Anal. Chem. 1994,66, 3587-3591
Cyclic Polyamine Ionophore for Use in a Dibasic Phosphate-Selective Electrode Cllfton M. Carey' and Wilson B. Rlggan, Jr.t American Dental Association Health Foundation, Paffenbarger Research Center, National Institute of Standards and Technology, Gaithersburg, Matyland 20899
A cyclic polyamine, 3-decyl-1,5,8-triazacyclodecane-2,4-dione (N3-cyclic amine), was used as the ionophore for a dibasic phosphate-selectiveelectrode. This electrode exhibited a linear responsebetween 1.0 pmol/L and 0.1 mol/L dibasic phosphate activity with a near-Nernstianslope of --29 mV per activity decade. The electrode selectivity for dibasic phosphate over other commonly occurring anions was evaluated. A mechanism for the selectivity of the electrode toward HOPd2- ions is postulated to be a function of the size and charge of the N3cyclic amine ionophore relative to the size and chargeof HPOdZions. The electrode's superior selectivity and sensitivity make possible the direct measurement of phosphateactivity in a wide variety of applications. Ion-selective electrodes owe their selectivity to the mutual specificity of the ionophore and the analyte to reversibly form a stable complex that can be transported into the electrode membrane. An ideal ion-selective electrode responds to a specific ion in a way that is proportional to the chemical activity of that ion in a solution. Further, this ideal electrode is not affected by the presence of any other ions in the solution. There are many commercially available ion-selective electrodes, some of which approach ideal theoretical behavior within certain limits of concentration. The popularity of these electrodes has come from the recognition of their advantages: (1) simple methodology, (2) fast, nondestructive analysis, (3) direct measurement of species activity, (4) sensitivity over a wide concentration range, ( 5 ) low cost, and (6) portability. Disadvantages of ion-selective electrodes include the following: (1) dominance of the electrode response by interfering species in the analyte solution' (One needs to know that the analyte solution does not contain significant amounts of interfering species.) and (2) possible limitation of the accuracy of the measurement due to electrode response drift.2 The ubiquitous nature of orthophosphates in biological systems and in the environment indicates the need for an ionselective electrode for phosphate species. In recent years there have been several reports of phosphate-selective electrodes that are based on organotin c ~ m p l e x e s ~and , ~ insoluble phosphate salt^.^-^ Unfortunately, these ion-selective electrodes can be characterized as having short lifetimes, high ~~~
~
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Present address: C & R Approaches, Inc., 3812 Northampton Rd., Durham, NC 27707-5082. (1) Durst,R. A. InIon-SelectioeElectrodes; Durst,R. A,, Ed.; Specialhblication 314; National Bureau of Standards: Washington, DC; 1969; Chapter 11. (2) Ross, J. W., Jr. In Ion-Selectioe Electrodes; Durst, R. A,, Ed.; Special Publication 3 14; National Bureau of Standards: Washington DC, 1969; Chapter 2. (3) Glazier, S.A.; Arnold, M. A. Anal. Cfiem. 1988, 60,2540-2542. (4) Glazier, G. A.; Arnold, M. A. Anal. Cfiem. 1991, 63, 754-759. This article not subject to U.S. Copyright. Published 1994 by the American Chemical Society
detection limits, and/or significant interferences from other commonly occurring anions. A new phosphate ionophore based on a cyclic polyamine has been identified that, when used in an electrode membrane, resulted in a phosphateselective electrode that has sensitivity and selectivity superior to those previously described in the l i t e r a t ~ r e .This ~ report presents the fabrication and characteristics of the electrode and investigates the effect of the cyclic polyamine ring size on the selectivity and sensitivity of the electrode.
EXPERIMENTAL SECTION Reagents. Poly(viny1 chloride) (high molecular weight), sodium ethoxide, dibutyl sebacate, diethyl malonate, 1-bromodecane, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, and pentaethylenehexamine (Aldrich Chemical Co., Milwaukee, WI), K2HP04,l .OO mol/L KOH, pH standards, KCl, K2SO4, lithium lactate, potassium acetate, KN03,H3P04, and KSCN (Fisher Scientific, Inc., Cincinnati, OH) and buffers such as HEPES, etc. (Aldrich Chemical Co.) were used as received. Doubly distilled water was used for all solutions. Synthesis of Diethyl a-Decylmalonate Precursor. Diethyl malonate (176.19 g, 167 mL, 1.1 mol) was slowly added to sodium ethoxide (373 mL of 2.68 mol/L, 1 mol) in 127 mL of absolute ethanol, while in a water bath at 45 OC. 1-Bromodecane (221.19 g, 207 mL, 1 mol) was then added slowly to this mixture. The color of the solution changed from clear brown-orange to opaque yellow. After a 2-h reflux period, the ethanol solvent was removed by distillation. The mixture was cooled to room temperature and was transferred to a separatory funnel for extraction into 550 mL of H2O. The resultant two layers were the same brown color, with the top layer containing the product. This top layer was collected and vacuum distilled to remove the remaining ethanol. A clear, slightly straw-colored liquid was obtained, weighing 280.86 g, representing a 93.5% conversion,and having a boiling point of 140-145 "C under 700 mmHg vacuum. 'H-NMR analysis (in CDC13 relative to TMS): 6,4.2 (quartet, OCH2CH3, 4 H); 3.3 (t, CH3(CH2)8CH2CHCOOEt)2, 1 H); 1.9 (quartet, C H ~ ( C H ~ ) S C H ~ C2HH); , 1.3 (m, OCH2CH3, CH3(CH2)&H2,22 H); 0.9 (t, CH3(CH2)8, 3 H) ppm. This compound, diethyl a-decylmalonate, was a precursor for all the macrocyclic polyamines synthesized. ( 5 ) Grabner, E. W.; Vermes, I.; Konig, K.-H. Elecrroanal. Cfiem. 1986, 214, 13 5-1 40. (6) GMiker, W.; Cammann, K. Anal. Lett. 1989, 22, 1237-1249. (7) Liu, J. H.; Masuda, Y.; Sekido, E. Anal. Cfiim. Acra 1989, 224, 145-151. (8) Liu, J. H.; Masuda, Y.; Sekido, E. J. Electroanal. Cfiem. 1990, 291,61-19. (9) Carey, C. M. US. Patent 5,180,481, January 19, 1993.
Analytiml Chemistry, Vol. 66, No. 21, November 1, 1994 3587
sequent analysis by gas chromatograph confirmed the contamination. R R Synthesis of N4-, Ns-, and Ns-Cyclic Amines. Synthesis of these additional macrocyclic polyamines (N4-cyclic amine = I I 3-decyl- 1,5,8,1 l-butaazacyclotridecane-2,4-dione(2) N5HN / N,H cyclic amine = 3-decyl-1,5,8,11,14-pentaazacyclohexadecane2,4-dione(3), Ns-cyclic amine = 3-decy1-1,5,8,11,14,17hexaazacyclononadecane-2,4-dione(4))was accomplished in 1. 2. a manner similar to that used for the N3-cyclic amine, substituting triethylenetetramine, tetraethylenepentamine, and pentaethylenehexamine, respectively, for diethylenetriamine used for the synthesis of N3-cyclic amine. These structures are shown in Figure 1. Electrode Construction. Electrodes were constructed by i formation of a PVC matrix membrane over the open end of HN NH/ / an electrode body by dipping the electrode body into a membrane-forming cocktail consisting of 80 mg (20 wt 7%) of ionophore, 130 mg (45 wt 7%) of PVC, and 140 mg (35 wt 7%) 3. 4. of dibutyl sebacate in 3 mL of tetrahydrofuran. The percent Flgure 1. 3Decyl-l,5,8-triazacyclodecane-2,4dione (N3-cyclicamlne, composition of ionophore, PVC, and plasticizer is similar to 1);3decyC1,5,8,1l-butaazacyclotridecane-2,4dione (N,-cyclic amine, many described in the l i t e r a t ~ r e ; ~1-13 > ~however, J it remains 2); 3decyl-l,5,8,11,14-pentaazacyclohexadecane-2,4dione(N5-cycIic amine,3);3decyl-1,5,8,11,14,17-hexaazacyclononadecane-2,4dione to be optimized for this electrode. To completely suspend the (Ne-cyclicamine, 4). ionophore in the cocktail and assure maximum saturation, the cocktail was stirred for 2 days at room temperature on a magnetic stir plate. Approximately one-quarter of the Synthesis of 3-Decyl-1,5,8-triazacyclodecane-2,4-dione ionophore mixed into the cocktail dissolves; therefore, much (Ns-Cyclic Amine, 1). The structure for this compound is less ionophore is required to make the membranes. Electrodes shown in Figure 1. The synthesis was accomplished in dilute made with excess ionophore exhibited the same performance solution to avoid polymerization and to encourage cyclization as electrodes made without excess ionophore. The dipped to form thedesired product.1° Diethyl a-decylmalonate (0.076 electrode body with the liquified membrane was allowed to mol, 22.80 g) and diethylenetriamine (0.076 mol, 7.74 g) were air-dry 24 h, resulting in a membrane of 15-30 fim in thickness. refluxed for 42 days in absolute ethanol. During this period The electrode body was then filled with 0.2 mol/L KH2P04 2-mL aliquots of the reaction mixture were removed by solution titrated to pH 7.2 with KOH and allowed to soak in hypodermic syringe on days 7, 15, 22, and 37 for 'H-NMR the same solution for 24 h before use. A calomel reference analysis to determine the completeness of the reaction. It electrode (Model K-4112, Radiometer America, Westlake, should be noted that this step does not need to run longer than OH) was inserted into the topof the electrode body to complete 3 days if high yields are not required. After termination of the electrode assembly. Electrodes made with all four cyclic the reflux, the ethanol was removed under reduced pressure. amines were constructed and evaluated in the same manner. The product was then recrystallized from cold acetonitrile to Characterizationof the Electrode Response. PO4 and pH yield 14.71 g of heavy white precipitate, representing a 62.1% electrode measurements were made simultaneously with a conversion. The 'H-NMR characterizations of this product dual electrometer (Model FD-223, World Precision Instruin CDC13, with signals reported in ppm downfield relative to ments, Sarasota, FL), with the output recorded on a two-pen tetramethylsilane, were as follows: 6,3.7 (quartet, CH2CH2strip-chart recorder. The reference electrode portion of the NHC=O, 4 H); 3.3 (m, CH,(CH2)8CH2CH, 1 H); 2.8 (m, combination pH electrode (290-330, Curtin Matheson SciC H ~ N H C H ZH); , ~ 1.8 (br s, CH3(CH2)&H2CH, NH, 5 H); entific, Jessup, MD) was used as the reference electrode for 1.3 (m, CH3(CH2)8CH2,16 H); 0.9 (t, CH3(CH2)8CH2,3 H) both the HPO& and the pH measurements. The electrode PPm. cell for potential measurements was as follows: Infrared spectra (in KBr) show strong absorbance at 165 1 cm-l for carbonyl and broad absorbance between 3500 and Hg~Hg,Cl,~KCl~,,,,~O.2 mol/L phosphate buffer (pH 7.2) 3300 cm-1, indicating water of hydration. DTA/TGA analysis llmembranellsample solutionlKCl(s,,JAgCllAg (Model ST-736, Harrop Industries, Inc. Columbus, OH) found that the compound lost 5.6 wt % at 75 OC, representing one water of hydration. A broad melting point between 160 and The response characteristics of electrodes made with each of 180 OC was found; however, decomposition appeared to start the four cyclic amine compounds were determined at room near the same range. The elemental analysis found 59.7% C, temperature (22 "C) by use of standard addition procedures 10.4% H, 13.6% N, and 16.3% 0, where the calculated at pH 7.2. In this procedure, small aliquots of concentrated elemental composition, with one water of hydration, is 62.0% C, 10.7% H, 12.8% N, and 14.6% 0, indicating as much as (1 1) Moody, G. J.; Thomas, J. D. R. Selecriue Ion Sensitiue Electrodes; Merrow Publishing Co. Ltd.: Watford, England, 1971; Chapter 7. 5% contamination by the reactant diethylenetriamine. Sub(12) Greenberg, J. A.; Meyerhoff, M. E. Anal. Chim. Acta 1982, 141, 57-64. /
, I
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(10) Umezawa, Y.;Kataoka, M.; Takami, W. Anal. Chem. 1988,60,2392-2396.
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(13) Ma, T. S.;Hassan, S . S.M. Organic Analysis Usinglon-selectiveElectrodes; Academic Press: New York, 1982; Vol. 1, Chapter 2.
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Log HP0:. Activity (mow) Figure 2. Response of 10 phosphate-selective electrodes made with the N3-cyclic amine ionophore to the activity of HP042-ions in solution near pH 7.2.
Log Anion Activity (mol/L) Figure 4. Response of the phosphate-selective electrode made with the N3-cyclicamine ionophore to the activity of some common anions in solution near pH 7.2: (solid Ilne) dibasic phosphate, (*) nitrate, (+) sulfate, (A)chloride, (0) acetate, and (+) lactate.
modified single-solution methodI4J5 at the midpoint of the observed linear range. Modification of the single-solution method balances the effect of charge and non-Nernstian slope in response to the interfering ion for the calculation of the Kifot. Thus, log K i F = (Ej- Ei)/mi
9
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PH Figure 3. Response of the phosphate-selective electrode made wRh the N3-cyciic amine ionophore to the activity of HP042-Ions in a 50 mmoi/L total phosphate solution as the pH is varied between 5 and 12. Each dashed line is the predicted response of an electrode that responds to the indicated phosphate species as calculated from the observed standard curve determined at pH 7.2 for that species. Because the phosphate species distribution is a function of pH, the relative amount of one phosphate species in solution will increase and the other will decrease as the pH is varied.
standard (KH2P04 titrated to pH 7.2) were added and the pH was readjusted with KOH to pH 7.2 as needed. In this way, the sample solution was moved through the desired phosphate concentration range, thus generating the data points for the electrode response curves (standard curves). Measurements were made at pH 7.2 because an anticipated use of the electrode is the measurement of phosphate in physiological samples. Physiological pH is between 7.1 and 7.4, where the buffer capacity of phosphate is greatest at pH 7.2 within this range. Additionally, preliminary experiments indicated that there was a substantial pH effect above pH 8. The effect of pH on the function of the electrode was determined by increasing the pH of a 50 mmol/L total phosphate solution from pH 5 to 12 by the addition of small aliquots of 1 mol/L KOH that contained 50 mmol/L total phosphate Concentration. The thickness of the membranes and the internal resistance of the ionophores tested were not constant, resulting indifferent standard potentials between electrodes. Because the standard potentials between electrodes were different, normalization of the potential was necessary to allow comparisons of electrode responses. Thus, for purposes of comparison the potentials plotted in Figures 2-4 were adjusted such that the potential for each test electrode in distilled HzO was set to 0 mV. of the electrode for dibasic The selectivitycoefficient phosphate ions over other anions was quantified by use of a
where Ei is the electrode potential in response to the primary ion at a fixed activity, e.g., the midpoint of the observed linear range; Ejis the electrode potential in response to theinterfering ion at the same fixed activity at which El was measured; and mi is the slope of the calibration curve for the primary ion without any interfering ions present. Calculationof the PhosphateSpeciesActivities. Calculation of the activity of the phosphate species in solution required values for the total phosphate concentration ([P04]Tot), the solution pH, and ionic strength. The ionic strength of the solution could not be calculated directly from [P04]Tot because it is a function of the solution pH due to phosphate species equilibria with the hydrogen ion activity. Thus, an iterative approach was required to calculate simultaneously the phosphate species concentrations and the ionic strength. The method used was to calculate the concentrations of the charged phosphate species (H2P04-, HP0d2-, P04'-) by use of known equilibrium constants, the measured pH, an estimate of the ionic strength, and an estimated concentration of the neutral phosphate species H3P04. The ionic strength was then recalculated from the calculated concentrations of the phosphate species in solution and sufficient singly charged cations (K+ ions) to achieve electroneutrality. The total phosphate concentration was then calculated: c a l ~ [ P O , ] ~=~[H3P0,] ,
+ [H,PO,] + [HPO:-] + [PO4'-] (2)
The brackets indicate the concentration of the species shown. The c a l c [ P 0 4 ] ~was ~ ~then compared to the known [P04]Tot that had been added to the solution. The estimate of H3P04 was then adjusted higher or lower, and the calculations were (14) Carey, C. M.A New Phosphate Ion Selective Electrode. The American University, Washington, DC, 1992. (15) Attiyat, A,; Christian, G. D. Anal. Sci. 1988, 4, 13-16.
Ph.D. Dissertation,
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repeated until the c a l c [ P 0 4 ] ~=~ ~[P04]Tot. The activity coefficients cf,) for the charged species were then calculated from the final ionic strength of the solution by use of the Davies modification of the extended DebyeHiickel equation.16 The activities of the charged phosphate species were then calculated:
(3) where i denotes a particular phosphate species and the braces indicate the activity of the species.
RESULTS AND DISCUSSION Electrode Responsein PhosphateSolutions. Figure 2 shows the response (EMF) for 10 electrodes to the activity of H P 0 & near pH 7.2 in aqueous solutions. The measured linear range was 1.0 wmol/L to 0.1 mol/L {HP042-}and the average slope was -28.9 f 0.4 mV per activity decade of HPOd2-. The upper limit of the reported linear range was the high end of the experiments rather than a loss of linearity of electrode response. The actual upper limit of theelectrode's linear range may be higher. When the observed EMF was plotted against the activity of H2P04- species in the same solutions, the average slope was -27.9 f 1.1 mV per activity decade. By itself, the slope of this electrode indicates that it probably responds to the dibasic phosphate species with a near-Nernstian slope of --29 mV per activity decade. However, the slope alone is not enough to demonstrate the species to which an electrode is selective because the electrode may be responding poorly to the monobasic species with a sub-Nernstian slope. Therefore, the effect of pH on the function of the electrode in a 50 mmol/L phosphate solution was evaluated, with the results shown in Figure 3. From the standard curves, determined at pH 7.2, a predicted response was calculated for both the H2P04- and HP042- species by use of the pH and [P04]Tot. Because the phosphate species distribution is a function of pH, the relative amount of one phosphate species in solution will increase and the other decreases as the pH is varied. The predicted response is shown as dashed curves in this figure, and the observed data are the solid line with some experimental points plotted for clarity. This figure shows that the measured response closely matched the predicted response for the dibasic phosphate species between pH 6 and 8 and was not far from the values predicted for this species between pH 5 and 10. Additionally, the electrode response clearly did not match that expected if the electrode was responding to H2P04-species. This method (varying the pH while keeping the total phosphate concentration near constant) is a practical method to determine the ability of a phosphate-selective electrode to distinguish between the phosphate species in solution. Further, this method can be used to help evaluate the selectivity of other electrodes that are sensitive to ions that have several ionic forms coexisting in solution. Having considered both factors, the near-Nernstian slope and the response to the pH variance, one can conclude that this electrode responds to dibasic phosphate ions. Electrode Selectivity. The selectivity of this electrode for dibasic phosphate ions over other anions at the midpoint of (16) Davies, C. W. Ion Association; Butterworth's: Washington, DC, 1962; Chapter 3.
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Table 1. Selectivity Coefflcients ( K p ) for the Dlbaslc Phosphate Ion-Selective Electrode. interfering anion Kip' interfering anion
chloride nitrate sulfate lactate
0.0045 0.0017 0.001 0.001
thiocyanate acetate BIS-TRIS Propane HEPES
Kip
0.005 0.0006 0.0007 0.0005
K , pot as determined by the modified single-solution method at the midpdtnt of the linear range (0.03 mmol/L (HP042-)) at pH 7.2 (see text). HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid; BISTRIS propane, 1,3-bis[tris(hydroxymethyl)methylamino]propane. (I
the linear range (0.3 mmol/L) is given in Table 1. Additionally, Figure 4 shows the electrode response to pure solutions of interfering anions and HP042-over the full response range of the electrode. Thus, the selectivity pattern for this electrode is HP0;-
>> SCN- = C1- > NO; > SO:- = lactate ion >> acetate ion
This selectivity pattern is consistent with the empirical series suggested by Hofmeister" based on the lipophilicity of the anion with the exception that the electrode responds better to C1- than N03-. Possible interactions between the chloride ion and membrane components such as the plasticizer, or the PVC, could generate preferential recognition of the chloride ion.18J9 Although the studies to demonstrate such interactions have not been done, it is possible that preferential reactions between C1- and the plasticizer could occur. Additional studies are needed to determine the full interaction of the components of the membrane with specific anions. Experiments have shown that the phosphate-selective electrode does not deteriorate for a period of more than 9 months. The uses of the electrode over this period of time included quantifying the phosphate activity in untreated human saliva, in complex saturated calcium phosphate solutions, and in numerous mixed standardsS20The ability to analyze phosphate concentrations accurately in complex biological samples without sample preparation is a challenge for any phosphate measurement system. Human saliva is an example of such a complex solution; it contains proteins, phosphate-binding metals, and potentially interfering anions.21 The analysis of 1-mL samples of saliva collected from seven individuals resulted in an average activity of 3.8 f 0.6 mmol/L HP042-and a total free phosphate (nonbound by proteins or cations) of 6.5 f 1.7 mmol/L. This value was not significantly different (paired t-test) from the total phosphate of these samples, 6.6 f 2.4 mmol/L determined with spectrophotometric methods.22 The activity of HP042- in more than 100 different samples of known HP0d2- activity was determined with an accuracy of less than 1%. The coefficient of variation at 1 mmol/L total phosphate was 3% (n > 10). (17) Hofmeister, F. Arch. Exp. Pathol. Pharmakol. 1888, 24, 247-260. (18) Wegmann, D.; Weiss, H.; Ammann, D.; Morf, W. E.; Pretsch, E.; Sugahara, K.; Simon, W . Microchim. Acta 1984, 3, 1-16. (19) Lewenstam, A,; Kulanicki, A. Selective Electrode Reu. 1990, 27, 161-201. (20) Carey, C. M. J . Dent. Res. 1992, 71, 258. (21) Nikiforuk, G. Understanding Dental Caries, Etiology and Mechanisms; Karger: New York, 1985; Chapter 9. (22) Murphy, J.; Riley, J. P. Anal. Chim. Acta 1962, 27, 31-36.
Table 2. Response Characterlstlcs of the Macrocycllc Polyamines to HP042-a
ionophore
slope (mV/decade)
N3-cyclic amine N4-cyclic amine Ns-cyclic amine Na-cyclic amine
-28.9 -24.3 -28.1 -27.4
linear range (mol/L) 1X 2X 1x 3X
lO-'-l X 10-' 1k5-9 X l t 3 1v-1x
1043 X
10-2
useable pH range 6-8 6-9 5-9 6-8
Slope and linear range determined at pH 7.2.
The response characteristics of the four macrocyclic polyamines tested as phosphate ionophores are shown in Table 2. An inverse relationship was observed between the ring size of the ionophore and the linear range for electrodes made with these ionophores. However, relationships between ionophore ring size and useable pH range or slope were not found. This indicates that the charge of all of these ionophores was +2, appropriate for binding HP042- ions. Umezawa et a1.10 have described a macrocyclic amine that was selective for A T P and had little selectivity for HP0d2-. This ionophore is similar to the Ns-cyclic amine described in this report with the exception that the carbonyl oxygens have been removed from their ATP-selective ionophore. The authors suggest that this macrocyclic amine takes up three protons, resulting in a positively charged membrane surface that forms strong ion pairs with the ATP4- anion. The ionophore ring size and the size of the ATP" anion along with the ionic attraction are probably the major factors that give this ionophore its selectivity for ATP" ions. Selectivity experiments with the N4-cyclic amine described here indicated only slight selectivity for HP042- over Sod2-and C1-ions. These relationships show that the high selectivity of the N3-cyclic amine ionophore for HP042- ions over other anions is a result of the size and charge of the ring structure relative to the size and charge of dibasic phosphate. This relationship between ring size of theionophore and the linear range and selectivity for HP042-ion~is evidence that there are steric factors that help the selection for HP042ions. The carbonyl groups attached to the ring structure are thought to stabilize the positively charged center of the ring and to direct the anion toward the center of the ring where hydrogen bonds or ionic bonds may be formed between the amine and the charged oxygen atoms of the HP042-ion. Modeling of the ionophore with space-filling and Dreiding models has confirmed that there is a ring-size effect and that
there is a conformational orientation preference when the ionophore binds to the HP042-ion.14 However, these modeling techniques are approximate at best and do not take into account the charge distribution over the ionophore. Therefore, computer modeling and verification by 31P-NMRstudies are under way to elucidate the mechanism for the phosphate ionselective electrode. This and other reportdo that describe the use of macrocyclic amines for use in ion-selective electrodes clearly indicate that further research is warranted in this area. Although the response to phosphate for a few of these amines has been described here, many others remain to be investigated. Also needed is further optimization of the ion-selective electrode system (the membrane, plasticizer, and construction) to yield the most sensitive and selective ion-selective electrodes for anion analysis.
CONCLUSION This dibasic phosphate ion-selective electrode (made with N3-cyclic amine) shows greater sensitivity and selectivity than any previously reported phosphate-selective electrode. The superior selectivity and sensitivity of this phosphate-selective electrode, along with its robust nature and long lifetime, make possible direct measurement of phosphate activity in a wide variety of applications. ACKNOWLEDGMENT This investigation was supported, in part, by USPHS Research Grant DE1085 1 to the American Dental Association Health Foundation from the National Institutes of Health-National Institute of Dental Research and is part of the dental research program conducted by the National Institute of Standards and Technology in cooperation with the American Dental Association Health Foundation. Certain commercial materials and equipment are identified in this paper to specify the experimental procedure. In no instance does such identification imply recommendation or endorsement by the National Institute of Standards and Technology or the ADA Health Foundation or that the material or equipment identified is necessarily the best available for the purpose. Received for review August 17, 1993. Accepted May 31, 1994.' #Abstract published in Advance ACS Absfrocrs, September 1, 1994
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