Anal. Chern. 7902, 5 4 , 821-823
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CORRESPONDENCE pH Sensor Based on Immobilized Fluoresceinamine Sir: We are interested in developing sensors based on immobilized reagents whose fluorescence characteristics change with pH and/or metal ion concentration. The first system that we have investigated in detail is a pH sensor based on immobilized fluorenceinamine. This system was chosen because it is easy to work with. Amino compounds are readily immobilized to solid supports, and fluorescein is an efficient fluorophor. In addition, since acid-base reactions are fast, there should be no complications due to slow kinetics. Using this system we have demonstrated the viability of sensors based on immobilized fluorigenic reagents and defined some of their characteristics. To our knowledge, a pH sensor based on fluorescence has not previously been reported in the literature. However, research on fluorescenlce sensors is in progress a t other laboratories and has been written up in the FOCUS section of the Dec 1981 issue of ANALYTICALCHEMISTRY.A nonelectric pH sensor based on fiber optics and dye absorption has been described ( I ) . EXPERIMENTAL SECTION Apparatus, Figure 11 diagrams the fluorescence sensor and ita associated instrumentation. It consists of a tungsten halogen lamp (250 W, 5000 lumens, Edmund Scientific),a bifurcated fiber optic threaded to fit the other components (diameter 4.5 mm), a photomultiplier tube (RCA 1P21 operated at 700 V), a photomultiplier tube housing with a variable slit width and the capacity to hold filters, a digital photometer/power supply (SPEX DPC -2), and two dielectric inlmference filters (1in. X in., Edmund Scientific). The excitation filter has peak transmittance at 480 nm and a bandwidth of 7.1 nm at half-maximum transmittance. The emission filter has peak transmittance at 520 nm and a bandwidth of 8.2 nm. The signal from the photometer is recorded on a Heath SR-255Bstrip chart recorder. The sample is contained in a 15-mL beaker covered by a lighttight aluminum casing with an injection port. A shutter in front of the detector excludes ambient light when the aluminum casing is removed. The immobilized fluoresceinamiineis attached to cellophane tape which is held on the end of the optic by a piece of Tygon tubing. An Orion Digital Ionalyzer/501 was used to measure pH. Reagents. Fluoresceinamine (isomer I) and cyanuric chloride were purchased from Aldrich. Glycophase G controlled pore glass (CPG/460) was purchased from Pierce. Powdered cellulose (microcrystalline for TLC, through 60 seive, Baker) was used as the cellulose support. Immobilization Procedure. The fluoresceinamine was immobilized on controlled pore glass by a modification of a previous procedure (2). Potassium periodate was used in place of sodium periodate to oxidize the glass support. The oxidized glass was soaked in a solution saturated with fluoresceinamine in borate buffer at pH 8.5 for 10 days at room temperature before washing. The sodium borohydride reduction step was omitted. The derivatized glass was dried by suction and stored at room temperature. The fluoresceinamine was immobilized on cellulose by a modification of a previous procedure (3). The cellulose was soaked in a solution of 1 M KOH for 15 min. The excess base was removed by filtration. ‘The cellulose was then immersed in a solution of cyanuric chloride in acetone (0.5 g/20 mL) and 20 mL of water was added immediately. After 15 s 20 mL of 20% acetic acid was added. The cellulose was then washed with 100 mL each of water and acetone and soaked in a solution of fluoresceinamine
in acetone (0.1 g/20 mL) for 1 h. The product was washed first with acetone and then several times with water until no fluoresceinamine was visible in the washings. Determination of Amount Bound. The amount of fluoresceinamine bound per gram of solid was found by titrating the neutral form of the immobilized fluoresceinamine with base. The immobilized fluoresceinamine was soaked in a solution 0.10 M in HC1 and then washed with water and dried. A weighed amount of the product was titrated with 0.00209 M KOH (standardized against KHP) under nitrogen in 5.00 mL of 0.1 M potassium nitrate. The titration was followed potentiometrically using a glass electrode. The end point was taken as pH 9. The amounts titrated were 0.0060 g for the cellulose and 0.0021 g for the glass. Blank titrations on equivalent amounts of underivatized support were also performed. The blank values were 7 % and 21% of the sample values for the cellulose and glass substrates, respectively. In this titration the neutral form of fluorescein is converted to the dianion. The pK values for forming the mono- and dianion are 4.4 and 6.7, respectively (4,5). Fluorescence Measurements. Fluorescence measurements were made by attaching the immobilized fluoresceinamine to the end of the optic and immersing the optic in a solution of 15 mL of 0.1 M acetic acid. Measurements were taken as the pH was varied by adding 4.0 M KOH. Blanks were run on solvents and the glass or the cellulose t o determine how much of the signal was due to scattering. The pH changes of the solvent system were also measured on a pH meter. The pH changes were measured both with and without immobilized fluoresceinamine (0.0010g). The presence of fluoresceinamine does not affect the pH. Fluorescence measurements were also made on fluorescein in solution for comparison. The optic was immersed in a solution of 14 mL of 0.1 M acetic acid and 1.00 mL of fluorescein solution M in M) making the solution 1.9 X in ethanol (2.9 X fluorescein. The pH was varied and measured as above.
RESULTS AND DISCUSSION Response vs. pH,, The fluorescence from the immobilized fluoresceinamine probe increases with pH as shown in Figure 2. The glass-bound and cellulose-bound fluoresceinamine behave similarly up to pH 8. Above pH 8 the glass-bound fluoresceinamine response increases only slightly and the cellulose-bound fluoresceinamine response decreases. In solution, fluorescein behaves similarly to the glass-bound fluoresceinamine. The fluorescence increases significantly from pH 3 to 6 and then increases slightly above pH 8.0. The fact that the pH response for immobilized fluoresceinamine and soluble fluorescein agree is evidence that the amino group has been tied up in the immobilization procedure. The variation of the fluorescence of dissolved fluoresceinamine with pH was found to differ considerably from the behavior shown in Figure 2. The observed variation in fluorescence with pH is consistent with previous studies of the pH dependence of fluorescein fluorescence (5). This pH dependence is complex because it is influenced not only by protonation and deprotonation but also by the fact that neutral fluorescein occurs as three different tautomers. The response of the pH sensor will depend on wavelength. At the excitation wavelength of 480 nm, the monobasic form of fluorescein is excited more efficiently than the neutral form. Thus the observed response reflects spectral shifts as well as changes in the fluorescence efficiency with PH.
0003-2700/82/0354-082 1$01.25/0 0 1982 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 4, APRIL I982 INTERFERENCE F I L T E R S
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Figure 1. Diagram of pH sensor based on fluorescence and associated instrumentation. s m
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Flgure 3. Typical raw data showing changes in fluorescence intensity
8
In response to added base. >
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Flgure 2. Relative fluorescence intensity as a function of pH for immobllized fluoresceinamine on glass (0)and cellulose (A).The values of fluorescelnamlne on glass have been multiplied by a factor of 10 so they fC on the same scale as the values for fluorescehamine on cellulose.
The reduction in fluorescence intensity for cellulose-bound fluoresceinamine at high pH is caused by hydrolysis of immobilized fluoresceinamine so that it is no longer attached to the solid support. This is accompanied by a momentary increase in fluorescence followed by a decrease. Operating Characteristics. The pH measurements were typically made with 1.0 mg of derivatized support material on the fiber optic surface. This amount of support holds about 2X mol of immobilized fluoresceinamine (as determined by titration). The area of the optic is 0.16 cm2. When pH is changed by injection of base, it takes about 15-30 s for the fluorescence intensity to reach a new steady state. Typical data are shown in Figure 3 . Since the acid-base reaction itself is fast, the response time reflects the rate of mass transfer of solution into the layer of immobilized fluorophor. This is not unreasonable since the layer is on the order of 0.1 mm thick. The signal-to-noise ratio is much poorer for immobilized fluoresceinamine than for dissolved fluorescein. The noise can be seen in the data of Figure 3. Two effects contribute to this. One problem is the presence of a significant background signal due to scattering for the immobilized fluoresceinamine. For cellulose-bound fluoresceinamine, the background is 40% of the signal at pH 3. The signal-tebackground ratio improves as pH increases because the background is constant with pH. For glass-bound fluoresceinamine the situation is poorer. The background is twice the signal at pH 3. The background due to scatter would be much lower if the excitation and emission wavelengths are further apart. For example, at an excitation wavelength of 420 nm and an
emission wavelength of 488 nm, both selected by interference filters, the background is barely detectable above detector noise. Unfortunately, in the case of immobilized fluoresceinamine the excitation and emission wavelengths need to be closer together to maximize the change in fluorescence with PH. The other problem is low fluorescence intensity from immobilized fluoresceinamine. The intensity per mole of cellulose-bound fluoresceinamine is about 2 orders of magnitude lower than intensity per mole of dissolved fluorescein in the volume of solution below the fiber optic. Although the comparison is crude, there is no doubt that we are getting much less light from the immobilized fluoresceinamine. Part of the decrease in intensity is due to attenuation of the source radiation by absorption and scattering. However, this effect by itself cannot account for such a large decrease in intensity since the thin layer of immobilized cellulose has considerable transmittance in water. The fluorescence efficiency must be lower for immobilized fluoresceinamine than for dissolved fluorescein. The fact that hydrolysis of the immobilized fluoresceinamine causes a momentary increase in intensity is further evidence for this. The decreased fluorescene efficiency may reflect the different environment of immobilized fluoresceinamine relative to dissolved fluorescein, or it may be a consequence of the changes in structure accompanying the immobilization process. The reduction in intensity is greater for fluoresceinamine on glass. Intensity per mole of glass-bound fluoresceiname is about 15% of the intensity for cellulose-bound fluoresceinamine.
CONCLUSIONS This study demonstrates the potential feasibility of chemical sensors based on immobilized fluorigenic reagents. The performance characteristics of the pH sensor could be improved in several ways. The background signal due to scattering can be reduced by using a clear film rather than a fine powder as the immobilization substrate and by finding a system in which excitation and emission wavelengths are further apart. The fluorescence intensity from immobilized fluoresceinamine can be increased by using a more intense excitation source and/or by finding an immobilization system which does not reduce fluorescence efficiency. However, in view of the availability of glass electrodes, we intend to direct our further efforts toward developing sensors that respond to metal ions that cannot be measured potentiometrically rather than attempting to improve the pH sensor. ACKNOWLEDGMENT The authors thank Julie Pflug for helping with the im-
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Anal. Chem. 1982, 54, 823-824
mobilization of fluoresceinamine on glass and the UNH machine shop for help in designing and constructing the fluorescence photometer.
LITERATURE CITED (1) Peterson, J. I.; Goldstein, S. R.; Fitzgerald, R. V. Anal. Chem. 1980, 52 864-869. (2) Pierce Catalog, 1979-1980. (3) Kay, G.; Crook, E. M. Nature (London) 1967. 218, 514-515. (4) Leonhardt, H.; Gordon, L.; Llvlngston, R. J . Phys. Chem. 1971, 75, 245-249.
(5) Martin, M. M.; Llndqulst, L. J. Luminescence 1975, IO, 381-390.
Linda A. Saari W. Rudolf Seitz* Department of Chemistry University of New Hampshire Durham, New Hampshire 03824
for review November 9, lg8I* Accepted January 15, 1982.
Comments om Variations in Concentrations of Organic Compounds I ncluding Polyc hIor inat ed Dibenzo-p -dioxins and Polynuclear Aromatic Hydrocarbons in Fly Ash f rorn a Municipal Incinerator Sir: Appropriate assessment of possible environmental effects arising from trace organic constituents present in combustion unit emissions require that published findings concerning such sources contain data which are carefully defined with regard to both accuracy and reliability (I). Therefore, we feel that it is necessary and appropriate to comment on the recently reported data of Eiceman, Clement, and Karasek regarding chlorinated dibenzo-p-dioxins (CDDs) observed in fly ash froim a particular municipal incinerator (2) because the described analytical procedure suffers from a systematic error which produces unreliable and biased quantitative data for CDDs. The source of this error involves the authors' procedure for determining CDDs (other than TCDDs), in particular, the gas chromatography mass sipectrometry selected ion monitoring (GC-MS-SIM) calibration technique. As stated, a HewlettPackard 5992 quadrupole GC-MS equipped with a silicone membrane separator operating under temperature programmed GC conditions was used to determine all CDDs. Instrumental calibration was accomplished by monitoring m/z 321.9 for a reference standard of 1,2,3,4-tetrachlorodibenzop-dioxin (1234-'I'CDD). Quantitative estimations in ng/g for pentachlorodibenzo-p-dioxins(PCDDs) monitored a t m / z 355.9, hexachlorodibenzo-p-dioxins(HCDDs) at m / z 389.9, heptachlorodibenzo-p-dioxins(H,CDDs) at m / z 425.8, and octachlorodibenzo-p-dioxin (OCDD) at m/z 459.7 were then determined in each sample using the response factor for 1234TCDD. These results are presented using two significant figures in the authors' Table II (2). Because CDDs of differing degrees of chlorination exhibit varying ionization efficiency, fractional abunclance of the monitored mass (3), mass-dependent nonlinear ion transmission through the quadrupole, and temperature-dependlent separator membrane transmission rates ( 4 ) , not only are the authors' reported data likely to be inaccurate but their proportions relative to TCDDs may also be incorrect. Although~the instrumental parameters mentioned affect quantitative accuracy for each of the CDD congeners differently, they would not be expected to significantly affect tho reproducibility of such data on a properly functioning GC-MS, as indicated by the authors' description of procedural reproducibility. During previous studies (5,6)we had conducted two simple experiments with a variety of CDDs which illustrate the nature and magnitude of the effects on CDDs quantitation accuracy arising from some of the GC-MS-SIM parameters mentioned. One experiment demonstrated that transmission efficiency of CDDs bearing different numbers of chlorine atoms through 0003-2700/82/0354-0823$0 1.25/0
Table I. Comparison of Relative Peak Ratios for CDDs through a Glass-Jet and Silicone Membrane Separator" re1 response no. of component. (I re1 std dev) replicates 1.00 i ... 1.00 '' ... 1234658-H7CDDmembrane 0.34 z 0.04 jet 0.63 z 0.10 OCDD membrane 0.21 I0.05 jet 0.38 I 0.04 123658-HCDD membrane jet
7 4 7 4
I 1
All values normalized to HCDD response, see text for
conditions. a silicone membrane separator was variable and not necessarily predictable. In order to isolate the effect of the separator, we employed a Kratos MS-30 GC-MS equipped with a packed column (5),similar in characteristics to the authors' column, operated under isothermal conditions. A test standard containing 100 ng/mL each of 123678-HCDD, 1234678-H7CDD, and OCDD was used to show variations in separator transmission characteristics when all other conditions were held constant. The MS-30 GC-MS was operated under SIM conditions at low mass resolution (- 1500) and the parent ions defined by the authors were monitored. Initially, 3-pL injections of the test standard were examined by using a single-stage glass-jet separator maintained at 250 O C . Next, a silicone membrane separator (also maintained at 250 "C) was substituted for the glass-jet interface and the test standard reexamined. The GC-MS-SIM data from this experiment appear in Table I. Comparison of the relative responses by peak area for H7CDD and OCDD, to HCDD, characterizes the transmission behavior of the membrane vis-a-vis the jet for different CDD congeners. Since it is known that permeation of a given CDD decreases with increasing temperature for silicone membranes (4), the authors' responses for higher chlorinated CDDs relative to 1234-TCDD would be expected to show even greater deviations in transmission because the interface in a Hewlett-Packard 5992 is located inside the GC oven and was therefore subjected to temperature programming conditions (2). A second experiment illustrated the degree of quantitative bias for higher chlorinated CDDs associated with the use of a Hewlett-Packard 5992 GC-MS when calibrated as described by the authors (6). A single-stage glass-jet separator was installed along with a packed column (again similar to the authors') which was operated under tempeature programmed @ 1982 American Chemlcal Society
