2369
Anal, Chern. 1984, 56,2369-2372
Calcium-Selective Polymeric Membrane Electrodes Based on Bicyclic Polyether Amide Derivatives Keiichi Kimura,* Kazuhisa Kumami, Sadaya Kitazawa, and Toshiyuki Shono*
Department of Applied Chemistry, Faculty of Engineering, Osaka University, Yamada-oka Suita, Osaka 565, Japan
Properties of the ttlle electrodes are described In detail. The Ca2+-seiectlveelectrodes are superior to those based on the correspondlng monocyclk analogues In the seiectlvtty, showing excellent selectlvlty coefficients lor Ca2+ relative to H+ as well as Na', K+, and Mg2+. Effects of concentration of the neutral carriers and a ilpophiilc satl in the polymer membrane upon the electrode properlies are also discussed. A practical application of the Ca2+-seiectlve electrode based on the blcyclic polyether amide derivatlve indicates that Il is promising for determination of Ca2+ actlvlty In blood serum.
Calcium-selective electrodes have gained increasing interest as convenient tools for determination of Ca2+activity in biological and environmental systems. In particular, considerable attention has been focused on the determination of biological Ca2+activity by ion-selective electrodes because free Ca2+ plays a n eminent role physiologically. Two types of Ca2+-selectiveelectrodes have been developed so far. One is an ion-exchange-type electrode based on organic phosphate (I-3),which offers good Ca2+selectivity, but generally suffers from severe interference by H+ (or H,O+). The other is a neutral carrier-type one, to which a large number of acyclic polyoxadiamide derivatives have been applied (4-7). In some of the neutral carrier-based Ca2+-selectiveelectrodes, high Ca2+ selectivities relative to Na+, K+, and Mg2+have been attained. There, though, seems to be a problem left, that the selectivity coefficients for Ca2+ relative to H+, when determined by a mixed solution method, are still rather large. Cyclic polyether amide derivatives have been also attempted as a neutral carrier of Ca2+-selective electrodes, taking advantage of their preferential complexation with alkalineearth-metal ions, especially with Ca2+ (8-10).The Ca2+-selective electrodes exhibited rather high Ca2+selectivity over ranging from to We recently commuMg2+,I&&, nicated that Ca2+selectivity was enhanced in bicyclic polyether amide derivatives such as 1 and 2, as compared with the corresponding monocyclic analogues ( I I ) (Figure 1). It was thought to be partly because the cyclic polyether amides are most likely to complex Ca2+with 2/1 stoichiometry of macroring and cation. In this article we wish to report the full details on the properties of the Ca2+-selective polymeric membrane electrodes based on the bicyclic polyether amide derivatives. A potentiometric determination of total Ca2+ concentration in blood serum using the electrode is also described.
EXPERIMENTAL SECTION Syntheses of Neutral Carriers. N,iV'-Bis(hydroxyethy1) Derivative of Cyclic Polyether Amide 3. Compound 3 was prepared by the cyclization reaction of 4,5-dimethyl-3,6-dioxaoctanedioyl chloride with 1,17-dihydroxy-3,15-diaza-6,9,12-trioxaheptadecane (12) in benzene at high dilution, with a modification of a previous procedure (8). The point is that the reaction temperature should be kept below 10 OC. Silica gel column chromatography (CHC13/MeOH,5/1)allowed a colorless oil of 3 (50%): IR (neat) 3350,1640,1100 cm-'; 'H NMR (CDC13) 6 1.20 (dd, 6 H, CH3CH),3.60 (m, 28 H, O(CH,),O, O(CH2)2N(C0003-2700/84/0356-2369$0 1.50/0
H2),0H, MeCH) 4.20 (m, 4 H, OCH,CO); mass spectrum, m / e 450 (M'). N-((A1kanoyloxy)ethyl)-N'(hydroxyethy1) Derivatives 4 and 6. The N,N'-Bis( (alkanoy1oxy)ethyl) Derivatives 5 and 7. The reaction of the N,N'-bis(hydroxyethy1) derivative 3 with an equimolar amount of octanoyl chloride in chloroform in the presence of triethylamine allowed a mixture of the N-((octanoy1)ethyl)-W(hydroxyethy1)derivative 4 and the N,N'-bis((octanoy1oxy)ethyl)derivative 6, which were separated by silica gel column chromatography (CHCl,/MeOH, l O / l ) . 4 (20%): colorless oil; IR (neat) 3350,1730, 1640 cm-'; 'H NMR (CDC13) 6 0.90 (t, 3 H, CH3CH,), 1.05-1.40 (m, 14 H, Me(CH2I4,CH,CH), 1.40-1.80 (m, 2 H, CH2CH2C02),2.25 (t, 2 H,CH,CO), 3.60 (m, 25 H, O(CH2),0, O(CH&N, N(CH2),0H, NCH2CH20C0,MeCH), 4.05-4.50 (m, 6 H, OCH,CON, CH2OCO); mass spectrum, m / e 576 (M+). 6 (30%): colorless oil; IR (neat) 1730, 1640 cm-'; 'H NMR (CDClJ 6 0.90 (t, 6 H, CH3CH,), 1.05-1.40 (m, 22 H, Me(CH2I4,CH3CH), 1.40-1.90 (m, 4 H, CH2CH2C02),2.25 (t, 4 H, CH2C02),3.60 (m, 22 H, O(CH2),0,O(CHb,N, NCH2CH20C0, MeCH), 4.05-4.50 (m, 8 H, OCH,CON, CHzOCO); mass spectrum, m / e 702 (M'). The similar reaction using dodecanoyl chloride instead of octanoyl chloride yielded the corresponding (dodecanoy1oxy)ethyl derivatives 5 and 7. 5 (25%): colorless oil; IR (neat) 3350,1730, 1640 cm-l; 'H NMR (CDC13) 6 0.90 (t, 3 H, CH3CH2),1.05-1.40 (m, 22 H, Me(CH,Je, CH3CH),1.40-1.80 (m, 2 H, CH2CH2C02), 2.25 (t, 2 H, CH2C02),3.60 (m, 25 H, O(CH2)20,N(CH2)20, N(CH2),0H, NCH2CH20C0,MeCH), 4.05-4.60 (m, 6 H, OCH,CON, CH,OCO); mass spectrum, m e 632 (M+). 7 (30%): colorless oil; IR (neat) 1730, 1640 cm-l; H NMR (CDC13)6 0.90 (t, 6 H, CH3CH,), 1.05-1.40 (m, 38 H, Me(CH& CH3CH), 1.40-1.90 (m, 4 H, CH2CH2C02),2.25 (t, 4 H, CH,C02), 3.60 (m, 22 H, O(CH2)20,N(CH,),O, NCH2CH20C0,MeCH), 4.05-4.50 (m, 8 H, OCH&ON, CH,OCO); mass spectrum, m / e 814 (M'). Bicyclic Polyether Amides 1 and 2. Compound 1 was prepared by the reaction of the N-((octanoy1oxy)ethyl)-N'-(hydroxyethyl) derivative 4 with half an equimolar amount of pimelic chloride in chloroform in the presence of triethylamine. The resulted crude product was purified by preparative gel permeation chromatography. 1 (30%): colorlem oil; IR (neat) 1730,1640 cm-'; '€3 NMR (CDC13) 6 0.90 (t,6 H, CH3CH2),1.05-1.40 (m, 28 H, Me(CH2I4,CH3CH), 1.40-1.80 (m, 10 H, CH2CH2C02,COCH2(CH2)3CH2CO),2.25 (t, 8 H, CH,CO), 3.60 (m, 44 H, O(CH2),0, N(CH2),0, NCH2CH20C0,MeCH), 4.05-4.50 (m, 16 H, OCH2CON, CHZOCO). Similarly, the reaction using the N-((dodecanoy1oxy)ethyl)N'-(hydroxyethyl) derivative 5 gave compound 2 (28%): colorless oil; IR (neat) 1730, 1640 cm-l; 'H NMR (CDC13)6 0.90 (t, 6 H, CHSCHJ, 1.05-1.40 (m, 44 H, Me(CH,),, CH3CH),1.40-1.80 (m, 10 H, CHzCH2C02, COCHz(CH2)3CHZCO), 2.25 (t,8 H, CH&02), 3.60 (m, 44H, O(CH&O, N(CHzI20, NCH2CH20C0,MeCH), 4.05-4.50 (m, 16 H, OCH,CO, CH20CO). Other Chemicals. Poly(viny1 chloride) (PVC, average polymerization degree of 1100, Wako, Japan) was purified by repeated precipitation from tetrahydrofuran (THF) in methanol. The plasticizer o-nitrophenyl octyl ether (NPOE) was prepared with a slight modification of a previous procedure (13). Potassium tetrakisb-chloropheny1)borate (KTpClPB) was produced according to the literature (14). AU of the metal salts were analytical grade. Water was deionized and distilled through a Widmer column. Electrode Construction. Unless otherwise specified the PVC membranes were prepared in the following way: PVC (50mg), NPOE (110 mg), a neutral carrier (ca. 5 mg, 3 w t %), and
I
0 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
2370
'R
1:R =
-2
'R C7H15CO2
: R = CiiH23C02
3 : R1
.
4 R,
5
i
i
RZ
OH
I
OH
, R2
= C7Hl~C02
R1 = OH , R 2 ' C l l H 2 3 C 0 2
-2
-1
t
s;?' ga2+
6 : R1 = R 2 C7H15C02 1 R1 = R 2 = C l l H 2 3 C 0 2 :
'
Figure 1. Bicyclic and monocyclic polyether amide derivatives employed here.
KTpClPB (50 mol % to the macroring) were dissolved in THF (2 mL). This solution was poured into a flat Petri dish of 23-mm i.d. The solvent was evaporated slowly at room temperature to give a transparent membrane of 0.15-mm thickness. A disk of 5-mm diameter was cut out from the PVC membrane and then incorporated into an electrode body of Philips Model IS-561. After the injection of 1 X M CaCl, as the internal solution, the electrode was conditioned by soaking in 1 X M CaClz overnight. The external reference electrode is a standard calomel electrode with 0.1 M NH4N03agar bridge. The composition of the electrochemical cell is Ag/AgC1[0.001 M CaCl,[PVC membrane[sample solutionlo.1M NH4N0,[KCl(saturated)[Hg,C12/ Hg. EMF Measurements. The measurements were performed at 25 0.1 OC by using a pH/millivolt meter with high input impedance (Corning pH 130). The sample solutions were magnetically stirred in a double-wallglass container connected with a circulating bath. The electrode systems and the millivolt meter were kept in a grounded wire-net cage to cut off any electrical noise. Unless noted otherwise the experimental EMF values were corrected for the changes of the liquid-junctionpotential between the external reference electrode and a sample solution, which was computed according to the Henderson equation (15). Selectivity Coefficients. The kF& values were determined by the mixed solution method (FIM) recommended by IUPAC (16). The constant concentrations of interfering ions were 0.5 M for Mg2+,Li+, Na+, and K+, 0.05 M for H+, and 0.005 M for S$+ and Ba2+. In salt concentrations of lower than 0.1 M, activity coefficients y were calculated by using the following Debye-Hiickel / ( l where I and z stand formalism: log y = - 0 . 5 1 1 ~ ~ I ~ / ~PIz), for the ionic strength and the charge of the ion. Some experimental values of activity coefficient (17) were also adopted in the higher salt concentrations. Blood Serum Measurements. A control serum (Control Serum I, Wako, Lot No. PEE97N1) was employed as the serum sample. The practically undiluted and 20-fold diluted samples were prepared by adding 0.3 mL of a concentrated acetate buffer (7.64 M acetic acid and 0.40 M NaOH) to 5 mL of the serum and by dilution of 0.5 mL of the serum with an acetate buffer (0.382 M acetic acid and 0.02 M NaOH), respectively. The Ca2+determination by the Ca2+-selectiveelectrode was conducted by Gran's plot method (18). For the practically undiluted sample, the sample solution volume was 5.3 mL and the standard addition solution was 0.252 M CaCl,, of which 0.05 mL was added each time. In the case of the 20-fold diluted sample, the sample solution volume was 10 mL and the standard solution was 1X lod2M CaC12 (0.1 mL for each time). For the Ca2+determination by flame photometry the control serum was diluted 20-fold with distilled water. The measurementswere carried out on a Jarrel-Ash AA-781 photometer, at 422.7 nm using an acetylene/air flame. The calibration of the instrument was achieved with CaCl, solutions possessing a constant background of 140 mM NaCI, 4 mM KCl, 1 mM MgCl,, 2.5 mM urea, and 1.0 g/L glucose. The value for the Ca2+concentration as determined by colorimetry was cited from the assay data of the control serum employed here.
*
+
RESULTS AND DISCUSSION As the neutral carrier of Ca2+-selective PVC membrane electrodes, two kinds of bicyclic poly(ether amide) derivatives were employed here: one is the N-((octanoy1)ethyl)derivative
I
LNa'
Na*
Flgure 2. Selectivity coefficients k'zh for Ca*+-selectiveelectrodes based on cyclic polyether amldes 1, 2, 6, and 7 as determined by the fixed interference method. (For details see Experimental Section.)
1 and the other is the N-((dodecanoy1oxy)ethyl one 2 which is more lipophilic than 1. Only 6 and 7 were chosen as the monocyclic analgues for comparison, because the other monocylic analogues, 3-5, did not offer good electrode property due to the lack of lipophilicity. Figure 2 shows selectivity coefficients for the PVC membrane electrodes of the cyclic poly(ether amide) derivatives under a typical membrane condition. Obviously, the bicyclic polyether amide derivatives gave better results than the corresponding monocyclic ones in the Ca2+selectivity of the electrode, especially relative to Na+, K+, and Mg2+. These results may reflect the high Ca2+selectivity of the bicyclic polyether amides 1 and 2 as expected from the 2/1 stoichiometry of macrocycle and Ca2+(9, 19, 20). In the Ca2+selective electrodes of 1 and 2 is negligibly small the interference by Na+, K+, and Mg2+which often exists in biological and environmental systems. Since the polyether amide derivatives possess rather strong affinities for Sr2+and Ba2+as well as Ca2+,their interference on the Ca2+electrodes cannot be ignored. However, even k& relative to Sr2+or Ba2+was improved on the electrodes of the bicyclic polyether amides, compared with those of the monocyclic analogues. Selectivities of polymeric membrane electrodes may be influenced by their membrane compositions. Figure 3 represents that the effect of the natural carrier content in the membrane upon the electrode selectivity is not so remarkable in the Ca2+-selectiveelectrode based on the bicyclic polyether amide. The electrodes incorporating PVC membranes with 1-5 wt % neutral carrier are almost the same in the kE& value. Neutral carrier contents of higher than 5 wt % were not attempted because of the limited solubility of the neutral carriers in PVC membrane. The contents of below 0.5 wt % did not seem to be sufficient for the normal electrode response. On the other hand, the adding effect of the lipophilic salt, KTpClPB, was drastic as shown in Figure 4. In the absence of the salt in PVC membranes containing the neutral carriers, the Ca2+ electrodes did not work well partly because of the high membrane resistance. Moreover, the electrode without the salt in the membrane showed rather modest Ca2+selectivity and sluggishness in the response for Ca2+activity change. Addition of a small quantity of KTpClPB to the neutral carrier membrane improved the electrode properties markedly, providing the electrodes with excellent k& values. This is not the case with the selectivity coefficients for Ca2+relative
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
2371
0 -1 -2 cL
0"s w
m
-3
#
-4
-5
::
Flgure 5. pH dependence of EMF readings in Ca2+-selecthreelectrode based on 1. (The pH values were controlled by using calcium hydroxkle and hydrochlorlc acid.)
-e 0.51
3
2
4
5
neutral carrier content / w t % Figure 3. Effect of neutral carrler on Ca2+selectivity of PVC membranes containlng bicyclic polyether amMe 1. (0)Mg2+,(A)Sr2+,( 0 ) Ba2+,(0)LI+, (A)Na+, (m) K+. (The amounts of the other membrane components are the same as shown In the Experlmental Section.)
200-
150-
> E \ w 100
0 -1
-2
50
+ oL e
% -3
-
0
Nernstian Slope
7
-4 -5
6
5
4
3
2
- LogaCa2+
1
0
Figure 6. Callbratlon plots for Ca*+-selectlve electrodes based on bicycllc polyether amlde derlvatlves under a typical membrane confor CaC12 solutions ditlon: (@) for CaCI, sdutbns by the 1 electrode, (0) by the 2 electrode, (A)In buffered soiutlons (0.382 M acetlc acM and 0.02 M sodlum hydroxide) by the 2 electrode.
-6
0
25
50
KTpClPB content / mol% to macroring
Flgure 4. Effect of KTpClPB content of Ca2+ selectlvity of PVC membranes containlng blcycllc polyether amide 2. (The symbols are identical with those In Figure 3. The amounts of the other membrane components are the same as shown in the Experimental Sectlon.)
to Sr2+and Ba2+,which were hardly altered either with or without the salt in the membrane. The optimal content of KTpClPB appears to be about 50 mol % to the macroring (100 mol % to the bicyclic polyether amide). Higher KTpClPB contents may produce free KTpClPB (not accompanying the neutral carrier) which itaelf acts as ion exchanger. The tendency regarding the effect of KTpClPB content on the Ca2+selectivity of the PVC membrane seems to resemble that for acyclic neutral carriers of Simon et al. (21). Another important thing is the effect of H+concentration on the Ca2+-selectiveelectrodes based on the bicyclic polyether amides. The @!& value for the Ca2+electrodes was around 1X as determined by a mixed solution method (FIM) which represents actual analytical conditions, being quite excellent. There is, however, little enhancement in the preference of Ca2+over H+ by changing the neutral carrier from the monocyclic derivatives to the bicyclic ones. The pH dependence of EMF readings in the Ca2+-selectiveelectrode
of 1 was also investigated by using three different Ca2+concentrations (Figure 5). It was found that the EMF values remain constant over wide pH ranges in the present electrode system. Both of the Ca2+-selectiveelectrodes based on 1 and 2 exhibited Nernstian response (30 mV per 10 times the Ca2+ to 1 X activity change) in the activity range of 3 X M CaC12,as illustrated in Figure 6. Any concentration change in the internal filling solution of the electrodes did not bring about enlargement of the activity range for Nernstian response in calibration plots. Calibration plots for buffered Ca2+solutions (in an acetate buffer) by the 2-based electrode are also presented in the figure, indicating that the electrode response in the buffered solution is very similar to that in the unbuffered solution. As a practical application of the Ca2+-selectiveelectrode based on the bicyclic polyether amide 2, an attempt was made to determine Ca2+ concentration in a control serum. It is known that bound Ca2+in blood serum can be fully replaced by free Ca2+ around pH 3.5 (7), so that potentiometric determination of total Ca2+ in the serum was attempted by adjusting the control serum samples to pH 3.5. Practically undiluted and 20-fold diluted samples of the serum were prepared here. Addition of a small quantity of concentrated acetate buffer to the serum gives the practically undiluted
2972
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
Table I. Potentiometric Determination of Total Caa+ Concentration in Control Serum by Using 2-Based Caa+-SeleotiveElectrode and Its Comparison with Results by Flame Photometry and Colorimetry Ca2+concn, mmol/L flame Ca2+-elecphototrodeb metry colorimetry
sample" practically undiluted
2.56 iC 0.06
20-fold diluted
2.56 & 0.09
2.48 iC 0.03
2.50
The preparation procedures are in the Experimental Section. bBy the Gran plots method. CFromthe assay data of the control serum obtained by using o-cresolphthalein complex.
LITERATURE CITED
I
140
> 120E \ 100-
(a)
1
r ?
t
r
t
8060
within 20 s. On the other hand, for a 20-fold diluted sample it took at least 2 min to obtain constant EMF values. Therefore, the practically undiluted sample is more suited than the 20-fold diluted one for the Ca2+determination in blood serum by the Ca2+-selectiveelectrode of the bicyclic polyether amide. This implies a possibility for direct determination of free Ca2+activity in undiluted blood sera which is very desirable in clinical analysis, by using the present Ca2+-selectiveelectrodes. Registry No. 1, 90906-78-8; 2,91632-04-1; 3, 90906-76-6; 4, 90906-77-7;5, 91632-05-2;6, 90906-79-9;7, 91632-06-3;Ca, 7440-70-2;4,5-dimethyl-3,6-dioxaoctanedioyl chloride, 66582-24-9; 1,17-dihydroxy-3,15-diaza-6,9,12-trioxaheptadecane, 88328-35-2; octanoyl chloride, 111-64-8; dodecanoyl chloride, 112-16-3; pimelic chloride, 111-16-0;calcium, 7440-70-2.
I
,
,
I
(1) Ross, J. W. Sclence (Washlngton, D.C.) 1087, 156, 1378-1379. (2) ROZIEka, J.; Hansen, E. H.; Tjell, J. C. Anal. Chlm. Acta 1073, 6 7 , 155-178. (3) Craggs, A,; Moody, G. J.; Thomas, J. D. R Analyst (London) 1070, 104.412-4ia. ., (4) Simon, W.; Ammann, D.; Oehme, M.; Morf, W. E. Ann. N . Y . Aced. Scl. 1078,307, 52-70. (5) Bisslg, R.; Pretsch, E.; Morf, W. E.; Slmon, W. He&. Chim. Acta 1078, 61. 1520-1530. (8) Oesch, W.; Ammann, D.; Pretsch, E.; Simon, W. Helv. Chlm. Acta 1070,6 2 , 2073-2078. (7) Anker, P.; Wieland, E.; Amrnann, D.; Dohner, R. E.; Asper, R.; Simon, W. Anal. Chem. 1081,53, 1970-1974. (8) Petrhk, J.; Ryba, 0. Collect. Czech. Chem. Commun. 1080,4 5 , 1587-1574. (9) PetrBnek, J.; Ryba, 0. Anal. Chlm. Acta 1081, 128, 129-134. (IO) Petrgnek, J.; Ryba, 0. Collect. Czech. Chem. Commun. 1083,4 8 , 1944-1949. (11) Kimura, K.; Kumami, K.; Kitazawa, S.; Shono. T. J . Chem. Soc., Chem. Commun. 1084,442-443. (12) King, A. P.; Krespan, C. G. J . Org. Chem. 1074, 39, 1315-1316. (13) Alien, C. F. H.; Qates, J. W. "Orpnlc Syntheses";Wiley: New York, 1955;Collect Voi. 3, pp 140-141. (14) Cassaretto, F. P.; McLafferty, J. J.; Moore, C. E. Anal. Chim. Acta 1081,32,376-380. (15) Henderson, P. 2.fhys. Chem. 1007,59, 118-127. (16) Pure Appl. Chem. 1078,48, 127-132. (17) Parsons, R. "Handbook of Electrochemical Constants";Butterworths; London, 1959;pp 20-29. (18) Gran, 0. Analyst (London) 1052, 7 7 , 661-671. (19) Klmura, K.; Maeda, T.; Tamura, H.; Shono, T. J . Nectroanal. Chem. Interfaclel Nectrochem. 1070,95, 91-101. (20) Kimura, K.; Tamura, H.; Shono, T. J . Chem. Soc ., Chem. Commun. iQS3. 492-493. (21) Meier, P. C.;-Morf, W. E.; Llubii, M.; Simon, W. Anal. Chlm. Acta 1084, 156, 1-8.
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RECEIVED for review April 2,1984. Accepted June 11,1984.
