731-1735.
ELSEVIER
controlled release Journal of Controlled Release 35 (1995) 155-163
Synthesis of pH-sensitive modified cellulose ether halfesters and their use in pH detecting systems based on fiber optics Tim F. Liebert a, David R. Walt b,, a Fritz-Kalisch-Strasse 3, 07743 Jena, Germany b Max TishlerLaboratoryfor Organic Chemistry, Department of Chemistry, Tufts Universi~, Medford, MA 02155, USA
Received 3 October 1994; accepted 24 February 1995
Abstract The syntheses of several derivatized cellulose ether halfesters with dissolution points above pH 7 are described. Fluorescent dyes were incorporated into these polymers, and the rate of fluorescent dye release as a function of pH was determined. Dye containing polymers were employed to create a new fiber optic sensor system for detecting pH changes over long time periods. In this study, sensors were prepared using polymers with high sensitivity in the pH region between 9.2 and 12. Sensors responded within 30 s after being exposed to basic solutions. Keywords: Modified cellulose ether halfester; Synthesis; Fiber optics; pH detection
1. Introduction
The detection of important chemical parameters such as CO2, 02, and pH in aqueous media is important for medical, environmental and process control applications. Optical sensors are a particularly promising approach because they can measure these parameters in real time, they are insensitive to electromagnetic interference, they can be internally calibrated and they can be operated remotely over long distances [ 1-3]. Such sensors operate on the principle of total internal reflection. Light is introduced into a fiber composed of two materials of differing refractive index - a core in which the light travels and a clad that provides a reflective surface for light traveling down the core. Light travels to the distal (far) tip on which is placed a sensitive indicating dye. The dye interacts with light in * Corresponding author. Tel.: (617)627-3470; Fax: (617)6273443; e-mail: [email protected] 0168-3659/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDIO 1 6 8 - 3 6 5 9 ( 9 5 ) 0 0 0 3 2 - 1
such a way as to give a change in the light proportional to the presence of a particular analyte species. For example, a fluorescent pH indicator could be disposed on the distal tip that emits a fluorescent signal proportional to the pH of the solution. To date, all optical pH sensors have been based on measuring absorbance or fluorescence changes between the ionized and the nonionized form of an indicator dye as a function of pH. Two configurations have been used previously to prepare optical sensors. In the first configuration, a reversible analyte-sensitive indicating layer is applied directly to the fiber. For example, the pH-sensitive indicator fluorescein has been attached covalently to a polymer coated on the fiber tip [4]. In the second configuration, reagents are released continuously from controlled release polymers [ 5,6 ]. In this approach, the indicators are released into the sample medium, where they react with solution analyte and are detected through an optical fiber. In this paper we describe a
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T.F. Liebert, D.R. Walt~Journal of Controlled Release 35 (1995) 155-163
third approach in which pH-sensitive polymers are used to provide the requisite sensitivity. Fluorescent dyes are incorporated into the pH sensitive polymers. When exposed to solutions that cause the polymer to dissolve, fluorescent dye is released in a pH-dependent manner. In this way, polymer release is coupled to fluorescence signal generation enabling precise pH measurements to be made via an optical fiber. A number of pH-sensitive polymers have been developed over the course of the last three decades for use as coating materials. These materials are used mainly to target drug release to specific organs [7-11 ]. The majority of these polymers undergo a fast irreversible response and their lifetimes vary from a few hours to up to two months. Three major dissolution mechanisms for pH-sensitive polymers have been established and include: ( 1) cleavage of the polymer backbone to smaller soluble units; (2) reaction of pH sensitive side groups and cleavage of the backbone; (3) simple reaction of pH sensitive units (such as ionization) [ 12]. pH-sensitive polymers combined with fiber optics for pH detection could solve major problems limiting the lifetime of existing sensors. Photobleaching processes resulting from the constant excitation of the dye can be diminished by isolating the unreleased dye away from the light source. In the present sensor, the chemistry is designed such that reaction occurs only if the solution being monitored deviates from a defined pH value. This kind of an 'alarm or threshold'-type system is a desirable tool in environmental analysis because it allows pH changes to be detected without giving constant background signals for the normal state of the system. If the release rate under neutral conditions is almost zero, a long lasting pH sensor becomes accessible. Ideally, if diffusional processes are ignored, polymer dissolution should be reversible so that the rate of polymer erosion is equal to the dye release. In such a system, it would be possible to calibrate the release rate versus pH. In principle such a system would enable the use of pH insensitive dyes and thereby extend the range of suitable fluorescent dyes. We report an optical pH sensor for measuring basic pH values. Our goal was to develop a reversible sensor based on the sensitivity of the polymer rather than the indicator. These requirements suggest the use of polyacids as ionizable polymers that dissolve under basic conditions. For example, studies on maleic anhydride
copolymers have shown that it is possible to tailor the dissolution behavior of the substance by partial esterification. In addition, the use of cellulose ether halfesters [13-15] as the polyfunctional backbone offers the potential for other derivatizations in order to prepare polymers sensitive to other analytes or other pH ranges. 2. Materials and methods
2.1. Materials Hydroxypropylmethylcellulose (HPMC), methylcellulose, fluorescein and the esterification reagents were from Aldrich Chemical Co. (Milwaukee, WI). HPMC phthalate (HPMCP, 23.2% phthalyl, 22.3% methoxy and 7.2% hydroxypropyl) was obtained from Eastman Kodak Chemical Co. (Rochester, NY). Sulforhodamine 640 (SR 640) was purchased from Exciton (Dayton, OH).
2.2. Syntheses Preparation of HPMC halfesters 4 g HPMC and 2 g sodium acetate were mixed with 0.45 ml acetic anhydride and 1.2 ml acetic acid. The mass was stirred for 5 min in a waterbath without heating. The system was agitated and heated to 80°C after addition of 2.6 g maleic anhydride and 3 g sodium acetate. Within 30 min it became a brown mixture of high viscosity. Isolation was carried out by addition of 100 ml water, 20 ml 10% HC1, filtration and washing with water. The HPMC pyromellitate, methylcellulose phthalate and polyvinylalcohol phthalate were prepared in the same manner.
Crosslinking of HPMC 0.5 g HPMC was dissolved in 25 ml dry pyridine and 0.39 g pyromellitic dianhydride was added. Gelation occurred within 20 min. The product was isolated after 2 h by precipitation in 200 ml water and addition of 30 ml conc. HC1. In the case of dye-containing samples, the dye (ca. 5 mg) was added before addition of dianhydride. The formed gel was sliced and purified by addition and washing with dilute HC1.
Crosslinking of HPMCP 100 mg HPMCP was dissolved in 9 ml dry pyridine and 61.3 mg tosyl chloride (4 mol/mol free acid group) was added. Gelation occurred within 20 min.
157
T.F. Liebert, D.R. Walt/Journal of Controlled Release 35 (1995) 155-163 fiber
The product was isolated by addition of 70 ml water and 10 ml conc. HC1. Crosslinking of methylcellulose phthalate and polyvinylalcohol phthalate were carried out in the same way. Crosslinking o f H P M C P with resorcinol
100 mg HPMCP was dissolved in 9 ml pyridine. 26.6 mg resorcinol (3 mol/mol free acid group) was added and after 5 min tosyl chloride ( 184 rag, 12 mol/ mol free acid group). Gelation occurred within 30 min. Isolation was carried out in the same way as in the previous case. The dye-containing material was prepared by adding the dye (approx. 1 mg) before addition of resorcinol. Isolation was carried out by slicing the gel and adding dilute HC1. The same procedure was applied for the crosslinking of polyvinylalcohol phthalate with resorcinol. 2.3. UV-VIS examinations
A Perkin Elmer Lambda 6 spectrophotometer was used for all spectroscopic measurements. Determination o f the dissolution rate:
The samples were ground and sieved to obtain a particle size <0.225 mm. Polymer (1 mg) was suspended in 3 ml 0.1 N NaOH. The absorption was measured at 288 nm and plotted versus time. Sensor configuration
The cross section of the sensor (configuration 1 ) is shown in Fig. 1. A polished fiber (core diameter 200 /~m, outer diameter 240/~m) was placed in the tube system after cleaving, washing with conc. sulfuric acid and rinsing with distilled water. The tube system consisted of a piece of polyethylene tubing (length approx. 1.5 cm, outside diameter 1.6 mm), a piece of siloxane tubing (length approx. 1 cm, outside diameter 5 mm), and the insulation material of the fiber. All pieces fit together tightly, thus no further fixation was necessary but the formed chamber was adjustable to the size of the polymer by simply shifting the position of the tubing. Two slices of polymer (approximately 4 mm X 0.5 m m X 0 . 5 mm) were inserted between the Siloxane tubing and the fiber and were fixed by swelling in 0.1 N NaOH for 10 rain. Polymer that had swelled over the tip of the fiber was removed mechanically.
tubing
polymer
Fig. 1. Cross-sectionof the fiber optic sensor (Configuration 1). 2.4. Measurements
The measurement system has been described previously [4,5 ] and consisted of an argon ion laser (Spex. air cooled), a dichroic filter for separating excitation and emission signals and a monochromater with photomultiplier tube as the detection unit. The excitation wavelength was 488 nm and the emitted light was detected at 525 nm for fluorescein and at 620 nm for sulforhodamine. 0.2 M phosphate buffer, 0.4 M phosphate buffer, 0.2 M carbonate buffer and aqueous solutions of NaOH and HC1 were used to vary the pH. Qualitative release experiments could be conducted without calibration by simply moving the sensor from one buffer solution to another with a brief water rinse between measurements. In the case of calibration experiments with sensor configuration 1, the fiber with the sensor tip was kept in the medium and the pH was adjusted by addition of aqueous solutions of NaOH and HC1. pH values were simultaneously detected by a glass pH electrode. The baseline (background signal) was obtained by stirring the medium between the measurements to distribute accumulated dye. 2.5. Flow cell experiments
A cross section of the flow cell (configuration 2) is shown in Fig. 2. The lower part was separated from the
158
T.F. Liebert, D.R. Walt~Journal of Controlled Release 35 (1995) 155-163
upper part by a layer of filter glass and black filter paper to avoid direct irradiation of the dye-containing polymer. The lower part was filled with a homogeneous mixture of dye-containing polymer (particle size 0.225-0.45 mm) and controlled pore glass (pores 500 Angstrom). The mixture was poured in the tube and was treated with distilled water, stirred and sealed with filter paper. The lower part was connected to a peristaltic pump that delivered a constant flow of 14 ml per min. The fiber and the glass electrode were inserted in the upper part. The buffer solutions and wavelengths were the same as in the sensor experiments with configuration 1.
1 water out pH electrod(
fiber
3. Results and discussion
Several halfesters of hydroxypropylmethylcellulose 1 (HPMC) were synthesized (Fig. 3) and examined to determine if they could be used to prepare dyecontaining polymers. The maleate 2 was obtained from a heterogeneous esterification reaction of HPMC with maleic anhydride. This polymer began to dissolve when exposed to solutions at pH 3.12. The pKa of the polymer was determined to be 3.9 by titration [ 11 ]. Because of this low value and the possibility of undefined side reactions on the double bond, no further experiments were carried out with this polymer. In order to increase the polymer pKa value, the HPMC pyromellitate 3 was prepared using partially hydrolyzed pyromellitic anhydride. The resulting polymer was insoluble in common organic solvents and soluble in basic media only by irreversible saponification. The pKa value of the polymer was determined to be approximately 6.3. Pyromellitate-modified polymers could also be synthesized by a homogeneous reaction of partially hydrolyzed pyromellitic anhydride with HPMC in pyridine but the resulting products show the same insolubility. The insolubility probably results from competitive network formation via intermolecular crosslinking processes by unhydrolyzed dianhydride. It is not possible to dissolve these networks without saponification. Although the polymer is not reversibly soluble in basic media it shows pH sensitivity by swelling and shrinking processes. Comparable observations were made for polymers obtained from the reaction of unhydrolyzed dianhydride with HPMC. For the first release experiments the dye Sulforhodamine was chosen because of its solubility both in
--
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polymer and controlled pore glass filter paper
wa=er in
T Fig. 2. Cross-section of the flow cell used for calibration studies (configuration 2).
water and pyridine and its pH insensitivity. Therefore, changes in fluorescence intensity should reflect the degree of polymer hydrolysis. Dye-containing samples of the polymer were prepared by dissolving the dye and HPMC in pyridine and adding pyromellitic anhydride. The resulting gel was sliced and then purified by precipitation with dilute HCI. After drying, the polymer was cut in strips. Strips were placed with a needle in the container around the fiber and fixed by swelling in dilute NaOH as shown in Fig. 1. The optical system for the release studies consisted of a laser based fiber fluorimeter reported previously [ 4,5 ]. Excitation was at 488 nm with emission at 620 nm. Measurements were carried out by changing the medium (phosphate and car-
T.F. Liebert, D.R. Walt~Journal of Controlled Release 35 (1995) 155-163
backbone
159
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R" = H, HPMC-unit, HPMCP-unit,
R" = H, HPMCP-units Fig. 3. Structures of the polymers and their derivatives. bonate buffers with defined pH values). The sensor responded to pH values above 9 within 30 seconds. Unexpected signals were obtained in acidic media and in solutions with pH values around 7. These results will be discussed below. The prepared sensor was applicable for at least 20 measurements (pH > 9) but released the dye within 2 weeks during storage in pH 7 phosphate buffer. Similar results were obtained with fluorescein-containing polymers.
The limitations of the maleate and pyromellitate, including their low pKas and rapid release prompted us to examine commercially available HPMC phthalate 4. This polymer has a pKa of 3.82 and starts to dissolve at pH 4.23. Sensors were prepared from HPMCP and Sulforhodamine by dissolving both substances in pyridine and evaporating the solvent. Such sensors respond to pH values above 5, however, they can be used only for one measurement because of their high dissolution
T.F. Liebert, D.R. Walt~Journal of Controlled Release 35 (1995) 155-163
160 2O
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rate and their constant release even in strong acidic solutions. Consequently, the rate of dissolution and the structure of the polymer matrix had to be changed. Attempts to vary the dissolution behavior of HPMCP by simple esterification of the acid residues were unsatisfactory. Crosslinking the polymer was considered to be a promising method, however, the formation of a network leads to a dramatic increase in the dissolution time. Crosslinking was carried out by intermolecular esterification of the free phthalic acid residues of the polymer with free hydroxyl groups on the cellulose backbone. Esterification was effected with tosyl chloride in pyridine. Reactions were carried out at room temperature in the absence of moisture resulting in formation of a gel. The resulting polymer 4a was isolated by precipitation with water and dilute HC1. The dissolution time was determined with a UV-Vis spectrometer. A plot of the absorbance at 288 nm versus time provides information about the dissolution rate as both phthalate esters and acids absorb at this wavelength. Four crosslinked samples were prepared using different amounts of tosyl chloride and the dissolution times in 0.1 N NaOH were determined (Fig. 4). The resulting polymers exhibited bimodal dissolution behavior characteristic of the starting HPMC [ 16]. Because of an observed stronger influence of the dye sulforhodamine on the release behavior caused by its free solubility in water and its ampholytic character, the less soluble and acidic dye fluorescein was used for
the release studies. The samples utilized were obtained from the reaction of tosyl chloride with HPMCP in pyridine in the presence of fluorescein. Measurements were carded out in the above-mentioned manner by using 488 nm light for excitation and determining the emission signal at 525 nm. The prepared sensor responded within 30 s to pH values above 9. The signals responded rapidly when the pH was changed to values below 7.5 however, within two min another signal appeared. Depending on the pH of the initially applied basic solution and the time of contact with basic solution, up to three additional signals appeared. This behavior can be explained by assuming that the basic medium is entrapped by the polymer matrix via fast formation of a thin stiff layer of protonated polymer around a highly swollen particle. This process is followed by the dissolution of this thin layer by the base from the inside out. An equilibrium can be established and a new layer of insoluble polymer is formed. The result is the occurrence of a new smaller signal. This conceptual model would explain the periodicity of the observed subsequent signals. One possible way to avoid this process would be to introduce spacer groups into the polymer structure to increase its accessibility and consequently slow down the formation of the insoluble layer. Resorcinol was used as a spacer unit to provide polymer 4b. Different amounts of crosslinking reagent were applied to the polymer to determine the dependence of the dissolution time on the degree of crosslinking. The reactions were carded out by dissolving the polymer, resorcinol and tosyl chloride at room temperature in pyridine. The molar ratio of tosyl chloride to the resorcinol spacer and crosslinker was 4" 1. The amount of free acid groups of the polymer per dehydrating reagent in the reaction mixtures was varied from 1:2 up to l:12. Dissolution experiments were carried out by placing the polymer in 0.1 N NaOH and monitoring dissolution at 288 nm. These experiments showed that an increase of the molar ratio of free acid to dehydrating reagent beyond one to eight does not result in a significant increase in the dissolution time. The maximum value achieved for the dissolution of 0.05 gram of polymer in l0 ml 0.1 N NaOH was 165 rain. A bimodal dissolution behavior is observed once again for these derivatives. Fluorescein- and sulforhodamine-containing polymers were then prepared. Fluorescein sensors responded within 30 s to pH values above 9 and the
T.F. Liebert, D.R. Walt~Journal of Controlled Release 35 (1995) 155-163
signal decay after changing the pH below 7.5 was almost as fast as in the previously discussed case but the subsequent signals disappeared almost entirely. Sulforhodamine containing samples were prepared to examine how this highly water-soluble, ampholytic dye is released over the entire pH range and whether it is possible to generalize the observations. The same crosslinking reaction was carried out with resorcinol. Measurements were performed in the above-mentioned manner with an excitation wavelength of 488 nm and emission at 620 nm. The response of this sensor was as fast as in the fluorescein case and the base line was equally stable. Usually the signal decay took longer (up to 2 min). When the sensor was exposed to basic medium, followed quickly by pH 7 and then with acid solution, a strong signal was obtained that disappeared almost entirely within a few min. We had already determined that dissolution of the polymer does not occur even in strong acidic solutions (UV-Vis experiments). This result can be explained by a 'squeezing effect'. When the pH is changed from slightly basic to acidic conditions the swollen polymer shrinks and the entrapped dye-saturated medium is squeezed out. A second possible reason for an unexpected release pattern could be the ionic strength of the medium. It was found that by changing the concentration of pH 7 buffer solutions from 0.4 M to 0.2 M and back, a signal is obtained for the sulforhodamine-containing polymer. The signal is due to osmotic effects. This influence on the release behavior occurs only in the region around pH 7-8. At higher pH values, it is not detectable because of a preferred erosion mechanism due to hydrolysis. The influence of temperature on the release was not investigated. The amount of dye has a strong influence on the release pattern. Dyes are generally hydrophilic and increasing the content of the water soluble dye increases the dissolution rate. Dissolution and release times for samples with different amounts of dye in 0.5 N NaOH were determined by UV-Vis analysis (Fig. 5). As can be seen, the lower loadings provided for greater release times. Since fluorescence detection is not limiting, we used samples with 1% dye loadings for our experiments. We next assembled a working demonstration of how such polymers could be used for continuous monitoring. A Sulforhodamine-containing resorcinol-crosslinked HPMCP sample was dried at room temperature.
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Amountof Dye(%) Fig. 5. Hydrophilizingeffect of the increasing amountof Sulforhodamine on resorcinolcrosslinked HPMCPillustrated by decreasing dissolution and release times. After three weeks' storage, it showed exactly the same release behavior as the freshly prepared polymer. A sensor was prepared with this material (Fig. 1) and kept in phosphate buffer at pH 7. After four months' storage, the sensor was exposed to base. A signal appeared after about three min of base exposure. The lag in response was probably due to a dye-depleted layer at the polymer surface as a consequence of slow leakage upon storage. Our attempts to calibrate the sulforhodamine-containing derivatized HPMCP were unsuccessful because no clear correlation was observed between the applied pH values and the signal intensities. The reasons could be the free water solubility of the dye or the capability of the ampholytic dye to buffer the interface layer to its own isoelectric point [ 17]. We next turned to the fluorescein-containing polymer in a sensor configuration (Fig. 1). Measurements were carried out by using aqueous solutions of 0.1 N NaOH and HC1 to adjust the pH of the medium. The baseline was obtained by constant stirring between measurements to distribute accumulated dye. As can be seen in Fig. 6, signals returned to baseline upon stirring, but rapidly went back to the steady state values when stirring was stopped. Although the sensor showed good pH sensitivity the signal levels were low, resulting in low precision. To obtain stronger and more stable signals and to avoid several irreproducible variables caused by the small interface between polymer and medium, it was
T.F. Liebert, D.R. Walt~Journal of Controlled Release 35 (1995) 155-163
162
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necessary to increase the reactive surface of the polymer. For this purpose a flow cell was constructed as shown in Fig. 2. It consisted of two chambers that were separated by a layer of filter glass. The upper chamber contained the fiber to detect the released dye, a glass electrode to measure the pH, and a black filter on the bottom to avoid photobleaching of the dye entrapped in the polymer. The lower chamber contained the polymer and was covered by a filter. A blend of polymer 100
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and controlled pore glass must be used, otherwise the polymer swells and occludes the flow cell thereby preventing flow through the system. Dilute solutions of NaOH or buffers were used as media and were pumped with a constant flow of 14 ml/min from the lower chamber through the upper chamber over periods of five rain. Usually the system responded at the same time a change in pH was detected by the pH electrode. Two measurements per pH value were carried out and the obtained signal intensities were plotted versus the pH values. The calibration curve obtained by this procedure for fluorescein-containing resorcinol-crosslinked HPMCP shows an excellent sensitivity to basic pH values (Fig. 7). The fluorescein intensity changes exhibited by the system are not due to the pH sensitivity of fluorescein because it is fully ionized at pH values above 8 [ 18]. Above this value no further ionization is possible and consequently no further increase of the fluorescence intensity is possible. We conclude that the dependence of the intensity on pH is due to a controlled dye release. Measurements performed over the course of three days still fit this curve. The system can be used to detect pH values above 10.5 in aqueous solution. The sensitivity of simple crosslinked HPMCP, was similar to the resorcinol-crosslinked polymer (Fig. 8). This polymer exhibits a small but measurable release at pH 9.2. In
T.F. Liebert, D.R. Walt/Journal of Controlled Release 35 (1995) 155-163
m e r matrix and not e x p o s e d continuously to solution, there is little chance that dye degradation or reaction with interfering substances will occur. Finally, it should be noted that p o l y m e r s with different pH sensitivity ranges as well as sensitivity to other analytes (e.g., organic solvents) can be prepared to broaden the applicability o f such a transduction m e c h a n i s m .
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pH Fig. 8. Calibration curve of crosslinked, fluorescem containing HPMCP obtained by using sensor configuration 2 and aqueous solutions. s o m e e x p e r i m e n t s with this p o l y m e r the p r e v i o u s l y m e n t i o n e d sporadic signals did o c c u r m a k i n g it a less desirable material.
4. Conclusion W e h a v e d e m o n s t r a t e d the use o f resorcinol crosslinked H P M C P in c o m b i n a t i o n with the dye fluorescein for construction o f an ' a l a r m / t h r e s h o l d ' - t y p e sensor to detect p H values in the basic region ( a b o v e p H 9) with a 30 s response time. O p t i m i z a t i o n of the sensor configuration will further i m p r o v e its p e r f o r m ance with respect to reduced response t i m e and i m p r o v e d signal levels. T h e overall principle o f this sensor differs significantly f r o m other p H sensors in that the p H sensitivity is a function o f the p o l y m e r , not the indicator dye. Seitz and c o - w o r k e r s h a v e reported p H sensors based on p o l y m e r s w e l l i n g [ 19 ] and changes in p o l y m e r opacity [ 2 0 ] . T h e present sensor can be d e s i g n e d so that it is always in an o f f state, unless a threshold p H is e x c e e d e d . Such sensors should be o f v a l u e in process control and e n v i r o n m e n t a l m o n i t o r i n g in w h i c h only o c c a s i o n a l deviations f r o m nominal p H values are expected. B e c a u s e the dye is trapped within the poly-
[ 1] M.A. Arnold, Fiber-optic chemical sensors, Anal. Chem. 64 (1992) 1015-1025. [2] C. Camera, M.C. Moreno, G. Orellana, For a comprehensive review on pH fiber-optic sensors, in: D.L. Wise and L.B. Wingard (Eds.), Biosensors with Fiber Optics, Humana Press, Clifton, NJ, 1991, pp. 37--43. [3] O. Wolfbeis (Ed.), Fiber Optic Chemical Sensors and Biosensors, CRC Press, Boca Raton, FL, 1991, Vols. 1 and 2. [4] C. Munkholm, D.R. Walt, F.P. Milanovich and S.M. Klainer, Anal. Chem. 58 (1986) 1427. [5] S. Luo and D.R. Walt, Anal. Chem. 61 (1989) 174. [6] S.B. Barnard and D.R. Walt, Science 251 ( 1991 ) 927-929. [7] R.M. Ottenbrite, in: L.G. Donaruma, R.M. Ottenbrite, and O. Vogel (Eds.), Anionic Polymeric Drugs, Wiley, New York, 1980, p. 21. [8] S. Matsumara and S. Yoshikama, in: J.E. Glass, G. Swift, (Eds.), Agricultural and Synthetic Polymers, American Chemical Society, Washington DC, 1990, p. 124. [9] C. Brand, C. Bunel, and M. Vert, Polym. Bull. 13 (1985) 293. [10] M.E.K. Krealing and W.A. Ritschel, Methods Find. Exp. Clin. Pharmacol. 14(3), (1992), 199. [11] J.H. Kou, D. Fleisher, and G.L. Amidon, Cosmet. Pharm. Appl. Polym. 1990 (Pub 1991), 201. [12] J. Heller, R.W. Baker, R.M. Gale, and J.O. Rodin, J. Appl. Polym. Sci. 22 (1978) 1991. [13] M. Schnabelrauch, T. Heinze, and D. Klemm, Acta Polymerica 41(2), (1990), 112. [14] P.C. Schmidt and F. Niemann, Drug. Dev. Ind. Pharm. 18(18) (1992) 1962. [151 P. Giunchedi, U. Conte, and L. Maggie, Int. J. Pharm. 85( 13) (1992) 141. [ 16] A.C. Shah, N.J. Britten, L.S. Olanoff, and J.N. Badalamenti, J. Control. Release 9 (1989) 169. [ 17] J.C. Shah and M. Maniar, J. Control. Release 23 (1993) 261. [ 18] C.-S. Chen, H. Nakumura, and Z. Tamura, Chem. Pharm. Bull. 27(2) (1979) 475. [ 19] S. Pan, V. Conway, Z.M. Shakhsher, S. Emerson, M. Bai, W.R. Seitz, K.D. Legg, Anal. Chim. Acta 1993, 279, pp. 195-202. [20] Z. Shakhsher, W.R. Seitz and K.D. Legg, Single fiber-optic pH sensor based on changes in reflection accompanying polymer swelling, Anal. Chem., 66, 1994, pp. 1731-1735.