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Anal. Chem. 1904, 56,2004-2000
was confirmed by analysis of the solid product isolated from the reaction mixture. The new method was also applied for controlling the condensation of phthaloyl-i-glutamic anhydride with several amino compounds used in synthesis of y-glutamyl amino acids (16) and y-glutamyl arylamides (17). I t was observed that heating of 0.2 M phthaloyl-L-glutamic anhydride with an equimolar amount of p-aminobenzoic acid in dry dioxane for 60 and 120 min at 40 "C caused the decrease of the anhydride concentration by 60% and 85%, respectively. The reaction with P-naphthylamine was much faster-after 10,60, and 120 min 50%, l o % , and 2% of the remaining anhydride was observed, respectively. During these two condensation procedures the anhydride concentrations were almost equal to the remaining free arylamines. Also condensation of phthaloyl-L-glutamic anhydride with L-alanine was monitored by the new spectrophotometric method. When 0.2 M anhydride in glacial acetic acid was heated at 60 O C with the amino acid, after 60 min a 50% decrease of initial concentration of the anhydride was observed and after 120 min 35% of the initial anhydride was found in the reaction mixture. Registry No. NaN3, 26628-22-8; Fe, 7439-89-6;acetyl chloride, 75-36-5;benzoyl chloride, 98-88-4; phenylacetyl chloride, 103-80-0; benzenesulfonyl chloride, 98-09-9; 5-(dimethylamino)-1naphthalenesulfonyl chloride, 605-65-2; hexanesulfonyl chloride, 14532-24-2; methanesulfonyl chloride, 124-63-0; phenylmethanesulfonyl chloride, 1939-99-7;p-toluenesulfonyl chloride,
98-59-9; acetic anhydride, 108-24-7;butyric anhydride, 106-31-0; phthaloyl-t-glutamicanhydride, 25830-77-7;propionic anhydride, 123-62-6; phenylmethanesulfonyl fluoride, 329-98-6; p-aminobenzoic acid, 150-13-0;@-naphthylamine,91-59-8; L-alanine, 5641-7.
LITERATURE CITED (1) Ashworth, M. R. F. "The Determination of Sulfur-Containing Groups"; Academic Press: London, 1972; Vol. 1, pp 51-70. (2) Buzlanova, M. M.; Shovortsov, N. P.; Mekhryusheva, L. I.Zh. Anal. Khlm. 1067, 22, 469-471. (3) Munson, J. W. J. Pharm. Scl. 1074, 63, 252-256. (4) Hasegawa, K.; Matsono, T.; Ikagawa, S.; Hoshoda, H. Vukagaku Zasshi 1081, 101, 1059-1064. (5) SiewiRski, M.; Kuropatwa M.; Szewczuk, A. Hoppe-Seyler's Z . Physlol. Chem., in press. Curtius, Th.; Haas, W. J. Prakt. Chem. 1021, 102, 85-112. Wallace, R. M.; Dukes, E. K. J. Phys. Chem. 1061, 65, 2094-2097. Brenna, 0.; Pace, M.; Pietta, P. G. Anal. Chem. 1075, 47, 329-331. Siewifiski, M.; Kubicz, Z.; Szewczuk, A. Anal. Chem. 1082, 5 4 , 846-847. Johnson, T. N.; Sprague, J. M. J. Am. Chem. Soc. 1038, 58, 1348-135 1, Honzi, J.; Rudlnger, J. Collect. Czech. Chem. Common. 1081, 26, 1333-1 344. Goid, A. M.; Fahrney, D. Biochemistry 1064, 3 , 783-791. King, F. E.; Clark-Levis, J. W.; Wade, R. J. Chem. SOC. 1057, 886-894. Gribova, E. A.; Dyatlova, V. V.; Sterlina, L. I.Zavod. Lab. 1080, 46, 597-598. Patchornik, A.; Rogozinski, S. E. Anal. Chem. 1050, 31, 985-989. Szewczuk, A.; Conneil, G. E. Can. J. Blochem. 1075, 53, 706-712. Orbwski, M.; Szewczuk, A. Clln. Chlm. Acta 1062, 7, 755-760.
RECEIVED for review April 20,1984. Accepted July 16, 1984.
Microdroplet Titration Apparatus for Analyzing Small Sample Volumes A. W. Steele and G . M.Hieftje* Department of Chemistry, Indiana University, Bloomington, Indiana 47405
A computer-controlled,automatlc titrator has been developed from a microdroplet dlspenslng system orlglnally deslgned for sample appllcatlon In electrothermal atomlc absorption spectrometry. The system Is capable of performlng hlghprecision titrations uslng total volumes of sample solutlon and titrant In the mlcrdlter range. With the devbe, titrant droplets are formed by the repetltlve Insertion and wlthdrawal of a 150-pm glass needle Into and from a small reservoir of titrant solutlon. Unlike other microtitration Instruments, thls apparatus permits operation wlth as llttle as 100 pL of bulk titrant solutlon. The control system employed for dlspenslng the droplets Is Inherently dlgltal, thereby simplifying computersupervised operation of the apparatus. Several chemlcal systems were used to evaluate the devlce In terms of preclslon and ease of operation. Potentlal appllcatlon to other analytlcal problems wlll also be dlscussed.
Automated titration systems range from simple push-button titrant delivery units to microprocessor-controlled instruments that handle titrant delivery, titration monitoring, end-point detection, sample changing, and data calculations. However, such systems generally must use large sample volumes, on the order of milliliters to tens-of-milliliters. There is considerable interest today in titrating smaller sample volumes, on the order of 5-100 pL.
One approach to titrating small sample volumes is simply to dilute the sample with a sufficiently large volume of solvent to allow the use of standard macroscale titration equipment. This method has two drawbacks. First, the chemistry of the titration reaction is often changed at high dilutions, and the titration results might be difficult to analyze. Second, most end-point detection devices are concentration dependent, so precision of the end-point determination decreases as the titrate becomes more dilute. As a result, the best approach to microtitration is usually to miniaturize the solution-dispensing apparatus and maintain the reagents at their original concentrations ( I ) . In order to achieve this goal, the analyst needs a device capable of delivering microliter to submicroliter aliquots of titrant with high accuracy and precision. Many instruments have been developed to perform microliter range titrations (2-7), but most are not easily automated and seldom offer precision better than 2-5% RSD. The device described in the present paper overcomes most of these limitations. It operates by producing precise droplets of titrant that are approximately 2 nL in volume. This type of operation is ideal for microtitration applications, since the titrant resolution is high and titrant delivery rate is adjustable over a broad range. In addition, operation of the device is inherently digital and is therefore easily automated. In the present paper, the operation of the device will be discussed and its performance on simple acid-base titration systems will
0003-2700/84/0356-2884$01.50/00 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
RESERVOIR
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RESERVOIR TUBE I
ELECTRO 1
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TITRATION VESSEL STAINLESS
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POSlTlONER
\ MOTOR
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Flgure 1.
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MINC 11/23 COMPUTER
Block diagram of microdroplet generator tltratlon system.
be evaluated and compared t o other microtitration methods.
EXPERIMENTAL SECTION Titrator and Procedure. Figure 1shows a block diagram of the instrument used in this study. The device that produces titrant microdroplets consists of a piezoelectric ceramic (bimorph) which has a miniature glass rod glued to it. The glass rod is pulled to a point and bent at a 90' angle, with the diameter of the tapered end being 150 pm. In operation, a 100-V peak-to-peak sine wave is applied to the bimorph a t the resonant frequency of the crystal-glass rod assembly (typically 160 Hz). As a result, the tip of the rod moves into and out of a reservoir of titrant, which is simply an inverted 8-mm glass tube connected to a supply vessel of titrant. Each time the glass needle is withdrawn from the titrant, it pulls with it a thin filament of titrant solution, which subsequently detaches from both the glass rod and the reservoir and collapses into a microdroplet approximately 2 nL in volume. The electronics that drive the system consist of a waveform generator, gate, gate controller, and high-voltage amplifier. The waveform generator produces a low-voltage sine wave which can be varied in frequency and amplitude. The amplifier raises this low-voltage waveform to a level sufficient to drive the bimorph. The gate and gate controller determine the number of cycles of the sine wave that reach the bimorph and thus determine the number of microdroplets that are dispensed. Because of inertia in the bimorph assembly, the initial cycles of the bimorph vary in amplitude, causing the first-formed microdroplets to be of varying size (8). Therefore, an air jet is employed to reject the initially formed microdroplets. This jet is controlled by a solenoid valve activated by the gate controller. At the beginning of the dispensing of each aliquot, the solenoid valve is opened for 100 cycles of the waveform generator. Thus, the first 100 microdroplets are expelled to a waste container, which results in far better precision for the remaining droplets dispensed in each aliquot. Further details on the droplet generator are available elsewhere (8, 9). All titrations performed here were on simple acid-base systems and were easily monitored by using conventionql pH electrodes (6023-03Ingold miniaturized pH electrode, Ingold Electrodes Inc., Andover, MA or 12228 Corning flat-surface pH electode, Corning Glass Works, Corning, NY) connected to an electrometer (Model 610A Keithley Instruments, Cleveland, OH). Figure 2 illustrates the mechanical portion of the titration apparatus built for this study. The bimorph assembly was bolted to a translation stage which allowed the assembly to be easily positioned under the reservoir tube. Two sample vessels were constructed, the first consisting of a 1.9-cm-diameter stainless steel rod 2.5 cm in length mounted on a small DC motor as shown in Figure 2. To monitor the titration, a flat-surface pH electrode was used. The electrode was positioned off center just above the top surface of the stainless steel cylinder such that the sample solution was held by capillary action between the top of the cylinder and the bottom of the electrode. This assembly was then positioned under the droplet generator so droplets would fall onto
Flgure 2. Schematic diagram of the mechanical components used wlth the small-volume sample vessel.
the top of the cylinder. Solution mixing was achieved by spinning the stainless steel cylinder a t a rotation rate of 120 rpm. This vessel was used for the titration of the smallest sample volumes used here, on the order of 20-100 pL, and is termed later the small-volume sample vessel. The second vessel was simply a 12-mm-diameter glass tube with a side tube connected to it in the form of a VEE. One arm of the vessel was used to hold a miniaturized pH electrode while microdroplets were directed into the other arm. Sample solution was placed in the vessel to a level that covered the salt bridge of the pH electrode. If the sample volume was too small, distilled water was added to raise the solution level. The solution was mixed by inserting a Teflon capillary tube into the vessel and bubbling compressed air through the solution a t a rate of 5 mL/min. This vessel was used for sample volumes on the order of 0.5-1 mL and was labeled the large-volume sample vessel. The aliquot size used for the large-volume sample vessel was 450 drops with a drop diameter of approximately 190 Nm, which yielded an aliquot volume of approximately 1.2 wL. Because the present device does not measure titrant directly, but rather dispenses titrant in the form of droplets, it must be calibrated. Such calibration is complicated by the small volume of individual droplets and of total titrant. Several methods exist for determining the volume of a single microdroplet, although they are unacceptably imprecise. The most common such method involves the measurement of the droplet diameter and, assuming the droplet to be a perfect sphere, the calculation of the droplet's volume. An example of this approach is the MgO-impression technique used extensively for small-particle-size measurements (10,11). To achieve adequate precision, the system was calibrated indirectly by titrating a known volume of standardized HC1 with standardized NaOH. The HCl was dispensed by using a micropipet (Gilson Model P-20D, Rainin Instrument Co. Inc., Brighton, MA) which had an accuracy of 1% and a precision of 0.2% RSD; a titration was then initiated using the standard NaOH as the titrant. When the concentrations of NaOH and HCl, the volume of HCl dispensed, the number of drops per aliquot, and the end point were known, the droplet volume was easily computed. The operation of the droplet generator can be controlled either by manual methods or by computer. For the work presented in this paper, a laboratory computer (Digital Equipment MINC 11/23, Digital Equip. Corp., Maynard, MA) was used to control the droplet generator during the titration and for collecting and analyzing the titration data. Data collection was performed by using a 12-bit successive approximation A/D module that is part of the MINC 11/23 laboratory computer system. After the droplet volume was calibrated, the computer would prompt the operator for the important parameters of the titration. These parameters included the type of sample being analyzed (acid or base), the concentration of the titrant, and the aliquot volume to be used. After the titration was performed, the computer would then display the titration curve and ask the operator whether the data should be stored, smoothed, or eliminated, whether a new titration
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
Table I. Titration Results Using the Large-Volume Sample Vessel, 60-pL Sample Aliquot titranttitrate NaOHHCl NaOHHOAc HCl-NaOH
% RSD
% abs
error
aliquot size, drops
drop diam, pm
aliquot vol, p L
1.6
+3.9
450
190
1.62
1.9
-4.5
450
182
1.42
1.5
+0.22
450
188
1.57
r,
Table 11. Titration Results Using the Small-Volume Sample Vessel, 60-pL Sample Aliquot titranttitrate NaOHHC1 NaOHHOAc HCl-NaOH
I
0
% RSD
error
aliquot size, drops
0.92
-0.85
750
151
1.35
2.1
-4.1
750
152
1.38
2.9
+1.2
750
151
1.35
% abs
drop diam, pm
vol, pL
20
aliquot
I
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40 60 VOLUME OF TITRANT (UI)
e
Figure 3. Titration of a 60-pL sample of 0.1 N HCI with 0.1 N NaOH using the large-volume sample vessel.
l3
t
Table 111. Titration Results Using the Small-Volume Sample Vessel, 20-pL Sample Aliquot titranttitrate NaOHHCl NaOHHCl" NaOHHOAc HC1-NaOH
% abs % RSD
error
aliquot size, drops
drop diam, km
vol, p L
aliquot
0.15
+1.7
450
151
0.81
0.24
+0.10
450
151
0.81
2.2
-3.1
450
153
0.84
4.7
-2.8
450
153
0.84
"Data in this row were taken 3 days after data in previous row to indicate long-term reproducibility. should begin, or whether the experiment should end. Titrations required 5-10 min to complete, depending on the sample size and aliquot volume used. A copy of the program used for this work can be obtained if requested. Reagents. All reagents were prepared by dilution of stock solutions made from reagent-grade chemicals. The solutions were all approximately 0.1 M, standardized by using a conventional 50-mL buret. The NaOH was standardized against primarystandard potassium hydrogen phthalate, and the acid solutions were then standardized against the base.
0
20
40 60 VOLUME OF TITRANT (UI)
I3
Figure 4. Titration of a 60-pL sample of 0.1 N HOAc with 0.1 N NaOH using the large-volume sample vessel.
I&
0
Io
RESULTS AND DISCUSSION The performance of the microdroplet titrator was evaluated by using three different titration systems. The first was a strong acid-strong base system using NaOH as the titrant and HC1 as the sample. The second was a weak acid-strong base system where acetic acid (HOAc) was used as the sample in place of HC1. The third system was simply the reversal of the first system, with HC1 as the titrant and NaOH as the sample. In the titration curves that follow, each point on the curves represents a single aliquot of titrant. The volume of each aliquot and the number of droplets dispensed per aliquot are listed in Tables 1-111, along with the volume of the individual droplets. The results obtained by using the large-volume sample vessel are shown in Figures 3-5 and in Table I. Figure 3 shows the titration curve obtained when a 60-pL sample of HC1 was titrated with NaOH. The sample was delivered to the vessel by using three aliquots from a 20-pL pipet, the same procedure
I I
I
I
3
Figure 5. Titration of a 60-pL sample of 0.1 N NaOH with 0.1 N HCI using the large-volume sample vessel. used for all samples of 60 p L in these experiments. The titration curve produced by the microdroplet titrator for the strong acid-strong base system is clearly well-defined and provides a sharp end point. The slight scatter of the data points is attributed to the method by which the sample solution was stirred. Stirring was by bubbling air through the solution; to reduce the amount of splashing, air flow through the vessel was minimized; mixing was therefore fairly slow and sometimes incomplete. Figure 4 shows a similar titration using HOAc as the sample. Again, evidence of poor sample mixing
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
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Figure 8. Titration of a 60-pL sample of 0.1 N HCI with 0.1 N NaOH using the small-volume sample vessel. exists, but the titration curve is well-defined and is the shape expected for a weak acid-strong base system. Figure 5 is the titration curve obtained when HC1 is used as the titrant and NaOH is used as the sample. Mixing problems are again apparent, but the titration curve is poorly shaped. The sloping nature of the curve as the end point is approached has been attributed to the entrainment of atmospheric COz in the sample solution as mixing air is bubbled through it. The reaction of COz with the titrant would be expected to cause some error in the calculated sample concentration. However, for titrations performed by using the large-volume sample vessel, this error was found to be no greater than other errors associated with the experimental procedure (see Table I). Of course, the COz problem could be eliminated entirely by using an inert gas for mixing instead of air. From Table I, the precision of the microdroplet generator titrator with the large-volume sample vessel is between 1.5% and 2.0% relative standard deviation (% RSD), comparable to previous microtitration methods, which achieved precisions of 3-5% RSD (1-7). The absolute error was computed as the percentage difference between the end point determined by the titration program and the end point expected from the concentrations of the reactants. The rather large absolute error shown in Table I is attributed to poor calibration of the droplet volume. A major factor in this miscalibration is judged to be drift in the waveform-generator output voltage. In turn, this voltage affects the amplitude of the bimorph movement and causes a change in droplet volume (8,9).Because drift in the waveform generator was relatively slow, the system required recalibration only once every hour. The small-volume sample vessel provided much better solution mixing than the large-volume cell and allowed much smaller sample volumes to be used. Figure 6 is a titration curve of a 60-pL sample of HC1 titrated with NaOH. The smoothness of this curve, compared with Figure 3, suggests more consistent mixing of the reactants. Similar improvements were obtained when HOAc was used as the sample. Figure 7 shows the titration curve obtained when NaOH is used as the sample instead of HC1. Compared to Figure 5, the curve in Figure 7 is much smoother but the interference from COS absorption is greater. This greater interference probably arises because of the increased surface-to-volume ratio of the sample solution in the small-volume sample vessel. During mixing, the solution is spread thinly over the top surface of the stainless steel rod, so a large area of the solution is exposed to the atmosphere. It would be possible to prevent this problem by designing a vessel in which the reaction area is immersed in an inert atmosphere, as was done by Spokane and Brill (7).
using the small-volume sample vessel.
1
0
IO
VOLUME
20
0
OF TITRANT (UI)
Figure 8. Titration of a 20-pL sample of 0.1 N HCI with 0.1 N NaOH using the small-volume sample vessel. Table I1 compiles the results obtained by using the small-volume sample vessel and 60-pL sample aliquots. As expected from observed improvements in mixing, end-point determination is more precise when the small-volume cell is employed and HC1 is the sample. In contrast, the precision for the titration of HOAc and NaOH is poorer. This loss in precision in both cases results from the increase in surface area of the sample exposed to the atmosphere. In the case of HOAc, more of the acid evaporates during the titration; for NaOH, the sample is more exposed to COz. With the present apparatus, the smallest usable sample volume is the amount necessary to span the pH-sensitive glass and the salt bridge of the flat-surface pH electrode used to monitor the titration. This minimum volume was determined experimentally to be 20 pL, conveniently a single aliquot from the available pipet. Using a single aliquot from the pipet eliminates cumulative errors during the sample deposition and therefore permits the determination of whether such errors contribute significantly to overall precision. Figure 8 is a titration curve obtained by using NaOH as the titrant and HC1 as a 20-yL sample aliquot. Again, the curve is quite smooth and very well-defined. Table I11 shows the results for several similar 20-pL titrations. The titration performed by using HC1 as the sample produced even better precision than in earlier titrations, suggesting that a large part of the reported error was caused by cumulative sample application. The degree of precision in this titration is significant in that it approaches what can be obtained by using a conventional macroscale titration apparatus. The titration was repeated after a few days, and the results were nearly identical
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Anal. Chem. 1984, 56,2888-2891
with those first obtained, illustrating the reproducibility and stability of the microdroplet titrator. The results for the titrations of HOAc and NaOH proved to be poorer than those obtained previously, which was expected based on the behavior those systems displayed earlier. With a smaller sample volume, the effects of evaporation of HOAc and the reaction of COz with NaOH would be even more pronounced and would lead to poorer precision.
CONCLUSIONS The microdroplet titrator used in this study can perform microscale titrations with a high degree of accuracy and precision. Its advantages over other microtitration instruments are that it is directly digital in operation, and thus easily automated, and offers greater precision. The only serious problems with the device are reactions that occur between the small-volume sample solution and the atmosphere and drift in the driving electronics which causes the calibration of the instrument to change. The first problem could be eliminated by designing a sample container that would allow an inert atmosphere to surround the sample solution. The second problem could be solved by electronic feedback from the bimorph to the driving electronics. Therefore, neither of these problems would be difficult to overcome and should not be viewed as serious disadvantages. Also, because the titrator is computer-controlled, other applications for the instrument could be easily impIemented by software modification. These
applications could include pH-stat experiments, kinetics experiments performed on a microscale, or microsample deposition.
ACKNOWLEDGMENT We are grateful for the technical assistance received from J. G. Shabushnig concerning the operation of the droplet generator and the help obtained from R. D. Deutsch in writing the software routines.
LITERATURE CITED Benedetti-Plchier, A. A. “Microtechniques of Inorganic Analysis”; Wiley: New York, 1942. Pecar, M. Mlcrochem. J. 1959, 3, 557-563. Rogers, D. W.; Lillian, D.;Chawla, I . D. Mikrochlm. Acta 1968 4 , 722-728. Knobloch, V.; Mudrova, B. Mlkrochlm. Acta 1970, 2, 235-239. Ceska, M.; Grossmuller, F.; Sjodin, A. V. I n t . J. Appl. Radiat. Isot. 1971, 22, 311-314. Waisby, J. R. Anal. Chem. 1973, 45, 2445-2446. Spokane, R. 8.; Brill, R. V. Anal. Biochem. 1980, 109, 449-453. Shabushnig, J. G.; Hleftje, G. M. Anal. Chlm. Acta 1981, 126, 167-174. Shabushnlg, J. G.; Hleftje, G. M. Anal. Chim. Acta 1983, 148, 181-182. May, K . R. J. Scl. Instrum. 1945, 22, 187-189. May, K. R. J. Sci. Instrum. 1950, 27, 128-131.
RECEIVED for review March 23,1984. Accepted June 8,1984. This research was supported in part by the National Science Foundation through Grants CHE 82-14121 and CHE 83-20053 by the Office of Naval Research.
Fluorescence Polarization Immunoassay af Phenytoin Employing a Sulfonamido Derivative of 2-Naphthol-8-sulfonic Acid as a Label Jeffrey S. O’Neal and Stephen G. Schulman* College of Pharmacy, Box 5-4,J.Hillis Miller Health Center, University of Florida, Gainesville, Florida 32610
The synthesls of a new fluorescent derlvative of 5,5-diphenylhydantoin (phenytoln) and Its application In a fluorescence polarization Immunoassay are described. This ftuorescent label undergoes excited-state proton transfer which resuns in a relatively large shM of the fluorescence spectrum to wavelengths much longer than the wavelength of excitatlon, thereby decreasing background serum matrix interference. Good precision was obtained for this immunoassay, demonstratlng the effectlveness of the label In this system.
Phenytoin (5,5-diphenylhydantoin) is a widely prescribed therapeutic drug for the treatment of epilepsy. The phenytoin plasma concentration range of 9-21 mg/L is considered to be clinically useful, concentrations outside of this range being either toxic or noneffective. It is therefore highly desirable to monitor phenytoin blood concentrations (1,2). Traditional methods of phenytoin quantitation include gas chromatography (3))high-performance liquid chromatography (4),radioimmunoassay (5),and homogeneous enzyme immunoassay (6). Fluorescence immunoassays for phenytoin have also recently been developed, including a double-antibody tech-
nique (7), a magnetizable solid-phase method (8), a reactant-labeled assay (9),and a fluorescence polarization immunoassay (10). Fluorescence immunoassays have almost exclusively relied upon derivatives of fluorescein as labeling compounds. Although relatively high molar absorptivities and quantum yields have been achieved, alternative fluorescent labels can potentially provide increased spectroscopic detectabilities and, hence, decreased limits of analyte detection. In this experiment a fluorescent derivative of phenytoin containing a sulfonamido derivative of 2-naphthol-8-sulfonic acid was synthesized and used in a fluorescence polarization immunoassay (FPIA) for phenytoin. This fluorescent label is one of a series of potential derivatizing reagents which fall into the class of hydroxyaromatic sulfonic acids. These probes undergo excited-state prototropic dissociation which is schematically summarized in Figure 1. This process, in hydroxyaromatics, results in large shifts to long emission wavelengths which are highly desirable when the analytical signal originates from a biological matrix. The large displacements between longest wavelength absorption and longest wavelength fluorescence maxima which accompany excited-state dissociation of hydroxyaromatics are
0003-2700/S4/0356-2888$01.50/00 1984 American Chemical Society
