Differential Double Pulse Voltammetry at Chemically Modified Platinum Electrodes for in vivo Determination of Catecholamines Ross F. Lane*

and Arthur T. Hubbard

Department of Chemistry, University of Hawaii, Honolulu, Hawaii 96822

Problems with film formation on platinum electrodes preclude the use of dlfferential pulse voltammetry for analytical determination of catecholamines in physiological media. A pulse electrochemical technique, differential double pulse voltammetry, utilizes two simultaneously varying unequal square wave potential pulses as an alternative to the linear dc ramp and allows these problems to be avoided. Theory and experiment are in good agreement with a reversible, two-electron Oxidation to the o-quinones, with no further complications from homogeneous chemical reactions Indlcated. Chemical modlfication of platlnum surfaces with aqueous iodide provides an electrode which is essentially devoid of electrochemical and chemical Interference over the potential range of interest. The modified electrodes exhibit a sensitivity toward the catecholamlnes unachievable with unreacted electrodes. The methodology presented Is discussed in regard to its potential usefulness for the determination of dopamine and norepinephrine in brain tissue.

The catecholamines, dopamine (DA) and norepinephrine

(NE), are essential participants in the neurotransmission process ( I ) . Accordingly, they are frequently implicated in neurological diseases of the brain (2-4). Advances in the treatment of such diseases have resulted from the study of these amines and their metabolites in the central nervous system (5). The rate of development in this area is limited by the experimental techniques available for the analysis and characterization of endogenous catecholamines. In spite of rapid progress in areas such as bioassay (6), fluorometry (7, 8), chromatography (9, IO), radiochemical analysis (11), fluorescence histochemistry (12),electron microscopy (13),mass spectrometry (14), and microelectrophoresis (15),an accurate method applicable in vivo to catecholamine determination has not been reported. Significantly, present techniques based upon freezing and dissection can result in significant error due to post mortem changes (4,16, 17)and, in general, only the average tissue level for the entire region is obtained (18). The potential applicability of voltammetric techniques to analytical and mechanistic characterization of biogenic amines in vivo has been clearly noted by Adams and co-workers (19, 20). The catecholamines react readily at electrodes, following well defined stoichiometric paths (21, 22). However, major experimental difficulties remain. Perhaps for this reason, only three voltammetric studies of the brain in vivo have been reported, and these were of a preliminary nature (19,20). The principal difficulties are a lack of sensitivity, and a susceptibility to electrode poisoning and interference from competing reactions. Recently developed pulse voltammetric (23-25) techniques are ideally suited to this application. In particular, differential pulse voltammetry (DPV) offers the advantage that measurement of the current is performed after the double layer charging process has subsided, yet at sufficiently short times

after application of the potentiostatic pulse that the Faradaic current is still large, leading to an unusually favorable signal-to-noise ratio for low concentrations of reactant. However, attempts to employ DPV for the present purposes failed for two reasons: First, the products of catecholamine electrooxidation undergo subsequent chemical reactions to form solid deposits on the electrode, resulting in a continual decline in the measured current with use, preventing quantitative interpretation of the results; and second, chemisorption of numerous species on conventional platinum electrodes caused marked and variable inhibition of the electron transfer process, which interfered with the analysis. These problems have been avoided by means of the approach described herein.

EXPERIMENTAL Preparation and Chemical Modification of Platinum Electrodes. Platinum electrodes were prepared by sealing high-purity Pt wire (Engelhard)in borosilicate “soft” glass and polishing the sealed end so as to expose only a disk of platinum to the solution. Cleanliness of the Pt surface was ascertained by the criteria presented elsewhere (26-28); briefly, the electrodes were heated to incandescence in a gas-oxygen flame, quenched in concentrated HC104, electrochemically oxidized (1.3 V vs. Ag/AgCl reference) and reduced (0 V) repeatedly in 1 F HC104, and cycled potentiostatically until resolution of the H deposition and Pt oxidation regions was apparent and constant. In all experiments, purity of the Pt electrodes, as judged by cyclic voltammetry (291,was established prior to use. Chemical modification.of the Pt electrodes was accomplished by exposing the electrodes, thus cleaned, to freshly prepared, deaerated solutions of KI in pure HzO or 1 F HClO4 (26-28). Electrochemical destruction of the surface layer does not occur until the potential exceeded 0.5 volt. When other modifying agents were employed, cleaned Pt electrodes were exposed to freshly prepared, deaerated aqueous solutions of the surfactant as described previously (27, 28).

Reagents. All solutions were prepared with pyro-distilledwater, purified by pyrolysis in pure oxygen over a Pt catalyst at 1000 “C (30). Buffer solutions were prepared from analyzed reagent grade chemicals without further purification. Unless noted otherwise, the supporting electrolyte was a 0.1 F phosphate buffer, pH 7.4, containing 0.9% (-0.15 F) NaCl. Dopamine hydrochloride and norepinephrine bitartrate monohydrate were obtained from Sigma Chemical Company (St. Louis, Mo.) and stored desiccated at room temperature. To avoid extraneous oxidation, accurately weighed samples of the catecholamines were added to buffer solutions which were thoroughly deaerated with prepurified nitrogen prior to use. All solutions were prepared fresh daily. Pulse Voltammetric Waveforms. In conventional differential pulse polarography (23,24),a fixed amplitude potential pulse, AE, is superimposed at periodic intervals on a slowly varying dc potential ramp (Figure 1A) and, if utilized with a dropping mercury electrode, is synchronized with the growth of the mercury drop. The dc current is measured immediately before application of the pulse and again towards the end of the pulse duration. The difference between the magnitudes of these two currents is measured and recorded vs. the dc potential. In contrast, the potential-time waveform employed in differential double pulse voltammetry (DDPV),Figure lB, consists of two square wave potential pulses of unequal amplitudes, El and E*, separated by a sufficient time interval and originating from a preset potential, E(O),where no Faradaic reaction occurs. No dc potential ramp is employed, so that the electrolysis is carried out only during the duANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976

1287

A

30TENTIAL

A

-AI

-FIRST

150 nA

SCAN

!

MEASURED

.

.

PULSE WIDTH

I -Ai

DELAY BETWEEN CYCLES

B

TiME

B

POTENTiAL +FIRST

CYCLE--f--------SECDND

CYCLE

-

&

Figure 2. DPV current-potential curves for 1 X clean Pt electrodes

A€ = 25 mV; At = 50 ms; A = 1.3 X cm2; scan rate = 2 mV s-'. (A) Repeated voltammetric scans. Successivescans are shown from top to bottom. (E) Effect of electrode potential. (a) Electrode held at € = -0.1 V vs. AglAgCl before recordingcurve. (b) Electrode held at € = 0.2 V

MEASURED

c------c-----IPULSE DELAY WIDTH BETWEEN PULSES

DELAY BETWEEN CYCLES

TIME Figure 1. Potential-time waveforms used in pulse voltammetric techniques (not to scale) (A) Differential pulse voltammetry. (E) Differential double pulse voltammetry. In the present studies: At = 15 ms; pulse width = 20 ms: delay time between pulses = 200 ms: time for one complete cycle = 1 s; A€ = 0 200 mV

-

ration of each pulse. Currents are sampled at identical times in each pulse; sufficient time is allowed so that the majority of the charging current has subsided. The difference between the two currents is obtained and recorded. The process is then repeated at time intervals of 1 s. Both pulse amplitudes are varied simultaneously between cycles by equal amounts, such that the difference between them, AE, is constant. With the present instrumentation, pulse heights are increased in increments of about 5 mV per cycle and A E was varied from 0 to 200 mV. Time intervals within the waveform are also variable; those employed for the studies described herein are given in Figure 1B. Current-potential data were obtained by means of a multipurpose electrochemical circuit based upon operational amplifiers and relays having mercury-wetted contacts. The circuit diagram of this instrument (available on application to the authors) will be presented later. Potentiostatic circuitry was conventional. The auxiliary electrode was a Pt wire; potentials are referred to a AgIAgC1 electrode prepared with 1 F NaC1.

RESULTS. AND DISCUSSION Conventional Differential Pulse Voltammetry. DPV current-potential curves obtained for 0.1 mM DA a t initially "clean" Pt electrodes appear in Figure 2. Although the initial trial produced a well-defined current-potential curve, significant poisoning of the electrode surface is evidenced by a marked decrease in oxidation currents with repeated voltammetric scans. A similar, although somewhat more gradual trend is observed for I--treated Pt electrodes. Under these experimental conditions, the DPV technique is clearly not recommended for use in catecholamine determination. 1288

M DA at initially

ANALYTiCAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976

Previous studies (26) have demonstrated that chloride ion chemisorbs on P t , in a form not removed from the surface apart from electrolysis a t strongly oxidizing potentials. Similarly, irreversible chemisorption of the catecholamines occurs on Pt to form a species which does not exhibit the conventional redox behavior (31). In order to test whether the observed decrease in the current was due to inhibition of the electron transfer process by gradual adsorption of solution components, or to interaction of oxidation products with the surface, dopamine DPV curves were recorded after the Pt electrode was held a t -0.100 V for 20 min. The experiment was then repeated a t +0.200 V, where appreciable DA oxidation occurs, Figure 2B. When the electrode was held a t the more positive potential, the sensitivity of the electrode to DA was almost completely destroyed, whereas poisoning did not occur a t the less positive potential, indicating that the loss of activity is due to contamination by the products of the oxidation process. Poisoning of a platinum electrode under DPV conditions in physiological solutions can be understood in terms of previously published studies of catecholamine oxidation (21,22). Electrooxidation yields the open-chain o-quinone, 11, Equation l.

R

R

I

I1 =

H, dommine, DA {OH ,norepinephrine, NE

Intracyclization of the o-quinone proceeds within seconds, producing the 5,6-dihydroxyindoline, 111, Equation 2.

R

Ai ,UA

- -.20

oE+NHz H

HO

0

I1

III

- -.I5

The cyclic intermediate, 111, is more easily oxidizable than the parent catecholamine, I, and accordingly, is rapidly oxidized by the o-quinone, 11,t o produce an aminochrome, IV, Equation 3.

- -10

R

R

Figure 3. Comparison of theoretical and experimental DDPV currentpotential curves for DA oxidation

Iv

(a

IV polymerizes readily to melanin-like products (32). The rates (21,22,32) of these reactions are such that they can occur to an appreciable extent on the time scales employed in the DPV experiment. I t is thus concluded that progressive build-up of an insulating film on the electrode surface is a direct consequence of the slowly increasing dc ramp inherent in the DPV potential-time waveform (Figure 1A). Supportive evidence for this conclusion is given below. The susceptibility of solid electrodes to poisoning is a well known fact of organic electrochemistry and is frequently attributable to polymerization of oxidation products (33). Limitations of the DPV waveform as applied to the analysis of reactants forming insoluble mercury compounds on Hg electrodes (e.g., sulfide and iodide) have recently been noted. Accordingly, normal pulse polarography (23) has been advocated for determinations involving this class of electrode reaction ( 3 4 ) , since it exhibits immunity to contamination comparable to the DDPV technique. Differential Double Pulse Voltammetry. The rates of formation of the 5,6-dihydroxyindoline derivatives of DA and NE (Equation 2) a t physiological pH are reported to be 0.26 s-l and 0.59 s-l ( t l l 2 -2.7 s and 1.2 s), respectively (21,22). Since, in the DDPV waveform, each pulse is applied for a comparatively short time (typically 1 2 0 ms, Figure lB), it is to be expected that reduction of the o-quinones back to the parent catecholamines should occur prior to appreciable intracyclization. Therefore, catecholamine oxidation under DDPV conditions should proceed according to Equation 1. Catecholamine Oxidation Process. For a “reversible” (diffusion-controlled)electrooxidation process, application of the first pulse in the DDPV waveform to a potential E l results in a dc current given by Equations 4 and 5 (35):

(4)

The symbols are defined in the appendix. Similarly, the current a t E2 is given by Equation 6.

.-

(--) Graph of Equation 7. Experimental results. The following parameters were employed in preparingthe theoretical curves: A € = 10 mV; Af = 15 ms; n = 2; A = 1.3 X cm2; = 3.0 X lo-’ mol ~ m - &A ~ ; = 7.1 x cm2 s-’; T = 297 K. It was assumed the D value of the oxidized catecholamine was the same as the reduced form a)

The differential current, Ai, is given by Equation 7.

Ai = nFACoR (%)‘I2

P-SP

+

(7) (1 SP)(l+P ) Providing the delay between pulses is sufficiently large that their effect on each other can be neglected (see Figure 1B) each pulse can be regarded as a separate perturbation of the electrode, such that DDPV is actually the correct waveform for which the DPV experimental equations were originally derived (23,25),Equations 7-9. rAt

S=exp(%AE) The maximum value of Ai, given by Equations 8 and 9, occurs a t E,, given by Equation 10.

P is defined by Equation 5. Graphs of Equation 7 are compared with experimental data for DA oxidation (corrected for background currents) a t Itreated Pt electrodes in Figure 3. The agreement between experiment and theory is evidence that the electrode reaction is a diffusion-controlled two-electron process, and that the reaction obeys Equation 1. Similar agreement between experiment and theory is obtained for NE oxidation. The “reversible” behavior observed under these conditions is not obtained a t untreated Pt or a t Pt electrodes coated with a variety of organic surfactants (28), for reasons discussed below. The dependence of peak current on pulse amplitude is shown in Figure 4. Ai, is a linear function of A E for A E 5 40 ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976

1289

-AI

B

AE (mv) Figure 4. Dependence of peak current on pulse amplitude for dation

CODA = 2.5 X

lod4 M; At = 15 ms; A = 1.3 X

4 oxi-

cm2; scan rate = 5 mV s-'

-Ai

Figure 6. DDPV current-potential curves for DA oxidation Experimental conditions as in Figure 3. (A) Flrst scan. (B) Tenth scan

L 03

02

01

0 -01

E , vou AgIAgCI

Figure 7. DDPV current-potential curve for DA oxidation

-

€(O) = 0.100 V: E, = 200 mV; A € = 50 mV; CoDA = 4.0X conditions as in Figure 4

03

02

01

E , VOLT

0 -01 AelApCl

Figure 5. DDPV current-potential curves for 4.0 X pulse amplitudes (A) A € = 25 mV; (B) A € = 100 mV; (C) A€

lod4M DA at various

= 200 mV. Other conditions as in

Figure 4

mV but deviates significantly a t higher values of the pulse amplitude, in accord with theoretical considerations (Equation 8) (23). Equations 7-10 indicate that a t small values of the pulse 50 mV) Ai, is essentially linear with AE (Figure amplitude (I 4).-As AE is increased further, significant broadening of the curves results, as shown in Figure 5. If AE is very large, the curves exhibit a rather flat peak, as predicted by Equation 7. This occurs because the magnitude of A E is such that the 1290

ANALYTICAL CHEMISTRY, VOL. 48,

NO. 9,

AUGUST 1976

M. Other

diffusion limiting region is reached over a larger range of potentials. Large values of A E have the further disadvantage in the present case that pulses carrying the potential beyond E , result in noticeable oxidative desorption of chemisorbed I. A detailed assessment of DDPV parameters as applied t o catecholamine determination is reserved for a future communication. However, it is worth noting here that, as expected, Ai, is linear with concentration (cf., Figure 3), varies predictably with changes in pulse duration (Figure 1and Equation 8), and variations with scan rate of the type recently reported (36) are not observed with the present instrumentation. Electrode Stability. The clear superiority of the DDPV technique with respect to electrode stability is illustrated by the results shown in Figure 6. Both current-potential curves in Figure 6 were obtained under identical conditions, except that curve B represents electrode response obtained after 10 successive potential scans. Appreciable decay of Ai, due to electrode poisoning occurs only when the potential is allowed to become unnecessarily positive of E,. A salient feature of DDPV is that the complete currentpotential curve, including data a t potentials substantially

I-ni

-AI

A

I

0.6

0.4

0.2

0

‘

-0.2

E

voLTys no,npa

curve for 3.0 X lop4 M DA at allylacetic acid coated Pt electrode (28).Experimental conditions as in Figure 3

Figure 9. DDPV current-potential

0

JOnA

I( 05

04

03

02

01

0 -01

C

I . \ I1

09

07

05

03

01 -01

E. VOLT Ag/AgCI Figure 8. DDPV current-potential curves in pH 7.4 phosphate buffer (A) Initially clean Pt electrode; (E) I--treated Pt electrode; (C) I--treated Pt electrode showing oxidative desorption of chemisorbed I. A€ = 50 mV; A = 1.3 X cm2; scan rate = 5 mV s-’

positive of E,, is not necessary for analytical measurement of peak current, thus prolonging the lifetime of the electrode. T h a t is, referring to Figure l B , El is easily adjusted to a potential just prior to the peak before the waveform is initiated. In this experiment, E1 was adjusted to a potential 40 mV negative of E,, and about ten data points were taken, Figure 7 . When utilized in this manner, there was no observable decrease in catecholamine peak currents when used continually over one day of experimentation. Chemical Modification of Pt Electrodes. Electrode contamination (Figure 2) represents only one factor limiting the usefulness of solid electrodes for analytical purposes. Pt, in particular, is prone to pH-dependent surface oxidation (33), which leads to high and irreproducible background currents and can critically affect the efficiency of electron transfer (cf., the behavior of NADH a t Pt ( 3 7 ) .The existence of surface oxides on pyrolytic and glassy carbons has likewise been demonstrated ( 3 8 4 0 ) . When used in biological media, adsorbed protein layers may form on Pt which can generate extraneous currents (41) and inhibit the electron-transfer process (42). The formation, nature, reactivity, and catalytic properties of irreversibly chemisorbed halide layers on Pt have been described previously (26, 28, 43, 4 4 ) . Specifically, exposure of the Pt surface to aqueous I- solutions results in a difficultly reactive, monoatomic chemisorbed layer which completely prevents H and 0 electrodeposition over the normal ranges of potential characteristic of the clean surface, and prevents (or markedly curtails) further chemisorption from taking place.

The double-layer modifying properties of chemisorbed I are revealed in DDPV experiments with initially clean and I--treated Pt electrodes in blank buffer solutions, Figure 8. At clean, unreacted electrodes, high background currents increasing with more positive values of the potential are observed, consistent with oxidation of the Pt surface, Figure 8A (26, 33). At the modified surface, background currents are significantly lowered and remain essentially constant apart from potentials a t which destruction of the surface layer occurs, Figure 8B. It is apparent that the suppression of H and 0 deposition by the chemisorbed layer reported elsewhere in acid solutions (26)persists a t physiological pH. Oxidation of chemisorbed I commences a t potentials roughly 300 mV more negative than those observed in 1F acid electrolytes, Figure 8C ( 2 6 , 4 4 ) . The effect of I chemisorbed on Pt shown in Figure 8B is that expected from background currents due primarily to dc double layer charging effects. That is, in the absence of double layer complications such as those stemming from surface oxidation and adsorption processes, the background is essentially a Aqm - E curve and is reflected in the variation of the differential double layer capacitance with potential (25, 34). Aqm is the change in surface charge density arising as a result of the necessarily different potentials a t which current measurements are made (see Figure 1).The differential capacitance of I-coated Pt electrodes is on the order of 20 pF ern+ and varies insignificantly with changes in potential where the coating is stable (45,46). In arriving a t the choice of aqueous I- as the modifying agent, a variety of surface-active molecules were tested (27, 28): Br-, S2-, 1-hexene, allylacetic acid, acrylic acid, allylamine, chlorotrifluoroethylene (the monomer of Kel-F) and tetrafluoroethylene (the monomer of Teflon). Although each surfactant a t coverages close to saturation expectably diminished the extent of oxidation of the Pt surface (28),catecholamine oxidations in the presence of the polyatomic organic coatings were, without exception, significantly irreversible. Electrochemical “irreversibility” under DDPV conditions has the effect of broadening the current-potential curve, lowering the peak current and shifting the peak potential to more positive potentials as illustrated by typical data shown in Figure 9. These observations are consistent with previous studies (28,43)in which it was shown that adsorbed, substituted olefins a t high coverages decrease the effective area of the electrode, due to steric hindrance of the approach of the reactant to the surface, and decrease the reaction rate; electroactivity a t such coated electrodes has thus been attributed to the presence on the surface of sites left vacant by incomplete or irregular coverage. The nature and functionality of the chemisorbed halide layer allowing the catecholamines to react a t a diffusion-limited rate is thus of some importance (Figure 3). It is thought that exposure of the Pt surface to ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976

1291

significant. As shown in Figure 10A, DDPV oxidation of DA M level yields a well-defined, analytically useful current-potential curve a t an I--treated Pt electrode. With an untreated electrode, however, no discernible DA oxidation peak is apparent (Figure 10B).The detection limit for the catecholamines will be reported separately. As noted in the introduction, preliminary applications of voltammetric techniques to studies of the brain in vivo have recently yielded some very promising results (19, 20). It is anticipated that the attainment of a voltammetric method permitting routine determination of brain catecholamines will require judicious appraisal of both the nature of the electrode sensor and the electrochemical methodology employed. We have recently presented evidence that chemically modified Pt electrodes allow the DPV detection of brain dopamine (49), although unstable electrode performance precluded the use of this technique for quantitative studies. The present work thus affords an alternative pathway for the determination of these amines in brain tissue, and these and other studies are in progress.

at the 8 X

.

A

’

B

APPENDIX NOTATION A = electrode area, cm2

0.3

0.2

0.1

E, VOLT !&

0

-0.1

A@gCi

Figure 10. DDPV current-potential curves for 8 X lo-’ M D A (A) I--treated Pt electrode: (B) untreated R electrode. A€ = 50 mV; scan rate = 5 mV s-‘

aqueous I- results in a mixed deposit of I and H as, for example, in Equation 11 (26-28). I

electrode

H

electrode

The degree of coverage (about 2 X mol cm-2) by the coadsorbed layer (26,43,44)(as compared with approximately 2X mol cm-2 of surface Pt atoms based upon geometric considerations) appears to preclude the existence of an appreciable fraction of Pt vacant sites for electron transfer. Extended-Huckel M.O. calculations, however, indicate that the stablest orientation for H chemisorbed on Pt(ll1) (the principal face exposed on cold-rolled Pt foil (47)and stablest of the low-index planes) is that in which atomic H is located interstitially in the plane of centers of Pt surface atoms, thus lying between three adjacent Pt atoms (48). Consequently, the adsorbed H would not be expected to exert appreciable steric hindrance on an incoming reactant, although rendering the site inactive toward further chemisorption because of (electronic) coordinative saturation. I t is possible that electron transfer takes place at these surface sites. I t has been noted earlier that steric deceleration by this surface layer appears negligible for dissolved couples, and the influence of chemisorbed I on electrode reaction rates is predominantly electrostatic (26,28, 43). In common with the DPV method, the sensitivity of the DDPV technique is enhanced for electrochemically “reversible” processes (23),and depends on the magnitude of background current. This complementary effect of I chemisorbed on Pt, as revealed in Figure 3 and Figure 8, is thus analytically 1292

ANALYTICAL CHEMISTRY, VOL. 48,

NO. 9, AUGUST 1976

C O R = initial concentration of reduced species, mol cmd3 Dox = diffusion coefficient of oxidized species, cm2s-l D R = diffusion coefficient of reduced species, cm2s-l E = electrode potential, volt Eo‘ = formal standard potential, volt E , = peak potential, volt A E = pulse amplitude, volt F = Faraday constant, equiv. mol-I i = current, ampere Ai = differential double pulse current, ampere Ai, = differential double pulse current, peak value, ampere n = number of electrons transferred per molecule R = gas constant, joules mol-1 K-l t = time, s At = time at which current is measured, s

LITERATURE CITED ( 1 ) J. Axelrod, Sci. Am., 230 (6),59 (1974). (2) 0. Hornykiewicz, in “Biogenic Amines and Physiological Membranes in Drua Theraw”, J. H. Biel and L. G. Abood, Ed., Part B. Marcel Dekker, New Yo&, N.Y.,’i971, p 173. (3) S. H. Snyder, S. P. Baneriee, H. I. Yamamura, and D. Greenberg, Science, 184, 1243 (1974). (4) M. A. Moskowitz and R. J. Wurtman, A.! Engl. J. M.,293,274 (1975);293, 332 (1975). (5) A. Pletscher, in “Frontiers in Catecholamine Research”, E. Usdin and S. H. Snyder, Ed., Pergamon, Oxford, 1973, p 27. (6)H. Weil-Malherbe, in “Methods of Biochemical Analysis”, E. Glick, Ed., Suppl. Vol., interscience, New York, N.Y., 1971, p 119. (7) H. Weil-Malherbe, in “Methods in Medical Research”, J. H. Quastel, Ed., Vol. 9, Yearbook Medical Publishers, Chicago, Ill., 1960, p 130. (8) S. Udenfriend, “Fluorescence Assay in Biology and Medicine“, Vol. 2, Academic Press, New York, N.Y., 1969, p 207. (9) S. Kawai, T. Nagatsu, T. imanari, and F. Tamura, Chem. Pbarm. Bull., 14, 618 (1966). (10) C. Refshange, P. T. Kissinger, R. Dreiiing. L. Blank, R. Freeman, and R. N. Adams, Life Sci., 14, 311 (1974). (1 1) K. Engalman, B. Portway, T. imanari, and F. Tamura, Am. J. Med. Sci., 255, 259 (1968). (12) K. Fuxe and G. Jonsson, J. Histocbem. Cytocbem., 21, 293 (1973). (13) F. E. Bloom, Proc. Assoc. Res. New, Ment. Dis., 2, 25 (1972). (14) S. H. Kosiow, in “Frontiers in Catecholamine Research”, E. Usdin and S. H. Snyder, Ed., Pergamon Press, Oxford, 1973, p 1085. (15) D. R. Curtis, in “Physical Techniques in Biological Research”, W. H. Nastuk, Ed., Vol. 5, Academic Press, New York, N.Y., 1964, p 144. (16) F. Grabartis, R. Chessick, and H. Lal, Biochem. Pharmacol., 15, 127 (1966). (17) T. S. Baich, T. N. Chase, and D. M. Jacobowitz, Brain Res., 52,419 (1973), and references cited therein. (18) H. M. Adam, in “Metabolism of Amines in the Brain”, G. Hooper, Ed., Macmillan, London, 1971, p 4. (19) P. T. Kissinger, J. 8. Hart, and R. N. Adams, Brain Res., 55, 209 (1973). (20) R. L. McCreery, R. Dreiling, and R. N. Adams, Brain Res., 73, 15, 23 (1974). (21) M. D. Hawley, S. V. Tatawawadi, S.Piekarski, and R. N. Adams, J. Am. Cbem. SOC., 89, 447 (1967). (22) A. W. Sternson, R. McCreery, B. Feinberg, and R. N. Adams, J. Nectroanal. Cbem., 46, 313 (1973). (23) E. P. Parry and R. A. Osteryoung, Anal. Chem., 37, 1634 (1965). (24) J. B. Flato, Anal. Chem., 44, ( 1 l ) , 75A (1972).

(40) V. L. Snoeyink and W. J. Weber, Prog. Surf. Mernbr. Sci., 5 , 63 (1972). (41) N. Ramasamy, S. Srinivasan, and P. V. Sawyer, Electrochim. Acta, 19, 137 11974). (42) N. Ossendorfova, J. Pradac, J. Pradacova, and J. Koryta, J. Electroanal. Chem.. 58. 255 11975). (43) A. L. Y. La; and A. T. Hubbard, J. Electroanal. Chern., 33, 77 (1971). (44) A. T. Hubbard, R. A. Osteryoung, and F. C. Anson, Anal. Chem., 38, 692 (1966). (45) K. Kinoshita and J. A. S. Belt, Carbon, 12, 525 (1974). (46) M. A. V. Devanathan and K. Ramakrishnaiah, Electrochim.Acta, 18, 259 (1973). (47) F. G. Will, J. Electrochem. SOC.,112, 451 (1951). (48) M. A. Leban and A. T. Hubbard, J. Electroanal. Chern., in press (1976). (49) R. F. Lane, A. T. Hubbard, K. Fukunaga, and R. J. Blanchard, Brain Res., in press (1976).

(25) J. H. Christie and R. A. Osteryoung, J. Electroanal. Chem., 49, 301 (1974). (26) R. F. Lane and A T. Hubbard, J. Phys. Chern., 79, 808 (1975). (27) R. F. Lane and A. T. Hubbard, J. Phys. Chem., 77, 1401 (1973). (28) R. F. Lane and A. T. Hubbard, J. Phys. Chem., 77, 1411 (1973). (29) H. Angerstein-Kozlowska, B. E. Conway, and W. B. A. Sharp, J. Electraanal. Chern., 43, 9 (1973). (30) B.E. Conway, H. Angerstein-Kozlowska, W. B. A. Sharp, and E. E.Criddle, Anal. Chem., 45, 1331 (1973). (31) R. F. Lane and A. T. Hubbard, unpublished experiments. (32) R. A. Heacock, Adv. Heterocycl. Chem., 5 , 205 (1965). (33) R. N. Adams, "Electrochemistry at Solid Electrodes", Marcel Dekker, New York, N.Y., 1969. (34) J. A. Turner, R. H. Abel, and R. A. Osteryoung, Anal. Chem., 47, 1343 (1975). (35) P. Delahay, "New Instrumental Methods in Electrochemistry", Interscience, New York, N.Y., 1954, p 55. (36) J. H. Christie, J. Osteryoung, and R. A. Osteryoung, Anal. Chem., 45, 210 (1973). (37) W.J. Blaedel and R. A. Jenkins, Anal. Chem., 47, 1337 (1975). (38) D. Laser and M. Ariel, J. Electroanal. Chem., 52, 291 (1974). (39) B. D. Epstein, E. Dalle-Molle, and J. S . Mattson, Carbon, 9, 609 (1971).

RECEIVEDfor review March 1,1976. Accepted May 7,1976. Acknowledgment is made t o the donors of the Petroleum Research Fund, administered by the American Chemical Society, and to the National Science Foundation for support of this research.

Buffers for the Physiological pH Range: Thermodynamic Constants of Four Substituted Aminoethanesulfonic Acids from 5 to 50 OC Carmen Amaralis Vega' and Roger G. Bates* Department of Chemistry, {Jniversity of Florida, Gainesville, Fla. 326 11

The pK2 values for four N-substituted aminoethanesulfonic acids have been determined at 10 temperatures in the range 5 to 50 "C. The dissociation constants were derived from the emf of cells without liquid junction, utilizing hydrogen electrodes and silver-silver bromide electrodes, and the thermodynamic quantitles AH",AS",and AC," were calculated from the temperature coefficients of the dlssoclatlon constants. The pK2 values at 25 "C are 6.270 (MES), 7.187 (BES), 7.550 (TES), and 7.565 (HEPES). N-Substitution lowers the pK2 of taurine and, with the exception of HEPES, pK2, AH",AS",and AC," form a regular series for the remainingfour compounds. TES and HEPES are especially useful for pH control and posslbly as standards in the pH region close to that of blood serum.

2-( N.morpholino)ethane-

HOC,H, >!:2H,SOJ-

HOC,H4 H@%\ H+ HOCHI-CNC,H,SO~ / HOCHi

N,N.bis( 2-hydroxyethy1)aminoethanesulfonic acid

BES

N-tris(hydroxymethy1)methyl. aminoethanesulfonic acid

TES

N-(2-hydroxyethy1)piperazine-

HEPES'

and

N'-ethanesulfonicacid

In their useful study of buffers for the pH range 7 to 9, Good e t al. ( I ) recommended a series of buffer substances compatible with most media of biochemical and physiological interest. In general, these compounds were amines or N-substituted amino acids. In earlier work, the pK values and associated thermodynamic quantities of several of these and other closely related acid-base systems have been determined. The substances that have been studied include tris(hydroxymethy1)aminomethane (Tris or THAM) (2, 3 ) ; 2-ammonium-2methyl-1,3-propanediol (AMP) ( 4 ) ; 2,2-bis(hydroxymethyl)-2,2',2''-nitrilotriethanol (Bis-tris) (5);and two N-substituted glycines: N,N-bis(2-hydroxyethy1)glycine (Bicine) (6) and N-tris(hydroxymethy1)methylglycine (Tricine) (7). We have now examined in detail the acid-base and thermodynamic properties for the second dissociation step of four Nsubstituted aminoethanesulfonic acids derived from the parent compound taurine. These compounds, selected from the list of Good et al. ( I ) ,are all ampholytes. Their zwitterionic structures, in order of increasing pK,, are as follows: Present address, Department of Chemistry, University of Puerto Rico, Mayagiiez, P.R.

MES

sulfonic acid

The second dissociation of each of these zwitterions Z* can be represented by Z*

H+ + Z-

(1)

EXPERIMENTAL MES, BES, TES, and HEPES, purchased from the Sigma Chemical Co., were recrystallized twice from 80% ethanol, dried, and assayed by titration with standard carbonate-free sodium hydroxide. The analyses averaged 99.97% for all four compounds, with a standard deviation of 0.05%. Three stock buffer solutions were prepared from weighed amounts of buffer compound, recrystallized sodium bromide, and sodium hydroxide solution in quantity sufficient to make the buffer ratio approximately unity. These stock solutions were diluted further with CO2-free water to yield additional solutions for the cell measurements. Before the cells were filled, the solutions were deaerated with purified hydrogen. The cells have been described elsewhere ( 8 ) ,as have the methods used in preparing the hydrogen electrodes (9) and the silver-silver bromide electrodes (10). Temperature measurements were made with a Hewlett-Packard quartz thermometer provided with calibrated probes. The emf measurements were made with a Hewlett-Packard Model 3460B digital voltmeter, checked occasionally against saturated Weston cells used as laboratory standards. ANALYTICAL CHEMISTRY, VOL. 48,

NO. 9,

AUGUST 1976

1293