Table 11. Selectivity Coefficients Membrane number Ion
Concn ( M )
Ca2+
10 - 2
Cdl+
10 - 4 10 - 2 10 - 4
Mgz+
10 - 2 10 - 4
ZnZ+
10 - 2
cut+
10 -2 10 - 4 10 - 2 10 - 4 10 - 4 10 - 2 10 - 4
10-4 ~
1+ 3
Fe3 + NiZ +
1
0.0027 0.24 0.0027 0.0044 0.0025 0.16 0.0064 0.39 0,0093 0.38 0,0029 0.18 0.42 0.006 0.40
0.0014 0.083 0.0013 0.065 0.0004 0.044 0.0007 0.032 0.0005 0.026 0.0038 0.057 0.48 0.003 0.032
0.0009 0.017 0.0034 0.022 0.0010 0.0066 0.0017 0.060 0,0019 0,066 0.0055 0.076 0.15 0.0017 0.061
A typical graph illustrating the effect of pH is shown in Figure 3. The optimum pH for reproducible emf measurements appears to be about 3. Above approximately pH 4, hydrolysis of the uranyl ion occurs and, below pH 2, the graph resembles that obtained by Griffiths et al. (7) in their work on the Ca2+ electrode; that is, the hydrogen ion contributes to the charge transport process across the membrane, and the slope becomes too steep to obtain reproducible measurements. In conclusion, electrodes prepared from the six membranes listed in Table I exhibit near Nernstian response to uranyl ion activities from -lo-’ to W 4 M . No major differences were observed among the more favorable membranes; however, our preference would be the first three. Electrodes incorporating these membranes exhibited the best all around stability and response characteristics as a function of time. The preferred media is chloride although small amounts ( 510- 3 M ) of NOa- and c104- can be tolerated. By combining the uranyl exchanger with a suitable solvent prior t o membrane casting, the useful lifetimes of the electrodes can be extended from days to weeks.
ACKNOWLEDGMENT The authors acknowledge helpful discussions with Gleb Mamantov of the University of Tennessee. The authors
0.0039 0.21 0,0031 0.14 0.0020 0.11 0.0014 0.041 0.0019 0.65 0,0026 0.060 0.67 0.007 0.033
+20
-
5
6
0.0023 0,096 0,0019 0.065 0.0014 0.065 0.0037 0.065 0.018 0.21 0.0014 0.14 0.92 0,001 0.042
0,0018 0.075 0.0015 0.048 0.0009 0,005 0.0025 0.045 0,0036 0.043 0.0034 0.050 0.38 0.003 0.061
4
3
2
t\
M E M B R A N E NO.
3
I
1
MEMBRAVE NO. 3
u 1
2
3
4
DH
Figure 3. Effect of pH on potential response of membrane No. 1 and 3.UOzCIz 10-3M.Fill solution, same at pH - 3
also thank Boyd Weaver of the Chemical Technology Division, Oak Ridge National Laboratory, for supplying many of the organophosphorus compounds. Received for review December 31, 1973. Accepted March 11, 1974. Research sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation. D.W.M. was an ORAU Summer Employee from Arkansas College, 1973.
Determination of Diphenylhydantoin by Phosphorescence Spectrometry Lee D. Morrison and C. M. O’Donnell’ Department
of Chemistry. Colorado State University. Fort Collins. Colo. 8052 7
Wallace et al. ( I ) introduced an assay for 5,5-diphenylhydantoin (DPH, Dilantin, Parke-Davis) which was later modified by Wallace and others (2-4). The latest modifiAuthor to whom reprint requests should be sent. (1) (2) (3) (4)
J. Wallace, J. Biggs. and E.V . Dahl. Anal. Chem. 37, 410 (1965). J. Wallace, Ana/ Chem.. 40, 978 (1968) P L. Morselli, Ciin Chim. A c t a . 28, 37 (1970). W A . Dill, L. Chucot, T. Chang, and A . J. Glazko. Clin Chem. 17, 1200 (1971)
cation of Dill et al. makes the procedure suitable for use in the clinical laboratory. Dill’s procedure involves an extraction of DPH from plasma into 1,2-dichloroethane, a subsequent extraction into alkaline medium followed by oxidation of the DPH with basic KMn04. The oxidation product (benzophenone) is extracted into a ,hydrocarbon solvent (i.e., isooctane) and analyzed by ultraviolet absorption. The blanks obtained from control plasma showed little absorption, ANALYTICAL C H E M I S T R Y , VOL. 46, NO. 8, J U L Y 1974
1119
Table I. Phosphorescence Intensities a t 446 nm of DPH Oxidation Product DPH concentration in plasma, rg/ml
Average intensity in amperesa
0.094 0.47 0.94 5.58 11.2 27.9 55.8 112
1.4 X 6.5 X 1 x 10-9 6.6 X 1.1 x 10-8 2 . 7 X 10-8 4 . 7 x 10-8 7 . 0 X lo-@
Error in intensities of 10%. WAVELENGTH ( n m )
Figure 1. Phosphorescence spectra of oxidation product (benzophenone) of 10-4M D P H (-X l ) , and oxidation product of 10-4M phenobarbital (------ X 20) in rnethylcyclohexane, Xexcite
= 260 nrn
and the presence of phenobarbital or other commonly used anticonvulsants did not interfere with the assay. We have modified Dill’s procedure, which has a 1 pg/ml limit of detection in order to incorporate phosphorescence analysis which enables the detection of DPH a t the 50 ng/ml level.
EXPERIMENTAL Reagents. All solvents used were spectroquality and all solids were reagent grade. 1,2-Dichloroethane was obtained from Fisher Scientific Co., Fair Lawn, N.J. Methylcyclohexane was obtained from Matheson Coleman & Bell, Norwood, Ohio. Potassium permanganate and NaOH were obtained from Mallinckrodt Chemical Works, St. Louis, Mo. Apparatus. The phosphorimeter consisted of the following components: A model C-50 complete arc lamp source with a 1000-Watt Xe Lamp (Oriel Optics Corporation, Stamford, Conn.); a B & L high intensity monochromator for excitation; an emission monochromator, model 82-410 .25M Ebert Monochromator (Jarrell-Ash Division, Fisher Scientific Co., Waltham, Mass.); a R446 multiplier phototube (Hamamatsu T.V. Co., Ltd.); a model 417 picoammeter (Keithley Instruments, Cleveland, Ohio); and a rotating sample cell assembly (5, 6). In addition, a CS7-54 excitation filter from Corning was used to remove second-order scattering. Procedure. Standards were prepared by dissolving a known amount of NaDPH in an alkaline solution a t p H 11.5-12 and adding this solution to a given volume of plasma. Dill’s procedure was used with the following modifications: The pH of 1 ml of plasma was adjusted to pH 6-7 in a Teflon capped centrifuge tube with an acid phosphate buffer. This pH is necessary to ensure the extraction of DPH into the 1,2-dichloroethane layer. The DPH was then oxidized by the method of Dill et al. and the oxidation product (benzophenone) was extracted into methylcyclohexane (MCH). M C H was used in the extraction procedure because of its ability to form a clear glass a t liquid nitrogen temperatures. Although this is not a necessary prerequisite for an ex-
(5) R Zweidinger and J D Wmefordner, A n a / Chem 42, 639 (1970) J D Winefordner, J Res Nat Bur Stand Sect A 76, 579
(6)
(1972)
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A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 8, J U L Y 1974
traction solvent, it is preferable because the use of a clear glassy matrix generally results in a significantly higher phosphorescence intensity. Approximately 0.1 ml of the sample dissolved in MCH is placed in a quartz cell (2-mm i.d., 4-mm 0.d.) fitted with a Teflon plug. This cell is cooled to liquid nitrogen temperatures and the intensity measured exciting a t 260 nm and observing a t 446 nm, i.e., the maximum phosphorescence wavelength for benzophenone.
RESULTS The phosphorescence spectra of the oxidation products of phenobarbital and DPH (benzophenone) are presented in Figure 1. The relative phosphorescence of the oxidation product of 10-4M DPH a t 446 nm was found to be 40 times as great as the phosphorescence of the oxidation product of 10- 4M phenobarbital. This indicates that phosphorescence detection is considerably more selective than the use of absorption spectrometry because the interferent must not only absorb, but phosphoresce with a high quantum yield. Standard curves were linear in the range of 0.1 pg/ml to 50 pg/ml of DPH (Table I). Blanks derived from control plasma subjected to treatment identical to standards showed virtually no phosphorescence background. The limit of detection was found to be 0.05 pg/ml with an error of less than 10%. The remarkable sensitiiity of the phosphorescence method enables the clinician to use sample sizes considerably less than 1 ml and still monitor submicrogram quantities of DPH which makes this method particularly applicable to pediatric care. The time required to make one reading of phosphorescence intensity and clean out the sample cell is less than 2 minutes. A standard curve may be generated in about 30 min. Since many samples may be run in a relatively short period of time, this method is suitable for screening purposes. Phosphorescence provides the most sensitive procedure for monitoring DPH yet developed and is even more specific than the ultraviolet absorption methods currently used. Received for review November 9, 1973. Accepted February 193 1974. This work was supported by Grant GP-38440X from the National Science Foundation.
