indication of the direction of the sequence but do provide valuable information about the relative positions of residues. The use of only the normal sequence ions identified in the spectrum will also cause one to deduce at least one incorrect sequence. Ions were observed at masses 130, 158, 335, and 378 which correspond to the following combinations of submolecular groups : OBu CO OBU (CO)?: Bzal OBu (CO)$ Bzal OBu (CO),
available information, the sequencing of small peptides and other sequence molecules by mass spectrometry should gain a new level of reliability. The submolecular group method seems especially well suited to structural analyses of mixtures of small peptides where the possibility of finding multiple sequences is particularly high (9).
ACKNOWLEDGMENT
Gly Gly Gly Ser Gly Ser Ala
These clearly support another sequence. However this sequence is much less probable since, unlike structure 4, few additional supporting ions were identified. Only seven ions in Table V are consistent with the alternative, and only two other ions were found which uniquely support the alternative. We conclude that with the Qdbmolecular group which conveniently permits the utilization of more of the
We thank Professor M. Bodanszky of Case-Western Reserve University for the protected peptide sample and Professor Harry Morrison of Purdue for the sample from his research program. RECEIVED for review December 30, 1C70. Accepted April 21, 1971. Work supported by the National Institutes of Health under grant FR-00354. (9) F. W. Mchfferty, R. Venkataraghavan, and P. Irving, Biocliem. Biophys. Res. Commun., 39, 274 (1970).
Mechanistic Studies on the Valinomycin-Based Potassium Electrode Ehud Eyal and G. A . Rechnitz Department of Chemistry, State Uniuersity of New York, Buffalo, N . Y . 14214
The potentiometric selectivity of the valinomycin electrode i s shown to arise from the relative complexing ability of valinomycin with univalent cations, so that the electrochemical selectivity ratio for cation pairs i s given by the quotient of the equilibrium formation constants of the respective complexes. I t is also shown, on the basis of experiments on “frozen” electrodes, that the transport mechanism is of the mobile site type in which valinomycin acts as a carrier molecule or transport catalyst. NEUTRAL CARRIER TYPE liquid membrane electrodes with selective response to monovalent cations have gained considerable acceptance in recent years because of their high selectivity coefficients. Neutral carriers include polyethers (I), polyesters ( 2 ) , and various cyclodepsipeptides including valinomycin (3). r ___----_____-
i
Typically, valinomycin, (D-Val-L-Lac-L-Val-D-HyIv)A, is dissolved in a n appropriate solvent such as hexane, octanol, or phenyl ether, placed in a conventional liquid membrane electrode assembly giving rise to an electrode with selectivity ratios of the order of 5000:l for potassium over sodium; such a selectivity ratio is considerably higher than those of about 8:l for the cation-sensitive glass electrodes. The high selectivity of this electrode makes it a very potent tool for analytical and biomedical measurements where potassium determinations are often required in the presence of comparable or larger concentrations of sodium. Thus, a n understanding of the electrode mechanism is important not only in order to make better electrodes, but also to our understanding of transport phenomena in biological membranes. (1) C. J. Pedersen, J. Amer. Chem. Sac., 89, 7017 (1967). (2) R. P. Scholer and W. Simon, Chimia, 24, 372 (1970). (3) M. S. Frant and J. W. ROSS,Jr., Science, 167, 987 (1970). 1090
ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
In the latter connection, it must be recognized that the electrode consists of a solution of valinomycin in an organic solvent that is immiscible with water, interposed between two aqueous solutions containing the ions to be measured, just as the membrane of a living cell or of mitochondria separates the internal from the external solution of physiological character. In both cases valinomycin has been shown to greatly enhance the permeability of the membrane to potassium ions ( 4 , 5). Indeed, the selectivity of the valinomycin electrode is comparable to that of biological membranes and it can be shown that some compounds with large antimicrobial activity are those for which complex formation constants with cations are high (6). Various theories have been proposed to explain the role of valinomycin in ion transport through lipid membranes. These mechanisms involve some complex formation between the valinomycin and the potassium ion. According to one model, the valinomycin molecule acts only at the phase interface to enable the ions to pass into the membrane wherein they move as Free ions. A second model explains the enhanced permeability on the basis of canal formation involving the passage of ions through a canal of ordered valinomycin molecules that span the thickness of the membrane. The third mechanism, which we also favor, involves initial complex formation between the valinomycin and the cation followed by the transport of the ion through the membrane in the cavity of the valinomycin ligand, which acts as a “carrier”
(4) B. C. Pressman, Antimicrob. A g . Chemoiher., 1969, 28. ( 5 ) H. K. Wipf and W. Simon, Helv. Chim. Acta., 53, 1732 (1970). (6) M. M. Shemyakin, Yu A. Ovchinnikov, V. T. Izanov, V. K. Antonov, E. I. Vinogradova, A. M. Shkrob, G. G. Malenkov, A. V. Evstratov, I. A. Laine, E. I. Melnik, and I. D. Ryabova, J. Membrane Biol., 1, 402 (1969).
- - - - - - I--IO‘
Pores
Canal
103
Kf
IO’ Carrier
Carrier Relay
Figure 1. Possible transport models 3
K +-valinomycin complex
0 valinomycin
+
K’ IO
(see Figure I ) (7, 8). This last model is also supported by the extensive work of Eisenman et al. (9-11) o n the actins. I n the present paper we propose to demonstrate quantitatively that for valinomycin the potentiometric selectivity of the electrode depends only o n the relative formation constants of complexes of the ions in solution with valinomycin and not o n any process inside the membrane. We also present qualitative experimental evidence in support of our view that transport of ions within the membrane takes place via a “carrier,” “mobile site” mechanism. I n order to check our hypothesis, we determined the selectivity ratios for different ions a t the valinomycin electrode and compared these to the ratios of complex formation constants of the same ions with valinomycin in homogeneous solution. To test the validity of the “carrier” model us. the “canal” model, we compared selectivity measurements with the membrane phase frozen, while the aqueous phases were still liquid, with those made in the wholly liquid three-phase system. The “freezing” should not change selectivities if the canal model were the right one, whereas it should change them significantly for the carrier mechanism by decreasing the mobility of the bulky valinomycin molecules in the organic phase. EXPERIMENTAL
Potassium chloride and ammonium chloride from Fisher and rubidium chloride from Matheson, Coleman and Bell were used without further purification. The electrodes used for our measurements were an Orion liquid membrane electrode body (Series 92) with potassium ion membranes, 0.01M KCI internal filling solution and 5 mg/ml valinomycin in phenyl ether as the liquid ion exchanger; a Corning monovalent cation glass electrode (Model 476220) and a Beckman saturated calomel electrode as a reference. The liquid membrane electrode, as prepared, gave an approximately Nernstian response at room temperature comparable to that of the Orion 92.-19 commercial version. Selectivity ratios were determined as previously described (12). ( 7 ) H. K. Wipf, A. Oliver, and W. Simon, Helv. Chirn. Acta., 53,
1605 (1970). (8) P. Lauger and G . Stark, Riochirn. Riophys. Acta., 211,458 (1970). (9) S . Ciani, G . Eisenman, arid G. Szabo, J . Membraue Biol., 1,
l(1969). (10) lhid.,p 294. (11) lbid., p 346. (12) K. Srinivasan and G . A. Rechnitz, ANAL.CHEM.,41, 1203 (1969).
L 01
L
- 1 . . - - - L - - I
0.3
02
Mole
frortion
0.5
0.4
H20
K f for valinomycin complexes
Figure 2.
0 K.‘ complex cl Rb“ complex e NH,’ complex
--
___-.
I
Table I. Potentiometric Selectivity of the Valinomycin Electrode (KK+/Mt) ~
Ion
This work
Selectivity ratio ___ (os. K+) Ref. i3 Ref. 14
Li+
--150()
-4700
-5000
Na
~.looOo
-3800 84 0.52
-4003 100 0.53
+
NHa+ Rbf
50
0.4
Ref. 3 ... -loo00 50
...
Freezing experiments were conducted at 5 “C where the phenyl ether in the membrane phase is frozen while the aqueous phases are still liquid. Selectivity measurements were made on the frozen electrodes as well. Formation constants for valinomycin with potassium, ammonium, or rubidium ions were measured by adding microliter amounts of the chloride salt of the cation to 2 ml of the solvent to construct a calibration curve, and adding valinomycin in the same solvent. Formation constants could then be calculated from the known total concentrations and the free concentration of the alkali metal ion as measured with the glass electrode. RESULTS AND DISCTJSSION
The selectivity coefficients of the valinomycin electrode for potassium over ammonium, rubidium, and sodium were 50, 0.4, and 10000, respectively, at room temperature. These values may be compared to ones obtained by other authors (Table I) and are seen to be in good agreement, taking into _____
-..
.---~
(13) W. Simon, Zurich, private communication, 1969. (14) 1,. A. R. Pioda, V . Stankova, and W. Simon, Anal. Lett., 2, 665 (1969). ANALYTI ‘AL CHEMISTRY, VOL. 43, NO. 8, JULY 1971 e
1091
Table II. Comparison of Electrode Selectivity and Complex Formation Constants (K+/NH,+ Pair) in Water-Methanol Mixtures Formation const Formation const Selectivity Mole fraction H 2 0 K+-valinom y cin ",+-valinomycin Formation const ratio ratio K+/NH,+ 0.491 0.360 0.284
16432 8 674 f 23 [1290]0 2830 & 260 7260 i 150
0.200 0.106. 6
19.9 f 2.7 i 20 77 i 21
-65 71 f 42 94 i 27
40
50
Calculated by interpolation.
Table 111. Comparison of Electrode Selectivity and Complex Formation Constants (Kf/Rb+ Pair) in Water-Methanol Mixtures Formation const Formation const Selectivity Mole fraction H 2 0 K+-valinomycin Rb+-valinomycin Formation const ratio ratio, K+/Rb+ 164* 8 674 rt 23 2830 f 260
0.491 0.360 0.200
436 f 45 1530 f 100 6040 f 950
0.38 f 0.06 0.44 0.04 0.47 f 0.12
*
0.40
Table IV. Comparison of Electrode Selectivity and Complex Formation Constants (K+/Na+ Pair) in Water-Methanol Mixtures Formation const Formation const Selectivity Mole fraction H 2 0 K+-valinomycin Na+-valinomycin Formation const ratio ratio, K+/Na+ 0.360 0.200 0.106
674 2830 7260
807
loo00
The fact that the formation constant ratios determine the selectivities of the membrane electrode means that the transport through the membrane must involve a carrier mechanism which depends principally o n the formation of the metal ion-valinomycin complexes. A canal mechanism is excluded since it would require a dependence on membrane parameters involving a special arrangement of valinomycin molecules across the membrane. The freezing experiments involved the measurement of potassium to sodium selectivity in pure phenyl ether and in valinomycin-phenyl ether solutions as the liquid membrane phase a t 5 "C. These experiments were compared to similar experiments carried out a t room temperature. Two main differences were noted. First, while the room temperature system yields Nernstian slopes, the frozen electrodes showed slopes of only 9-13 mV/decade of cation activity. Second, the potassium to sodium selectivities dropped from -IO4 t o -2 for the valinomycin electrode and from 500 to -1 for the pure phenyl ether electrode. Comparable results are obtained also for NHd+ (Table V). These experiments support the view that the transport mechanism could not be a canal mechanism which would not be impaired by freezing the solvent surrounding the canals, but are consistent with a carrier mechanism in which the bulky complex molecule loses most or all of its mobility in the frozen membrane phase. The residual potentiometric response appears to be due to solvent impurities since the properties of the frozen membranes are essentially the same for the pure solvent and the valinomycin containing solution. Our conclusions are also supported by the recent tracer experiments of Wipf et at. (7) o n layered membranes. According to the work of Eigen et al. ( 1 3 , the specific rates of formation of the valinomycin complexes with monovalent cations should depend only on the cations and not o n the ligand, Eigen et at. measured rate constants for different (15) H. Diebler, M. Eigen, G. Ilgenfritz, G. Maass, and R. Winkler, Pure Appl. Chem., 20, 93 (1969).
ligands and report values in the 3 X 108 M-1 sec-' range-Le., nearly diffusion controlled. The calculation of dissociation rate constants for these complexes from the equilibrium formation constants and the formation rate constants yields values in the 108 sec-1 range for the most stable complexes. Valinomycin is thus seen t o be a n ideal carrier for such ions as potassium and rubidium in membrane electrodes because it
offers both attractive equilibrium selectivity and adequate interfacial kinetics to yield a rapidly responding electrode. RECEIVED for review March 19, 1971. Accepted April 27, 1971. We gratefully acknowledge the generous support of the Environmental Protection Agency, the National Science Foundation, and the National Institute of Health.
I
NOTES
Gas Chromatographic Resolution of OpticalIy Active Alcohols as 3 p-Acetoxy-A5-Etienates M. W. Anders a n d M. J. Cooper Department of Pharmacology, University of Minnesota, Minneapolis, Minn.55455
GASCHROMATOGRAPHY has been employed for the determination of absolute optical purity. Two approaches have been followed. Alcohols or amines may be converted into diastereomeric esters and amides, respectively, which are then separated on conventional packed gas chromatographic columns. Resolving agents used for the optical analysis of alcohols and amines include N-trifluoroacetyl-(S)-prolyl chloride ( I ) , N-trifluoroacetyl-(S)-phenylalanyl chloride (2), 2-acetoxypropionates (3), and (R)-(-)-menthyl chloroformate (4). Alternatively, derivatives of amino acids may be separated by chromatography on capillary columns coated with a n optically active liquid phase such as N-trifluoroacetyl-(5')-valyl-(S)-valine cyclohexyl ester (5). 3/3-Acetoxyd5-etienic acid has been employed for the resolution of asymmetric alcohols by fractional crystallization (1) B. Halpern and J. W. Westley, Biochem. Biophys. Res. Commun., 19, 361 (1965). (2) J. W. Westley, B. Halpern, and B. L. Karger, ANAL.CHEM.,40, 2046 (1968). (3) R. L. Stern, B. L. Karger, W. J. Keane, and H. C. Rose, J. Chromatogr., 39, 17 (1969). (4) J. W. Westley and B. Halpern, J. Org. Chem., 33, 3978 (1968). (5) S.Nakaparksin, P. Birrell, E. Gil-Av, and J. Or6, J. Chromatogr. Sci., 8, 177 (1970).
(6). This suggested the possibility that etienate esters of
optically active alcohols could be resolved by gas-liquid chromatography. This report describes the gas chromatographic separation of diastereomeric esters formed from asymmetric alcohols and 3&acetoxy-AS-etienic acid. EXPERIMENTAL
Chemicals. 3P-Acetoxy-A5-etienic acid (Mann Research Laboratories, New York, N. Y., [alD-20 "C [c = 1, acetone]) was converted t o the acid chloride as described by Djerassi and Staunton (6). Etienic acid has the S configuration at carbon 17. (R)-(+)-and Q-( -)-phenylmethylcarbinol were prepared in this laboratory by the pathway: mandelic acid + phenylethylene glycol + phenylethylene glycol-Ztosylate + phenylmethylcarbinol. Polarimetric examination of (R)-(+)and (a-( -)-phenylmethylcarbinol thus prepared showed [a]g +45.9" (c = 3.32, methanol) and -45.6' (c = 1.48, methanol), respectively. Literature : [a]2go - 44.2 O (neat) (7). Rotations were measured on a Bendix model 969 polarimeter in a 0.2-dm cell. (S)-(+)-and (R)-(-)-methyln-hexylcarbinols (Aldrich Chemical Co.) had [a]:: +9.5 " (neat). (6) C. Djerassi and J. Staunton,J. Amer. Chem. Soc., 83,736(1961). ( 7 ) E. Downer and J. Kenyon,J. Chem. SOC.,1156 (1939).
Table I. Gas Chromatographic Resolution of Optically Active Alcohols as 3/3-Acetoxy-A5-Etienatesand Menthyl Carbonates. 3!3-Acetoxy-A5-etienates Menthyl carbonates f R of Diastereomers, min t R of Diastereomers, min OV-17 DC 200 OV-17 Temp., Temp., (R)-(+) Temp., @)-(+I Carbinol "C (94-1( R ) - ( + ) (SI-(-) O C 694-1 ( R ) - ( + ) (94-1 "C (94-1( R ) - ( + ) (94-1 Phenylmethylcarbinol 260 16.0 17.5 1.09 230 73.5 83.0 1.13 150 11.0 12.0 1.09 Phenylethylcarbinol 260 19.0 21.5 1.13 230 11.0 1.16 102.0 160 9.5 91.0 1.12 Phenyl-n-propylcarbinol 260 22.0 24.0 1,09 230 14.0 1.12 160 12.5 110.0 120.0 1.09 Phenyl-n-butylcarbinol 260 27.5 29.0 1 ,05 20.5 1.08 230 140.0 150.0 1.07 160 19.0 Phenylcyclohexylcarbinol 250 93.5 98.5 41.5 1.08 250 1.06 110.0 110.0 170 38.5 1.00 Methyl-n-hexylcarbinolb 260 9.0 10.0 1.11 9.5 1.05 230 58.5 62.0 1.06 9.0 140 a Configuration and rotation refer to the alcohol. (S)-methyl-n-hexylcarbinol is dextrorotatory.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
1093
