Anal. Chem. 1987, 59, 2331-2334
233 1
Lithium Ion Selective Electrodes Based on Crown Ethers for Serum Lithium Assay Keiichi Kimura,* Hideki Oishi, Tsutomu Miura, and Toshiyuki Shono*
Department of Applied Chemistry, Faculty of Engineering, Osaka University, Yamada-oka, Suita, Osaka 565, Japan
Prevlous Ll+-selective polymeric membrane electrodes based on 14-crown-4 derlvativel have been Improved in the Li' selectivities against Na' and K+ by using 2 and 3 as the neutral carrler. Examinationof the membrane solvents for the electrodes allowed further enhancement In the Ll+ seiectivities. An excellent value of 1.3 X was attained wlth the poly(vlny1 chlorlde) membrane containing 2 as the neutral carrier and o -nltrophenyl phenyl ether/trls( 2-ethylhexyl) phosphate (9812) as the solvent. Serum Li+ assay was successfully achieved wlth an electrode containing a comMnatbn of the optlmlzed membrane based on the crown ether and a dialysis membrane, whlch eliminated interference by proteins In the samples.
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Considerable efforts are presently being directed toward Li+-selective electrodes. Specifically, applications of the ion-selective electrodes to Li+ assay in biological and environmental systems necessitate high Li+ selectivities against Na+ and K+ that are apt to coexist with Li+ in fairly high concentrations. In order to attain excellent Li+ selectivities for the ion-selective electrodes, neutral carriers such as noncyclic ligands (1-6) and crown ethers (7-12) have been studied extensively. We have attempted to use a number of crown ethers with small cavities as the Li+ neutral carriers (10). As a result, derivatives like 14-crown-4 (1,4,8,11-tetraoxacyclotetradecane) 1 have proved quite promising for the neutral carriers of Li+-selective polymeric membrane electrodes. The applicabilities of the 1-based Li+-selective electrodes have been substantiated (11,13,14). Still higher Li+ selectivities of the crown ether based electrodes against Na+, however, may be desired for their application to Li+ assay in biological systems such as blood sera. We have, therefore, designed a great variety of 14-crown-4 derivatives bearing substituents to enhance the Li+ selectivity of the parent macrocycle with respect to Na+ (12). One type of 14-crown-4 derivative synthesized consists of the derivatives carrying bulky substituents such as a benzyl group which are expected to suppress the formation of a 2:l (crown ether/cation) complex with Na+ and thereby to diminish the affinity of the crown ring for the ion as compared to Li+ forming the 1:l complex. The other is 14-crown-4 derivatives incorporating potential binding sites, such as an amide group, which raise the Li+ affinity of the macrocycle. We have thus found that 2 and 3, when incorporated into membranes, exhibit improved Li+ selectivities against Na+ and K+ as compared to the previous derivative, 1.
In this publication we wish to report on improved and optimized Li+-selective electrodes based on 2 and 3 and their application to serum Li+ assay. A Li+-selective electrode incorporating a combination of the crown ether based membrane and a dialysis membrane is also described.
EXPERIMENTAL SECTION Chemicals. The syntheses of the crown ethers employed here, dodecylmethyl-14-crown-4 (1) (IO), dibenzyl-14-crown-4(2), and
1 . R i = C H 3 Rz=CizHz5 2 R ~ = R Z = C H ~ ~
3:
R i =CHzCON(C2Hdz R z = C i ~ H 2 5
(diethylcarbamoylmethyl)dodecyl-14-crown-4 (3) (12),have already been described elsewhere. The membrane solvents or plasticizers, o-nitrophenyl octyl ether (NPOE) (1.9, o-nitrophenyl phenyl ether (NPPE) (I@, and o-fluorophenyl o-nitrophenyl ether (FPNPE) (17)were prepared according to the previous procedures. Commercially available (Wako) bis(2-ethylhexyl)sebacate (DOS), tris(2-ethylhexyl) phosphate (TEHP), and trioctylphosphine oxide (TOPO) were purified by vacuum distillation. Dioctyl phenylphosphonate (DOPP) was employed as received from Dotite. Poly(viny1chloride) (PVC, average polymerization degree of 1100, Wako) was purified by reprecipitation from tetrahydrofuran (THF) in methanol. The lipophilic salt, potassium tetrakis(pchloropheny1)borate (KTpClPB) was obtained according to the reported procedure (18). Alkali and alkaline-earth metal and ammonium salts employed were the best grade. Water was deionized and distilled. Membranes and Electrodes. Unless otherwise specified,the electrode membranes were cast from the THF solutions according to the procedure described previously (11). The membranes consist of 28 w t % PVC, 70 wt % membrane solvent (plasticizer), ca. 1 wt % crown ether, and KTpClPB (50 mol % to the crown ether). A disk (7 mm diameter) of the PVC membrane was incorporated into the electrode body of a Philips IS-561. The internal filling solution was 1 M LiCl aqueous solution. The resulting membrane electrodes were conditioned by soaking in the LiCl solution overnight. The construction of the Li+-selective electrode with the combination membrane containing an exclusion membrane is as follows: Disks (7 mm diameter) of the particular Li+-selective PVC membrane and an appropriate exclusion membrane were soaked overnight in 1M LiCl aqueous solution and distilled water, respectively,for the membrane conditioning. The external solution or water of the membranes was wiped off. The exclusion membrane was carefully attached to the surface of the PVC membrane and the combination membrane was then incorporated into the electrode body. Employed as the exclusion membrane was a dialysis membrane (Visking membrane 36/32, Union Carbide Corp.) or an ultrafiltration membrane (Ultrafilter UK-10, Toyo Kagaku Sangyo, molecular weight cutoff of 10000). emf Measurements. The measurements were made at 25 OC with a pH/mV meter of high input impedance. The external reference electrode was a double-junction-type Ag-AgC1 electrode or standard Ag-AgC1 electrode with an electrolyte bridge. The electrochemical cell is Ag-AgC1/ 1M LiCl/membrane/measured solution/O.l M NH4N0,/4 M KCl/AgCl-Ag. The selectivity coefficients, k&, were determined by a mixed solution method (fixed interference method) (19). The constant background concentrations were 5 X M for alkali metal ions, and 5 X lo-' M for alkaline-earth metal ions, H', and NH,'. The calculation details for ion activities and selectivity coefficients are as described previously (10). The practical response times (19)were determined by changing the measuring solution from serum samples without Li+ to those with appropriate amounts of Li+, irrespective of the need of ultrafiltration pretreatment.
0003-2700/87/0359-2331$01.50/00 1987 American Chemical Society
2332
ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987 0
0
-_
LI+
--
Ll*
--
Ll+
-_
"
-1
zs
;;I a3 -L -2 0
a2
K'
07
-
-1
CS'
CS'
N a'
K' N a' NH: "
R b*
-3
Na'
R b'
NH;.
CS'
K' Rb'
N"
-2
-t
- -3 0 0
-4
Ba4*
-5
-6
NPOE
FPNPE
NPPE
DOS
TEHP
NqPE
NpPE
NpOE
DOPP
TEHP
TOP0
-5
1
2
3
Flgure 1. Comparison of Li+ selectivities as neutral carriers among crown ethers 1, 2,and 3: membrane solvent, NPOE. For details see Experimental Section.
Figure 2. Effects of membrane solvents on Li' selectivities of 2-based
PVC membrane electrodes.
Table I. Selectivity Coefficients of Li+-Selective Electrodes Based on PVC-NPPE/TEHP Membranes of 1 , 2 , and 3
Serum Lithium Assay. The artificial serum samples contained 145 mM NaC1,4.5 mM KC1,2.5 mM CaCl,, 0.8 mM MgCl,, 2.5 mM urea, 4.7 mM glucose, and appropriate amounts of LiCl. Employed as the real blood serum was a control blood serum (Nescol-X, Kaketsuken, normal human blood), to which appropriate amounts of LiCl were added. It was confirmed by flame photometry that the added Li+ concentrations in both of the artificial and real sera were in agreement with the actual Li+ concentrations. When protein removal was needed, the serum samples were subjected to ultrafiltration by using Millipore filters (Immersible PTTK CX-30 with molecular weight cutoff of 30000 and PTGC CX-10 with molecular weight cutoff of 10000). On the serum Li+ assay, Li+ activities were read simply from the calibration curves and the ion concentrations were then computed by using an equation (10) based on Debye-Huckel theory.
"NPPE:TEHP = 9:l Experimental Section.
R E S U L T S A N D DISCUSSION Improved Neutral Carriers. The 14-crown-4derivatives, 2 and 3, when utilized as neutral carriers of the Li+-selective electrodes with the PVC-NPOE membrane system, certainly showed enhanced Li+ selectivities against Na+ and K+ as compared to the previous one, 1 (Figure 1). The substituent effects caused by the bulky group (benzyl) and the additional binding site (amide), although not very drastic, seem to function for the increase of the Li+/Na+ (or K+) selectivity ratio. The selectivity coefficients of Li+ with respect to alkaline-earth metal ions, NH4+,and Hf are still very small in the 2 membrane system. Yet, in the 3 membrane system, Li+ selectivities against alkaline-earth metal ions and H+ are appreciably diminished as compared to the 1 system. Specifically, the interference of H+ is remarkable in the 3 membrane system. It is probably due to the high affinity of the amide group attached to the crown ring toward these cations with high charge density. Thus, 2 is generally superior to 3 as well as 1 in Li+ selectivity as the neutral carrier of the ion-selective electrode, although 2 and 3 resemble each other in Li+ selectivity against Na+. Optimization of Membrane Solvents. Several plasticizers and their binary mixtures were tested as the membrane solvent for the PVC-2 membrane to further promote the Li+ selectivity of the resulting electrodes, especially with respect to Na+ (Figure 2). In general, the phenyl ether type solvents are better for the electrode membrane than the diester- and phosphate-type solvents. Employment of the mixtures of the phenyl ether type solvents and the phosphorus-containing ones (90/10) augmented the Li+ selectivities of the 2-based membrane against Na+, as is the case with other crown ether based Li+-selectivemembranes (11,13,20). Most of all,the mixtures of N P P E and T E H P seem excellent as a membrane solvent for the 2-based Li+-selective electrodes in terms of the ion selectivity. The NPPE/TEHP mixture solvent also improved
the Li+ selectivities of the 1- and 3-based membranes more or less, as shown in Table I. Even under this membrane condition, 2 is still superior to 1 and 3 in the Li+ selectivity against Na+. It is, however, preferred to use NPPE/TEHP mixtures with as low a fraction of T E H P as possible, since addition of TEHP to the PVC membrane is likely to decrease the Li+ selectivity against H+. From further examination about the fraction of the mixture solvent, NPPE containing 2 vol % of TEHP was found to be the best as the membrane solvent for the Li+-selective electrodes based on dibenzyl14-crown-4 2. The selectivity coefficients for the 2-based electrode obtained under the optimum solvent condition are listed in Table I as well, being the most excellent so far. The selectivity coefficients of Li+ with respect to Na+ and K+ are 1.3 X and 6.5 X lo4, respectively. The Li+ selectivity with respect to Na+ was not improved any more by the variations of the neutral carrier and the lipophilic salt concentrations in the membranes. The interference of alkaline-earth metal ions, NHJf, and H+ are extremely low, the selectivity coefficients of Lit with respect to the ions being on the order of either or lo4. The Li+-selective electrodes incorporating the PVC-NPPE/TEHP (98/2)-2 membrane exhibited near-Nernstian response (58 mV decade-') in the wide Li+ activity range as shown in Figure 3. The logarithm of Li+ activity in the detection limit (19) was -5.3 for the Li+-selective electrode. Electrode Response to Serum. The Lif-selective electrode with the PVC-NPPE/TEHP (98/2)-2 membrane was tested for its usefulness in Li+ determination in blood sera. At first, artificial serum samples containing Na+, K+, Ca2+, Mg2+,urea, and glucose as the background were employed for the purpose. The calibration curve for the artificial serum is also depicted in Figure 3. Obviously, there is some interference by Na+ that is contained at a concentration of 145 mM
interfering ion
Na+ K+ Rb+ cs+ Mg2+ Ca2+
Sr2+ Ba2+ NH4' H+
kLiMPot 2"
1
4.5 x 10-3 1.7 x 10-3 (1.3 x 2.6 x 10-3 1.3 x 10-3 (6.5 x 9.8x 10-4 1.7 x 10-3 (5.0 x 4.7 x 10-3 5.6 x 10-3 (1.8 x 3.2 X lo-" 1.7 X 10-5 (2.5 X 1.4 X lo-' 4.3 X (3.0 X 8.1 X lo-' 3.8 X (3.1 X 1.2 x 10-4 3.5 x 10-5 (3.2 x 1.7 x 10-4 1.1 x 10-3 (2.4 x 1.1 X lo-' 2.6 X lo-' (8.5 X (*
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3
10-3)* 10-4)* 10-4)* 10-3)*
io-^ 10-4)*
2.2 x 1.6 x 1.9 x 3.9 x 1.0 x 1.9 x 7.4 x 5.4 x 3.2 x 2.5 X
= 982). For
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details see
ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987
2333
T
> E w
. i20mv
It” -6
-5
-4
-3
-2
-1
Figure 3. emf response of Li+-selective electrode based on 2: (a) artificial serum sample, (b) aqueous LiCl solution; membrane solvent, NPPE/TEHP (98/2).
-4.0
-5
-4
log
-3
-2
aLI’
in the sample. The detection limit for the serum samples by the 2-based electrode is -3.9 in the logarithm of Li+ activity, being improved as compared to that by the previous 1-based electrode (11). The emf measurements of real serum samples with added Li+ were also made by use of the 2-based Li+-selective electrode. On the direct measurements of the serum samples, the Li+ detection limit of the electrode was increased as compared to that of the artificial samples, as demonstrated in Figure 4. This abnormal electrode response can be attributed to the proteins contained in the serum samples (21). In order to avoid the protein interference with the Li+ electrode response, the serum samples were subjected to ultrafiltration. Two ultrafiltration membranes with different values of molecular weight cutoff were attempted for the protein removal from the serum samples. Protein interference was still found in the serum samples ultrafiltrated with molecular weight cutoff of 30000. On the contrary, the inferference with the electrode response was alleviated drastically by the ultrafiltration of the sera with molecular weight cutoff of loo00 (Figure 4). This suggests that serum proteins with molecular weights of more than 10000 mainly interfere with the Li+ electrode response. The Li+ calibration curve of the 2-based electrode for the serum samples ultrafiltrated with molecular weight cutoff of 10 000 is quite similar to that for the artificial samples not containing any protein. The calibration curve for the ultrafiltrated sera around the clinical range of Li+ activity is depicted in Figure 5, which shows that the slope is still steep. The practical response times of the 2-based Li+-selective electrode ranged from 20 to 30 s in the clinical activity range.
-3.0
-2.5
ultrafiltrated serum samples around clinical activity range. The membrane condition is the same as for Figure 3.
0
-1
Flgure 4. emf response of 2-based Li+-selective electrode for sera with and without ultrafiltration: (a)without ultrafiltration, (b) ultrafiltrated with molecular weight cutoff of 30 000, (c) ultrafiltrated with molecular weight cutoff of 10 000. The membrane condition is the same as that given in Figure 3.
-3.5
log aLi’ Flgure 5. Calibration curve of 2-based Li+-selective electrode for
I
L j
-6
L
J
log aLi+
;-li
10
20
30
Time/ min FI ure 6. emf-time profile on measurements of serum samples by Li -selective electrode with combination of PVC-2 membrane and dialysis membrane.
B
Thus, serum Li+ assay by the crown ether based Li+ electrode is considered feasible with a prerequisite of the protein removal. Electrode Containing Exclusion Membrane. In order to avoid the troublesome pretreatment for the serum protein removal, Li+-selective electrodes with combination membranes, in which an appropriate protein exclusion membrane was attached simply to the PVC-NPPE/TEHP (98/2)-2 membrane, were fabricated unless otherwise identical conditions. Employed as the exclusion membrane were conventional dialysis and ultrafiltration membranes. Figure 6 shows the emf-time profile for the Li+-selectiveelectrode based on the combination membrane with a dialysis membrane. The electrode responded to the Li+ activity gradually, when the real serum sample without Li+ was replaced by the samples with appropriate amounts of Li+. As anticipated, the electrode with the combination membrane possesses longer response times than those with the single PVC membrane containing the crown ether. The response time for the Li+-selective electrode containing a dialysis membrane depends on the Li+ activities, being shorter in the lower activity ranges. A similar emf-time profile was observed in the Li+-selective electrode containing the PVC-2 membrane combined with an ultrafiltration membrane. However, the resulted electrode containing an ultrafiltration membrane exhibited much slower response to Li+ activity change than the electrode with the dialysis system. Attempts were made to assay Li+ in real sera to which Li+ was added in the clinical activity range, by using the Li+-selective electrode with the combination of PVC-NPPE/TEHP (98/2)-2 membrane and dialysis membrane. The results are given in Table 11, showing that the potentiometric serum Li+
Anal. Chem. 1907, 59, 2334-2339
2334
T a b l e 11. Lithium A s s a y in S e r u m S a m p l e s by E l e c t r o d e I n c o r p o r a t i n g PVC-2 M e m b r a n e C o m b i n e d w i t h D i a l y s i s Membrane
Li+ concentration, mM serum sample
actual
founda
coeff o f var, 7%
1 2 3 4 5
0.32
0.35 0.38 0.83 1.08 1.61 3.08
4
6
0.40 0.80 1.00 1.50 3.00
6 4 5 7 3
n M e a n of four repeat measurements. (Measurement o f a set o f t h e six samples was repeated.)
assay is rather reliable. In conclusion, the Li+-selective electrode incorporating the PVC-2 membrane in combination with the dialysis membrane seems useful for serum Li+ assay in spite of its somewhat prolonged response time. Registry No. 1, 91539-72-9;2, 106868-21-7;3, 106868-32-0; NPOE, 37682-29-4; NPPE, 2216-12-8; FPNPE, 93974-08-4; DOS, 122-62-3;TEHP, 78-42-2;TOPO, 78-50-2;DOPP, 1754-47-8;PVC, 9002-86-2; KTpClPB, 14680-77-4;Li, 7439-93-2.
LITERATURE CITED (1) Guggi, M.; Fiedler, U.; Pretsch, E.; Simon, W. Anal. Lett. 1975, 8 , 857-866. (2) Zhukov, A. F.; Erne, D.; Ammann, D.; Guggi, M.; Pretsch, E.; Simon, W. Anal. Chim. Acta 1981. 131, 117-122.
(3) Metzger, E.; Ammann, D.; Asper, R.; Simon, W. Anal. Chem. 1988, 58, 132-135. (4) Gadzekpo, V. P. Y.; Hungerford, J. M.; Kadry, A. M.; Ibrahim, Y. A,; Christian, G. D. Anal. Chem. 1985, 57, 493-495. (5) Gadzekpo, V. P. Y.; Hungerford, J. M.; Kadry, A. M.; Ibrahim, Y . A,; Xie. R. Y.; Christian, G. D. Anal. Chem. 1988, 58, 1948-1953. (6) Gadzekpo, V. P. Y.; Moody, G. J.; Thomas, J. D. R. Analyst(London) 1985, 170. 1381-1385. (7) Aalmo, K. M.; Krane, J. Acta Chem. Scand., Ser. A 1982, A36, 227-234. (8) Olsher, U. J . A m . Chem. Soc. 1982, 104, 4006-4007. (9) Gadzekpo. V. P. Y.; Christian, G. D. Anal. Lett. 1983, 16, 1371- 1380. (IO) Kitazawa, S. Kimura, K.; Yano, H.; Shono, T. J . Am. Chem. Soc. 1904, 106, 6978-6983. (11) Kitazawa, S.;Kimura, K.; Yano, H.; Shono, T. Analyst (London) 1985, 170, 295-299. (12) Kimura, K.; Yano, H.; Kitazawa, S.;Shono, T. J . Chem. Soc., Perkin Trans. 2 1986, 1945-1951. (13) Xie, R. Y.; Christian, G. D. Analyst (London) 1987, 112, 61-64. (14) Gadzekpo, V. P. Y.: Moody, G. J.; Thomas, J. D. R. Anal. R o c . (London) 1986, 2 3 , 62-64. (15) Allen, C. F. H.; Gates, J. W. Organic Synthesis; Wiley: New York, 1955; Collect Vol. 111, pp 140-141. 16) Brewster, R. Q.; Groening, T. G. Organic Synthesis: Wiley: New York, 1943; Collect Vol. 11, pp 445-446. 17) Ryba, 0.; Petranek, J. Collect. Czech. Chem. Commun. 1984, 4 9 , 2371-2375. 18) Cassaretto, F. P.; McLafferty, J. J.; Moor, C. E. Anal. Chim. Acta 1965, 32, 376-380. 19) "Recommendations for Nomenclature of Ion-Selective Electrodes" Pure Appl. Chem. 1976, 4 8 , 129-132. (20) Imato, T.; Katahira, M.; Ishibashi, N. Anal. Chlm. Acta 1984, 765, 285-289. (21) Gadzekpo, V. P. Y.: Moody, G. J.; Thomas, J. D. R. Analyst (London) 1986, 711, 567-570.
RECEIVED for review March 2, 1987. Accepted June 1, 1987.
Direct Electron Transfer Reactions of Cytochrome c at Silver Electrodes David E. Reed and Fred M. Hawkridge* Department of Chemistry, Virginia Commonwealth University, Richmond, Virginia 23284
The dlrect, heterogeneous electron transfer reactions between horse heart cytochrome c and silver electrodes have been shown to be stable for periods of time exceedlng 12 h. The kinetics of these reactions are quasi-reversible at polished silver surfaces and at ekctrochemically roughened silver surfaces. These results demonstrate that neither electrode surface modification nor the inclusion of mediators is necessary to study the electron transfer reactions of cytochrome c at silver electrodes.
Direct electron transfer reactions of cytochrome c at bare silver electrodes have been reported to proceed by slow, irreversible heterogeneous kinetics ( I , 2). The slow ra'tes of electron transfer have been attributed to electrode fouling by irreversible adsorption of protein ( 3 , 4 ) . Strong irreversible adsorption of cytochrome c has been indicated by surfaceenhanced Raman scattering (SERS) measurements a t this surface ( I , & 5-7). In several cases, prior modification of the silver surface ( 2 , 5 )by the adsorption of surface promoters has facilitated electron transfer reactions between cytochrome c and silver electrodes ( I , 2). In our studies, quasi-reversible electron transfer kinetics have been obtained for the reaction 0003-2700/87/0359-2334$0 1.50/0
of cytochrome c a t silver electrodes without the presence of mediators, modifiers, or surface promoters. This was accomplished by using cytochrome c samples that had been chromatographically purified, but not lyophilized, prior to the voltammetric study. In a previous report (8) attention had been drawn to the effects of sample purity on the voltammetric response of cytochrome c at solid electrodes. In that report it was clear that impurities found in high-quality commercial samples of cytochrome c (9) could affect the heterogeneous electron transfer kinetics a t electrodes. In a more recent report ( I O ) , it was indicated that the process of lyophilization (this is how the protein is usually stored after purification) denatured a small amount of the ferricytochrome c. This denatured form of cytochrome c does not return to its native state when reintroduced into aqueous media as indicated by various chromatographic bands with mobilities both slower and faster than the native protein. Therefore, these recent results have led us to consider the importance of sample purification as it applies to the direct heterogeneous electron transfer reactions of cytochrome c at bare silver electrodes. In this report, cyclic voltammetry (CV) ( I I , 1 2 ) , derivative cyclic voltabsorptometry (DCVA) (8, 13-15),and single potential step chronoabsorptometry (SPS/CA) (16,17) have been 1987 American Chemical Society
