Anal. Chem. 1989, 61,174-177
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Fiber-optic Sensors Based on Reagent Delivery with Controlled-Release Polymers S h u f a n g L u o and David R. Walt*
Max Tishler Laboratory for Organic Chemistry, Department of Chemistry, Tufts University, Medford, Massachusetts 02155
Two configurations of long lasting flbersptlc sensors have been prepared and used to measure pH values continuously. These sensors are based on contrdled-reiease polymers, whlch pennit the sustained release of dyes over long periods of time. I n the fkot approach, 8-hydroxypyrens1,3,6-trlsulfonic acid (HPTS) was entrapped into an ethylene-vinyl acetate (EVA) polymer matrix and released upon contact with aqueous solution. The ratio of fluorescence emlsslon intensitles at 515 nm resutting from excitatfon at 405 and 450 run was employed to measure pH values from 5.5 to 8.0 with a minimum precision of fO.07 pH units In the pK, region. I n the second approach, a mixture of two dyes was employed: HPTS, whlch is pH sensitive, and sulforhodamine 640 (SR640), which is not pH senoltlve. I n this system, both dyes were released from the EVA polymer at virtually the same rate upon contact with aqueous solution. The ratio of fluorescence emlsdon intmsltks at 530 and 610 rm resulling from excitation at 488 nm was used to quantify pH from 5.5 to 8.0 with precision of at least f O . l pH unit determined in the pK, reglon. Both types of sensors have been demonstrated to last for at least 3 months in the laboratory.
INTRODUCTION Fiber-optic sensors are becoming established analytical tools for remote and in situ sensing (1-6).An ideal optical sensor must have the ability to measure the concentration of an analyte continuously and reversibly through changes in the optical properties of the sensing reagent. To date, this ability has been based on the availability of suitable, long-lasting, reversible chemistries. Many colorimetric or fluorometric techniques are irreversible because they form a tight binding complex or utilize reagents that generate an irreversibly colored adduct. Irreversible sensors can be used if they operate in an integrating mode; however, they must be replenished frequently with fresh sensing reagent. We have now solved this problem by developing a polymeric delivery system capable of delivering fresh sensing reagents for long periods. Although the sensing reagent we employed responds to the analyte reversibly, the principle of delivering fresh sensing reagent applies equally to irreversible reagents. Polymeric delivery systems have been useful vehicles for the sustained release of macromolecules such as polypeptide hormones (e.g., insulin), polysaccharides, antigens, antibodies, and enzymes (7).Their application in immunization and in vivo inhibition of tumor growth has also been explored (8, 9). This paper describes long lasting pH fiber-optic sensors based on controlled release of pH-sensitive fluorescent dyes. The dyes are incorporated into ethylene-vinyl acetate (EVA) copolymers and released slowly upon contact with aqueous solution. Two methodologies have been developed to demonstrate the adaptation of the sensor to different indicating systems. In the first approach, a pH-sensitive fluorescent dye, 8-hydroxypyrene-l,3,6-trisulfonic acid (HPTS),was incorporated into an EVA copolymer and the ratios of fluorescence intensities (A, = 515 nm) resulting from excitation at 405 and 0003-2700/89/0361-0174$01.50/0
450 nm were used to quantify pH. The second approach utilized a mixture of two fluorescent dyes: HPTS, which is pH sensitive, and sulforhodamine 640 (SR-640), which is not pH sensitive. Upon excitation by an argon laser at 488 nm, the ratios of the two fluorescence intensities measured at 530 and 610 nm were used to measure the pH of the solution. Both approaches can measure pH values between 5.5 and 8.0, and do so continuously and with good precision for more than 3 months.
EXPERIMENTAL SECTION Materials. Ethylenevinyl acetate copolymer (40% by weight vinyl acetate, Elvax 40),in pellet form, was obtained from Du acid, Pont, Wilmington DE. 8-Hydroxypyrene-l,3,6-trisulfonic trisodium salt (HPTS) was purchased from Molecular Probes, Eugene, OR. Sulforhodamine 640 (SR-640) was purchased from Exciton, Dayton, OH. Teflon tubes were obtained from ColeParmer, Chicago, IL. Apparatus. Two different instrumentation systems were used in the two experimental approaches described below. The first system, described previously (IO),employs a 488-nm argon-ion laser as the excitation source. Light is conducted through a series of lenses and filters into an optical fiber (CorningCore Guide glass numerical aperture = 0.28). The fluorescence is conducted back through the same fiber and reflected by a dichroic mirror to a photomultiplier tube. The intensity of the fluorescence is measured as a function of emhion wavelength with a photon-counting detection system (Pacific Instruments, Model 126). The second system was used to measure flourescence intensity at different excitation wavelengths with a fixed emission wavelength and consists of four basic components: a variable wavelength light source for excitation, an optical system for conducting light into the sensor and to the detector, an emission detection system, and computer control and data acquisition system. The excitation light source consists of a 75-Whigh-pressure xenon arc lamp (Osram Co.) which gives a continuous spectrum from 190 to 750 nm and a Spex 1680 0.22-m double monochromator for selecting any specified wavelength light. The use of a double monochromator ensures low stray light levels in the excitation. The optical system consists of lenses and mirrors that focus the excitation light onto the fiber, retrieve the emission light from the fiber, and focus it onto the entrance slit of the emission detection system. The emission detection system is comprised of a second Spex 1680 0.22-m double monochromator with a 300 lines/mm grating and an RCA 31034A-02 photomultiplier tube. The detected signal is then processed by a photometer. Finally, a PC-AT with an AD (analog to digital) and DA (digital to analog) board is used to acquire and display the data and to control the movements of the stepping motors in the excitation and emission spectrometers. Sensor Construction. The cross section of a pH sensor based on the polymer delivery system is shown in Figure 1. One end of a glass fiber (200/250 pm) was cleaved with a carbide tip to ensure a smooth surface, washed in concentrated sulfuric acid, and rinsed with distilled water. To prepare a sensor, the fiber wm first inserted into a 1.5 cm long Teflon capillary tube (A) with inside diameter (i.d.) of 1/32 in. and outside diameter (0.d.) of 1/16 in. and was then inserted into a 1-cm Tygon tube (B)of in. i.d. and 3 / l e in. 0.d. This Tygon tube was then connected to a polymer reservoir made of another Teflon tube of 3/la in. i.d. and in. 0.d. A 60-mg polymer slab with dimensions 9 mm X 4 mm X 1 mm was placed around the wall of the reservoir. The third 0 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 2, JANUARY 15, 1989
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. L H
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15r
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5
15
25
20
30
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H
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Flgure 2. Kinetic study of the release rate of HPTS from polymer matrices. The concentration of HPTS was determined by measuring UV absorption at 405 nm. Table I. Effect of Source Intensity Variation ([HPTS] = 10" M, pH 8) current, A
Figure 1. Cross section of a pH sensor: (A) Teflon capillary tube in. i.d., '/le in. 0.d.); (6)Tygon tube in. i.d., 3/la in. 0.d.); (C) Teflon tube (3/rs in. i.d., in. 0.d.); (D) Teflon tube ('/le in. i.d., 1/4 in. 0.d.) with tiny holes (0.25-0.36mm); (E) plastic coating; (F) polymer matrix; (0)Parafilm; (H) glass fiber. Teflon tube (1/16in. i.d., 1/8 in. o.d.), having many holes (0.25-0.36 mm) drilled through its wall (D), was sealed at one end with Parafilm and wrapped with Parafilm at the other end to allow it to fit snugly into the polymer reservoir. These holes allow the exchange of solutions on both sides of the tube. The sensor was filled with buffer before sealing. No bubbles were introduced in the sensing region. All pH measurements were performed in phosphate-citric acid buffers. The sensor was always stored in buffer when not in use. Polymer Matrix Preparation. Ethylene-vinyl acetate copolymer (EVA) pellets were first washed at least three times in distilled water with constant stirring. The EVA pellets were then extracted in a Soxhlet extractor with high-quality acetone for at least 3 days. The EVA pellets were then quickly removed from the paper thimble at the end of the extraction while the acetone was still hot. Finally the EVA pellets were dried in a desiccator under house vacuum for at least a week. Drying was complete when all acetone had evaporated. The EVA copolymer was dissolved in methylene chloride to give a 10% (w/v) solution. A weighed amount of HPTS was added to 15 mL of polymer solution in a glass vial to give a dye loading of 33% (in the case of the HPTS and SR-640 mixture, a one to one ratio was employed, and the total loading in the polymer was 33%). The mixture was vortexed to yield a uniform suspension. After vortexing, the mixture was poured quickly into the center of a glass mold (5 X 5 X 2 cm), which had been cooled previously in a freezer for 20 min. The mold was covered and remained in the freezer until the mixture froze (about 30 min). The frozen slab was easily pried loose with a cold spatula, transferred onto a wire screen, and kept in the freezer for 2 days. The slab was dried for an additional 2 days at room temperature in a desiccator under house vacuum. Dye Release. The dried slab was weighed and cut into nine small pieces. Each piece was weighed and placed in a test tube containing 10 mL of 0.015 M phosphate buffer, pH 7. These test tubes were shaken continuously at 50 rpm and kept at room temperature. Periodically, the dye-containingpolymer pieces were transferred with forceps into test tubes containing fresh buffer. During the transfers, excess solution on the matrix surface was removed by gentle blotting on a tissue. The concentration of HPTS in the test tubes was determined by UV absorption at 405 nm.
RESULTS AND DISCUSSION The construction of the sensor device is a critical element in its performance. Prior to the pH sensor design shown in
4 5 6 OX,,
= 405 nm; A,
A,O
kcps
41.5 68.5 70.0
B,b kcps
ratio A I B
164 279 275
0.253 0.246 0.255
= 515 nm. b X e , = 450 nm;
km = 515 nm.
Figure 1,a sensor was designed with the polymer placed on the bottom of the sensor tube. Some disadvantages associated with this arrangement were readily apparent. When the polymer was below the fiber tip, the emerging light from the fiber was reflected by the polymer surface and part of this reflected light reentered the fiber resulting in a systematic error in the signal. Therefore a longer sensing region was employed to minimize this back-reflected light. This configuration, however, resulted in longer sensor response times. Another disadvantage with this design was the buildup of a large concentration gradient of dyes around the polymer. Thii concentration gradient constantly changed with time, causing instability even in the ratio measurement. The sensor construction that overcame these drawbacks was prepared by placing the polymer in a reservoir-like section above the fiber tip. In this way, reflected light from the polymer was not a problem. In addition, variation in the concentration gradient of the dyes around the fiber tip was minimal. An additional advantage of this configuration was the ability to enlarge the polymer reservoir section, thus making the sensor last longer. Based on the results of Langer et al. (7), an optimal total dye loading of 33% was used. Above this loading, a rapid release of dye was observed probably resulting from the higher porosity of the polymer matrix, facilitating the rapid diffusion of the dye out of the matrix. The sensor containing both HPTS and SR-640 employed a one to one ratio of the dyes; no experiments were performed to optimize the dye ratio in the polymer. This ratio was sufficient to obtain good sensitivity for pH measurements. The resulta of total HPTS release from a polymer matrix are shown in Figure 2. The initial rapid release was presumably due to HPTS dissolution from the polymer surface. After this initial period, the release of HPTS became constant and continued for an extended period. The sensor containing HPTS alone produced the pH data shown in Figure 3. Two excitation wavelengths at 405 and 450 nm were used to excite the acid and base forms of HPTS, respectively (11,12).Both forms of the dye emit a t 515 nm. The measured emission intensity ratio was insensitive to the source intensity (see Table I) or the amount of HPTS in the
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 2, JANUARY 15, 1989
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Ffgws 3. Data produced by pH fiber-optic sensor based on HPTS. Signels were produced with excitation at 405 and 450 nm, observation was at 515 nm. Each point represents the mean of 20 measurements. Standard deviations were less than 5 % of the respective mean values.
PH Figure 4. Data produced by pH fiber-optic sensor based on two dyes: HPTS and SR-640. Signal was produced with laser excitation at 488 nm; observations were at 530 and 610 nm. Each point represents the mean of 20 measurements. Standard deviations were less than 10% of the respective mean values.
Table 11. Fluctuation of HPTS Sensor at pH 6.5 (Representative Data)
Table 111. Response Time of HPTS Sensors (min/pH unit)
time, days
A: kcps
B,b kcps
ratio AIB
7 18 22 28 38
191.5 367 142 417 677
57.5 110 42 123 201
3.33 3.34 3.38 3.39 3.37
sensing length 3 mm
sensing length 6 mm
hole diameter
a
b
a
b
0.25 mm 0.36 mm
35 10
160
50 15
300 90
60
= 405 nm; brn = 515 nm. b X e x = 450 nm; he, = 515 nm.
"Data shown were measured with stirring. bData shown were measured without stirring.
sensing region at the time of measurement. Table I1 contains some representative data of an HPTS sensor. On each day of measurement the concentration of HPTS in the sensor was different and produced changing absolute readings of fluorescence intensities; however the measured ratios remained constant. The useful concentration range of HPTS in the sensor was found to be to lo4 M as determined from standard solutions of HPTS. Below lo4 M, the ratio varied due to the relatively high background readings of the instrument. Above lo4 M, the intensity of fluorescence was too high to be readable on the photon-counting detector. Therefore, we measured the intensity ratio only when the direct intensity readings exceeded 30 kcps (thousand photon counts per second) in order to minimize experimental deviation. From these data, pH values in the range of 5.5-8.0 can be measured with a minimum precision of f0.07 pH units determined in the pK, region. The second method employed a mixture of HPTS and SR-640 and produced the intensity ratio M pH response curve shown in Figure 4. SR-640 was chosen as a reference dye because it can also be excited by the 488-nm line of the argon ion laser and has a longer emission at 610 nm. The emission intensity ratio of 530 nm to 610 nm represent the fluorescence from the base form of HPTS to the fluorescence from SR-640, respectively. The release rate of the two dyes is critical in this case since SR-640 serves only as an internal reference. Variations in the release rate of the dyes would cause significant changes in the accuracy of the ratios. Therefore, before both dyes were entrapped into the EVA polymer, they were dissolved in water and thoroughly mixed; water was then removed by lyophilization. With premixing, the dyes should be uniformly incorporated into the polymer and be released at virtually the same rate. However, the precision (fO.l pH units measured in the pKa region) of this method in the pH range of 5.5-8.0 is not as good as the sensor that uses HPTS alone. On the other hand, the use of a single excitation wavelength in this sensor offers the potential for a simpler
instrument design for field applications. On comparison of Figure 3 and Figure 4, the apparent pKa of the HPTS sensor is shifted toward a lower value. This result can be explained by inner filter effects at high dye concentrations associated with absorption of both acid and base forms of HPTS as discussed in ref 11. Since our sensor has a slow response time, the concentration of HPTS in the sensing region is in the regime where inner filter effects are significant. The fluorescence intensity ratio measurement does not compensate for this effect due to the difference in absorptivity of the two forms at different pH values. To illustrate this point, we consider the ratio at a pH value where the base form (450 nm) has a small but significant absorption. Because the absorption is small, little inner filtering exists leading to efficient excitation and an unperturbed emission intensity. On the other hand, the acid form (405 nm) exhibits significant inner filter effects at low pH resulting in inefficient excitation leading to lower emission intensity. Therefore the resulting ratio is decreased relative to more dilute solutions. The two-dye sensor does not exhibit this shift because only one excitation wavelength is utilized. The response time per pH unit change of a sensor is a function of its design and the conditions of the solution being measured. Here we define the response time as the time required to reach 95% of the steady-state signal. Stirring greatly enhanced the rate for establishing equilibrium between the analyte solution and solution inside the sensor; the response time decreased by a factor of 6 (from 60 min to 10 min, as shown in Table 111). When the hole in the sensing region was enlarged from 0.25 to 0.36 mm, the response time decreased by a factor of more than 3 (from 35 min to 10 min). Furthermore, shortening the sensing region length by half decreased the response time by a third (from 15 min to 10 min). The response time of this type of sensor is longer than for other pH sensors with immobilized reagents on the fiber tip. However it is acceptable when sensors of this type are used in remote sensing applications for extended periods of
Anal. Chem. 1989, 6 1 ,177-184
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time. These sensors have worked in the laboratory for more than 3 months without any change in sensitivity or precision. Fiber-optic chemical sensors based on continuously released dyes have been prepared by trapping reagents into an EVA polymer matrix. This technique is simple and straightforward. It overcomes the limitation of using only reversible indicating systems to prepare fiber-optic sensors. This approach should allow optical sensors to be used in remote sensing of groundwater or hazardous environments by linking a spectrophotometer to the remote environment through optical fibers. Although we have developed a pH sensor to prove the feasibility of this technique, the more fundamental goal was to prove the suitability of this type of continuous sensor as a means of releasing reagents capable of detecting other chemicals or metal ions. Finally, the use of continuous release reagents should have other analytical applications such as in flow injection analysis and chromatographic detection.
preparing the polymer matrix, and Ms. Christiane Munkholm and Professor Rudolf Seitz for helpful discussions. Registry No. (EVA) copolymer, 24937-78-8; HPTS (trisodium salt), 6358-69-6; SR-640, 60311-02-6.
ACKNOWLEDGMENT We wish to thank Dr. Fred Milanovich for his assistance in designing and constructing the optical systems, Professor Robert Langer (MIT) and Ms. Liz Albert for their help with
RECEIVED for review July 28,1988. Accepted October 26,1988.
LITERATURE CITED Angel, S. M. Spectroscopy 1987, 2 , 38-48. Hliilard. L. A. Anal. R o c . 1985, 22, 210-224. Bolsde, G.; Blanc, F.; Perez, J. J. Talanta lg88, 35. 75-82. Wolfbeis, 0.S. Pure Appl. Chem. 1987, 59. 663-672. Seitz, W. R. Anal. Chem. W84, 56,16A-34A. Seitz, W. R. CRC Crit. Rev. Anal. Chem. 1988, 19, 135-173. Rhine, W. D.; Hsleh, 0. S. T.; Langer, R. J . Pharm. Sci. 1980, 69, 265-270. Langer, R. Meth. Enzymol. 1981, 73,57-75. Langer. R. CHEMTECH 1982, February, 98-105. Munkholm, C.; Walt, D. R.; Mllanovich, F. P.; Klalner, S. M. Anal. Chem. 1988, 58,1427-1430. Zhang, 2.; Seitz, W. R. Anal. Chim. Acta W84, 160, 47-55. Offenbacher, H.; Wolfbeis, 0. S.; Fiirllnger, E. Sens. Actuators 1986, 9 . 73-84.
This work was supported by a grant from the Environmental Protection Agency through Tufts Center for Environmental Management.
Conductometric Titrations of Polyprotic Acids in Nonaqueous Mixed Solvents. Effects of Temperature and Composition of the Solvent Mixture Giancarlo Franchini, Andrea Marchetti, Carlo Preti, Lorenso Tassi, and Giuseppe Tosi*
Department of Chemistry, University of Modena, Via G. Campi 183, 41100 Modena, Italy
The effects of temperature and composltlon of the solvent mixtures on the shape and on the analytical recoveries of conductometric tltratlons of polyprotlc aclds were studled for the solvent system 2-methoxyethanoVethane-1,2-dloi operating at -10, 25, 50, and 75 ‘C. The experimental evidence indicates that the N- or chair-shape of the tltratlon curves depends on the solvent physicochemical propertles, on the temperature, and on the acld solute to be tltrated; in particular the Influence of the distance between the carboxylic groups Is discussed. Titrations performed In 2-methoxyethanoi are associated in general wlth N-shaped curves, which however turn to chalr-shaped as the temperature Increases. Ethane1,2-dloi always produces chair-shaped curves as a consequence of its more dlssoclating ability toward the tltratlon formed adducts. The study of the tltrations of phthalic acld In some mixtures of the above solvents exhibited behaviors consistent wlth previous observations regarding the exlstence of a partlcuiar “ilmltlng mlxture” whlch separates the solvent system under study Into two well-defined groups (0 I x . ~ < “limiting ~ ~ mixture” ~ ~and “limiting , ~ mixture” ~ ~< x ~ I1). The ~ feaslblilty~ of the resolution ~ of ~acid mixtures has been demonstrated by tltrating cltrlc acld in the above solvents.
INTRODUCTION In the last years alcohols have received good attention as solvents for acid-base determinations, although not many 0003-2700/S9/0361-0177$01.50/0
conductometric studies appeared about their mixtures (1-3). The technique of the titrimetric determination of weak electrolytes in nonaqueous media can provide useful information about the formation of ion pairs or other ionic aggregates during the titration (1,2); in particular for conductance measurements, a careful choice of the solvent system can enable the differentiation of single functions present either in different molecular units ar in the same molecule. In our previous works (4-7) conductometric titrations of monoprotic and polyprotic acids, phenols, and aromatic nitro derivatives in pure 2-methoxyethanol (Gliem) and ethane1,2-diol (Gliet) using N,”-diphenylguanidine (DPG) titrant solutions in the above solvents were presented; the choice of solvents and titrant was suggested by their physical and chemical properties (4-8). In a recent paper (8) we reported the results obtained by studying the effects of the solvent properties and of the temperature on the conductometric titrations of monoprotic acids and phenols. In the same work a detailed study was done on the titrations of picric acid at various temperatures in a series of binary mixtures of Gliem and Gliet covering the 13-49 dielectric constant range. of our studies deals with the , The present ~ development ~ investigation of the effects of the above-mentioned factors on the titration curves of polyprotic acidic species. The shapes of the titration curves of bivalent acids and bases in nonaqueous media depend on temperature, solute, solvent, and titrant used and it is clear that the form of the titration curve is important for practical purposes. For certain bivalent compounds N-shaped curves can be obtained in some solvents, 0 1989 American Chemical Society
