Anal. Chem. I 9 8 & 58, 2282-2285
2282
ACKNOWLEDGMENT The authors thank Joel Harris for his helpful comments regarding the treatment of the data. This paper is dedicated to Cheves Walling on the occasion of his 70th birthday. LITERATURE CITED (1) Pons, S.; Ghoroghchian, J.; Fleischmann, M., patents pending. (2) Ghoroghchian, J.; Fleischmann, M.; and Pons, S.,in preparation. (3) Bond, A.; Fleischmann, M.; Robinson, J. J . Nectroanal. Chem. 1984,
I f , 172. (4)Howell J. 0.; Wightman, R. M. Anal. Chem. 1984, 56, 524. (5) Bond, A. M.; Fleischmann. M.; Robinson, J. J . Nectroanal. Chem. 1884, 168, 299. (6) Bond, A. M.; Fleischmann, M.;Khoo, S. B.; Pons, S.; Robinson, J., submitted for publication in J . Electrochem. Sac
(7) Cassidy, J., Khoo, S. B., Pons, S., and Fleischmann, M., J . Phys. Chem. 1985, 89, 3933. (8) Dibble, T.; Bandyopadhyay, S.; Ghorcghchlan, J.; Smith, J. J.; Sarfarazi, F.; Fleischmann, M.; Pons, S., submitted for publication in J . Phys. Chem .
(9) Bond, A. M.; Fleischmann, M.; Robinson, J. J . Electroanal. Chem. 1984, 180, 257. (IO) Fleischmann,M.; Bandyopadhyay, S.; Pons, S. J . Phys. Chem. 1985, 89,5537. (11) Harris, J. M. Anal. Chem. 1982, 5 4 , 2337. (12) Ghoroghchian, J.; Fleischmann, M.; Pons, S., in preparation.
RECEIVED for review February 27,1986. Accepted May 12, 1986. We thank the Office of Naval Research and Dow Chemical Co. for support of this work.
Neutral Carrier Based Ca*+-Selective Electrode with Detection Limit in the Sub-Nanomolar Range U r s Schefer, Daniel Ammann, Ern0 Pretsch, U r s Oesch, a n d Wilhelm Simon* Department of Organic Chemistry, Swiss Federal Institute of Technology (ETH), Universitatstrasse 16, CH-8092Zurich, Switzerland
I n a 1:3 cation/ligand complex the Ionophore N,N,N',N'-
tetracyclohexyl-3-oxapentanedlamldeforms an almost ideal coordination sphere of nlne oxygen atoms for the uptake of Ca". By use of thls ionophore and current membrane technology, solvent polymeric membranes with extremely high Ca2+selectlvlties are obtained. Na+ and K+ are reJected by a factor of and IO8, respectively, and the detection limit of the Ca2+electrode response function In an intracellular ion background Is at about 100 pM Ca2+.
Considerable effort is invested in the design of calcium ion selective electrodes with improved selectivity and detection limits (1-5). The most attractive sensors in this respect are based on the neutral carrier (-)-(R,R)-N,N'-bis[ll-(ethoxycarbonyl)undecyl]-N~'-4,5-tetramethyl-3,6-dioxaoctanediamide (ETH1001) (2) introduced in 1975 (6). These sensors have been widely accepted for clinical and electrophysiological Ca2+-activity measurements (7). Studies of resting and changing intracellular Ca2+levels are however performed close to the sensor's detection limit, which is dictated by K+ interference (8). Similarly, it has been claimed that the usefulness of these sensors in soil sciences is limited by cation interferences (9, 10). The neutral carrier N,N,N',N'-tetracyclohexyl-3-oxapentanediamide (ETH 129) (11) was shown to form extremely lipophilic 1:3 cation/ionophore complexes with Ca2+and 1:2 complexes with Mg2+ (12). It is therefore not surprising that the corresponding solvent polymeric membranes show a high rejection of Mg2+ relative to Ca2+. A high selectivity for calcium over monovalent cations may be obtained through the incorporation of lipophilic anionic sites into the membrane phase of neutral carrier based liquid membranes for divalent cations (13). Thus, with adequate membrane technology, a further improvement of the selectivity of Ca2+ electrodes should be feasible. Here we report on a sensing system involving ETH 129 with clearly improved selectivities and detection limits, which .-?as obtained through such interventions.
EXPERIMENTAL SECTION Reagents. For all experiments, doubly quartz distilled water and chemicals of puriss. or p.a. grade were used. Membranes. The solvent polymeric membranes were prepared according to ref. 14 using 1wt % ETH 129 or ETH 1001 carrier, 66 wt '70 o-nitrophenyl octyl ether (0-NPOE;puriss. p.a., Fluka AG, Buchs, Switzerland), and 33 w t % poly(viny1 chloride) (PVC high molecular; purum p.a., Fluka AG). As lipophilic anionic site, potassium tetrakis(pchloropheny1)borate (KTpCLPB;purum p.a., Fluka AG) was used (for concentration see below). The syntheses of the carriers ETH 1001 and ETH 129 are described in detail in ref 15 and 11, respectively. Electrode System. Cell assemblies of the following type were used: Hg; Hg2C12,KC1 (satd)l3 M KCllsample solution11 membrane110.01 M CaC12,AgC1; Ag The external half cell was a free flowing free diffusion liquidjunction calomel reference electrode (16). The solvent polymeric membranes were mounted in electrode bodies Philips IS-561 (N.V. Philips Gloeilampenfabrieken, Eindhoven, Holland). Emf Measurements (General). The emf values were measured at 20 f l "C unless otherwise specified. The 16-channel electrode monitor used was equipped with FET operational amplifiers AD 515 KH (input impedance 1013 Q/2 pF; bias current < 150 fA; capacity neutralization; Analog Devices, Nonvood, MA). The signal was digitized by a 14-bit analog-to-digital converter DT 1744 (successive approximation, full scale i 500 mV, Data Translation, Natick, MA). Data were acquired with a single board computer (SBC 80/20-4; SBC 116A; Intel Corp., Santa Clara, CA) programmed in assembly language, using an AMC 95/6011 arithmetic processor (Advanced Micro Computers, Santa Clara, CA). Further processing of the data was performed off-line on a HP-85 calculator (Hewlett-Packard, Palo Alto, CA), programmed in BASIC. The measured emf values were corrected for changes in the liquid-junction potential according to Henderson (17 , l B ) . Using the Debye-Huckel theory, single-ion activities were calculated with the coefficients given in ref 19. Selectivity Determinations. Separate Solution Method. The selectivity factors, log Kg&, were obtained in 0.1 M unbuffered solutions of the corresponding chloride salts. Fixed Interference Method. The selectivity factors were evaluated from the electrode response functions for the primary
0003-2700/86/0358-2282$01.50/062 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58.
NO. 11. SEPTEMBER 1986 2283
Table I. Comwsition of Caw Buffer Solutions
ETH 1001
ETH 129
Flgum 1. Ganstnutlons of the Ca2+ c a m k s discussed.
ion containing a fued concentration of the respective interfering ion The solutions were buffered with pH buffers and Caw huffera, using 94 mM Na+ and 125 mM K+ as background electrolytes, respectively. The preparation of the buffer solutions is described in ref 20 (Na+)and 21 (K+). The solutions with pCa = 9 and pCa = 10 containing 125 mM K* were prepared according to ref 21. The comoositions of the solutions are eiven in Table I. The accuracy ;f the Caz' activity in the hullerkith pCa = 10 is limited by the buffer range of EGTA. The differences between the measured emf and values COrreEted for liquid-junction potentials amount. e.g., to -1.39 mV for pCa = -, -1.39 mV for pCa = 6 and -1.36 mV for pCa = 3. Interference by Hydrogen Ions. About 50 mL of a CaC1, 1W2,or 10.' M) was used solution of a given concentration (W3, for the measurements. By stepwise addition of small amounts of NaOH and HCI solutions, the pH was gradually changed to a b u t 13 and 1,respectively. The emf values were corrected for changes in the liquid-junctionpotential and in the activity of Caw due to the ionic strength. For example, the differences between the measured emfa and the values corrected for liquid-junction potentials are 1.94 mV for IO-' M CaCI,, -3.83 mV for lo-, M CaCI,, and -4.51 mV for M CaCI, (worst case). Practical Response Time. The electrodes were conditioned M CaC1, solution for 1-2 min. Then 1 mL in 40 mL of 3 X of 0.3 M CaCI, solution was added and a magnetic stirrer was turned on for approximately2 8. Emf values were taken at 100-ms intervals. Stnbility/Drift. Emf stabilities and emfdrift were examined by use of a cell assembly in a thennostated open beaker at 21 1 "C (room temperature) containing a lV3M CaCl, solution. The total measuring time was 48 h.
RESULTS AND DISCUSSION Results of the analpis of the crystal structures of MgRJCS), and of Ca(NCS), complexes with E T H 129 (Figure 1) are shown in raster graphica in Figure 2 (12). These pictures corroborate the high lipophilicity of the 1:3 CaZf/ETH 129 complex relative to the 1:2 Mg2+/ETH 129 complex. Indeed, an almost ideal fit with a cavity radius of 102 pm is obtained for Ca2+(radius, 106 pm (22)),if nine oxygen atoms (radius
components
pCa = 9 (125 mM K+)
pCa = 10 (125 mM K+)
PH HEPESD(EL-') EGTA' (gk') CaC12.2H20(8L-I) KOH 1 M ( m L W KCI 2 M (mLL-')
8.34 2.3831 3.8035 0.7351 45.8 39.6
8.97 2.3831 3.8035 0.7351 48.95 38.03
OHEPES: 4-(2-bydroxyethyl)piperazine-l-ethane-2-sulfonic acid. EGTA ethylene glycol his@-aminoethylether)tetraacetic acid.
'
Table 11. Influence of the Concentration of LipoDbilic Anionic Sites in ETA 129 Based Membranes" on the Ca*+ Selectivity KTpCIPB' IogKE
0%
23%
37%
61%
93%
107%
M = Na+ M = K+
-3.0 -3.3
-3.3 -3.6
-3.5 -3.9
-3.7 -4.0
4.9 +1.8
+2.1
4.9
'Membrane composition: 1 wt % ETH 129,66 w t % o-WOE, 33 wt % PVC. 'Additions of KTpClPB in mol % relative to the Caz+carrier concentration.
140 pm) are brought together with a local symmetry of D3h (23).This is very close to the ohserved fit (see Figure 2)with a Ca2+-oxygendistance of 239263 pm (mean distance amide oxygen to Ca2+,242 pm; mean distance ether oxygen to Ca2+, 258 pm) formed by three nonmacrocyclic ligands (12). Since Mg2+is not located in an optimal, octahedral array of coordinating atoms, a high rejection relative to Ca2+has to he expected in ETH 129 based ion-selective electrodes (11). A high preference of divalent relative to monovalent cations in neutral carrier based potentiometric sensors is observed, if the ionophore is used in a membrane with plasticizers of high dielectric constant in the presence of lipophilic anionic sites (e.g., potassium tetrakis(p-chloropheny1)borate (KTpClPB)). The highest pcsihle carrier-induced selectivity for divalent cations is expected at concentrations of anionic sites of 162.73, and 46 mol ?& with respect to the total carrier concentration and 1:1, 1:2, and 1:3 cation/ionophore stoichiometries, respectively (13). This trend can be recognized from the data in Table 11. In agreement with the 1:3 Ca2+/ETH 129 stoichiometry an optimal rejection of Na+ as well as of K+ is observed at around 50 mol % site addition. Since the selectivity data in Table I1 have been assessed by
Flgwe 2. Raster display of the srmcture of ths Ca" (left) and W*+ complex (right) of ths imnd ETH 129. Oxygen atoms in contact wiih Ca2+ and Mg2+are marked wim asterisks (for clarity. pichlres show 60% of the van der Waals radii).
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 11, SEPTEMBER 1986
Table 111. Selectivity Factors, log @A, for Solvent Polymeric Membrane Electrodes Based on the Ca2+ Carriers ETH 1001 and ETH 129
log K b Z
M = H+
1.0 wt 70 ETH 1001 0.5 wt % KTpClPB (69 mol %) 64.0 wt % 0-NPOE 34.5 wt % PVC
1.0 wt % ETH 129 0.6 wt % KTpClPB (53 mol % ) 65.6 wt % O-NPOE 32.8 w t % PVC
-4.4'
-1.6a
EMF
[mvl Ca" -BUFFERED SOLUTIONS ( 94 rnM Na') ETH 1001
1
-0.lb
M = Li+ M = Na+
-2.8" -3.4" -6.1' -3.8' -6.6d -4.4" -5.1e
M = K+ M = Mg2+
pvc
P-
-3.3" -3.7" -7.4' -4.0" -8.0d -4.90
I
o-NPOE PVC
" Separate solution method using 0.1 M unbuffered metal chloride solutions. bFixed interference method (pH 2), no Ca2+buffers. 'Fixed interference method & = 94 mM) using Ca2+-buffered solutions. dFixed interference method (CK = 125 mM) using Ca2+-bufferedsolutions. e Fixed interference method (cMg= 0.33 M), no Ca2+buffers. EMF [mvl Ca2'-BUFFERED SOLUTIONS (125 mM K*)
+$,
I
1
[mvl
11
ETH 129 KTpClPB
I:- - -
I
o-NPOE
-
10-' M CaC12
mV
,,-d' I
EMF
i,,
A
~
P
B
ETH 1001 KTpClPB o-NPOE
log a,,
Figure 4. Electrode response of membrane electrode cell assemblies based on the carriers ETH 1001 and ETH 129 in Ca2+-bufferedsolutions Containing 94 mM Na'. The detection limits are at -8.4 (ETH 1001) and -9.7 (ETH 129) log a units.
ETH 129 KTpClPB o-NPOE PVC
I
1
I
I
0
I
I
-12 -11 -10 -9 -6 -7 -6 -5 - 4 log aca
Figure 3. Electrode response of membrane electrode cell assemblies based on the carriers ETH 1001 and ETH 129 in Ca2+-bufferedsolutions containing 125 mM K+. The detection llmlts are at -8.7 (ETH 1001) and -10.1 (ETH 129) log 8 units.
the separate solution method, they may be biased by the presence of alkaline-earth metal cation impurities in the alkali metal solutions. The data therefore may mimic selectivities that are too poor. A comparison of the selectivities of membranes based on the Ca2+carriers ETH 1001 and ETH 129 (Figure 1)is given in Table 111. In both membranes, the addition of a welldefined concentration of lipophilic anionic sites has led to a considerable increase of the Ca2+selectivity. These extremely high Ca2+selectivities can only be measured reliably when using Ca2+-bufferedsolutions with a high background of interfering ions. In Ca2+-unbufferedsystems the emf measured for the 0.1 M interfering ion solutions is determined by the Ca2+ impurities within the reagents used, e.g., an impurity of lo4 M Ca2+determines the emf to the same extent as lo-' M Na+ for a KPAa = This explains the drastically different selectivities observed in Ca2+-buffered and Ca2+-unbuffered systems. Figures 3 and 4 show Ca2+ electrode response
l
1
l
2
,
,
,
,
,
,
,
,
,
,
,
3
4
5
6
7
8
9
10
11
12
13
,
14pH
Flgufe 5. Dependence of the emf of the Ca2+electrode cell assembly based on ET!i 129 on the pH of the sample solution for different CaCI, concentrations: points, experlmental values (uncorrected);thlck line, emf res nse after corrections for changes in liquid-junction potentials and Ca'activity Coefficients.
functions obtained in Ca2+-bufferedsolutions containing a constant typical intracellular K+ concentration of 125 mM or a relatively high Na+ background (94 mM). Membranes based on ETH 129 exhibit clearly improved detection limits at pCa = 10.1 (K+ background) and pCa = 9.7 (Na+ background), respectively. The optimum pH working range of the Ca2+electrodes with ETH 129 was evaluated by the fixed primary ion method (Figure 5). The data, which have been corrected for changes in the liquid-junction potential and in the activity coefficients, clearly show that even in millimolar Ca2+solutions no relevant H+ interference is present at pH values of about 2. For a stepwise change in the Ca2+concentration from 3 x M to M, the response time is 2.5 s (tW%,Figure 6). Stability measurements using thermostated, unstirred M CaClz solutions in an open beaker yielded emf drifts of 150 jtV/h. According to its lipophilicity (log Pmc = 7.2 (24))the Ca2+carrier ETH 129 should be a suitable component for the design of membrane electrodes with high lifetime. The results presented here clearly demonstrate that very high selectivities may be induced with nonmacrocyclic ligands. As predicted earlier (25),macrocyclic systems such as cryptands (26,27)and crown ethers (28,29) are not a prerequisite
l
Anal. Chem. 1988, 58,2285-2289
10 rnV
4
S STIRRING
0
5
10
15
20
[s]TIME
Flgure 8. Emf response of a cell assembly based on ETH 129 to a change in concentration from 3 X lo3 M to io-' M CaCI,.
for a high selectivity. Using the nonmacrocyclic neutral carrier ETH 129 and current membrane technology, solvent polymeric membranes exhibiting the highest so far reported membrane selectivities for metal cations have been realized. The extreme ion selectivity (108) and low detection limit (100 pM Ca2+)open several new aspects in Ca2+analysis. If these properties can be taken advantage of in microelectrodes, intracellular studies of sub-micromolar Ca2+activities will become more reliable and versatile. Furthermore, the sensor is especially suited for other low-level Ca2+measurements, e.g., the detection of
