Purging the reaction solutions with nitrogen prevented any kinetic complications arising from independent reactions with dissolved oxygen. Hydroxylamine in neutral solution is oxidized by dissolved oxygen to a variety of products which are reduced at platinum electrodes (15). Removing dissolved oxygen was also necessary from the standpoint of preventing irreversible oxygen reduction at the working cathodes, The potential at which this process occurred was found to be less reproducible than hydrogen reduction. From the two systems explored, some of the basic capabilities of the method of DCCP can be stated. Care must be exercised in choosing the systems to which the technique is to be applied. It is essential that the products of the desired reaction are not themselves capable of reacting at the working electrodes. If the analyte is electroactive, the potential at which it reacts at the working electrode must be significantly different than the potential at which the species added to start the kinetic run interacts with the working electrode.
(15) P. C. Noews, Jr. and L. F. Audrieth, J . lnorg. Nucl. Chem., 11, 242 (1959).
The lower determination limit for the electrodes used in these applications is about 10-BM. Because of the pseudofirst-order requirement on the reaction (pseudo-zero-order on analyte concentration), the smallest analyte concentration determinable by the method would be about 10-4M. For cases where initial increasing concentrations of product rather than decreasing concentrations of reagent are monitored, this limit could be lower. Increasing electrode area would increase sensitivity, but the associated increase in the contribution of undesirable electrode surface reactions to the overall signal is often intolerable. Maximum concentrations determinable depend on rates at which electrode surface effects permit electrode response to solution concentrations. This same criterion limits the selection of applicable systems to those where rate of reaction allows adequate time for the electrode response. Systems with slow reaction rates are prohibitive only when competing solution reactions are significant. RECEIVED for review October 20, 1969. Accepted December 1, 1969. Presented at the 3rd Great Lakes Regional ACS Meeting, DeKalb, Illinois, June 1969. Part of a thesis for the M.S. degree by J. R. S.
Direct Current and Alternating Current Polarographic Response of Some Pharmaceuticals in an Aprotic Organic Solvent System Albert L. Woodson Food and Drug Administration, Chicago District Laboratory, Room 1222, Post Ofice Building, Chicago, III. 60607
Donald E. Smith Department of Chemistry, Northwestern Uniaersity, Ecanston, 111. 60201 Extensive investigations of electrode reaction mechanisms in aprotic organic solvents have revealed that considerable potentialities and advantages should attend application of such solvent systems to voltammetric analysis of organic compounds in general. Because they have received little exploitation, the present work was undertaken primarily to demonstrate these potentialities in a more explicit manner than provided by earlier mechanistically-oriented investigations. Interest was focused on organic compounds of significance in the pharmaceutical field. t h e dc, fundamental harmonic ac, and second harmonic ac polarographic responses of 24 pharmaceutically-important compounds in acetonitrile-tetrabutylammonium perchlorate media were investigated. Barbiturates, salicylates, corticosteroids, alkaloids, sulfa drugs, and estrogens are among the compound classifications included in this work. The majority of the compounds yielded ideal responses for analytical purposes with one or more of the techniques employed. Many gave ideal, one-electron reversible (diffusion-controlled) waves, whereas the corresponding aqueous solution response is irreversible or nonexistent. ONEof the most significant advances in modern electrochemistry has involved the study of electrode processes in aprotic organic solvents. The past decade has witnessed this field develop from infancy to a state of incipient maturity as a result of rapid evolution of knowledge in three principal areas: instrumental techniques, usable solvents and appropriate 242
schemes for their purification, and electrode reaction mechanisms. The measurement problems attending the high resistance characterizing most aprotic solvent-supporting electrolyte systems (1, 2) have been overcome to a large extent by the development of potentiostats and galvanostats which permit automatic potential and current control with threeelectrode cell configurations (3-10). This development began in the late 1950’s with the efforts of Booman (3),DeFord (3, and Kelley and coworkers (6, 7). With the latest refinements
(1) J. Heyrovsky and J. Kuta, “Principles of Polarography,” Academic Press, New York, N. Y., 1966, pp 61-63. (2) P. Delahay, “New Instrumental Methods in Electrochemistry,” Interscience Publishers, New York, N. Y., 1954, pp 132-135, pp 166-168. (3) G. L. Booman, ANAL.CHEM., 29,213 (1957). (4) G. L. Booman and W. B. Holbrook, ibid., 37,795 (1965). (5) D. D. DeFord, Division of Analytical Chemistry, 133rd Meeting, ACS, San Francisco, Calif., April 1958. (6) M. T. Kelley, D. J. Fisher, and H. C. Jones, ANAL.CHEM., 31, 1475 (1959). (7) M. T. Kelley, D. J. Fisher, and H. C. Jones, ibid., 32, 1262 (1 960). (8) D. E. Smith in “Electroanalytical Chemistry,” A. J. Bard, Ed., Vol. 1, M. Dekker, Inc., New York, N. Y.,1966, Chapter 1. (9) E. R. Brown, D. E. Smith, and G. L. Booman, ANAL.CHEM., 40,1411 (1968). (10) E. R. Brown, H. L. Hung, T. G. McCord, D. E. Smith, and G. L. Booman, ibid., 40,1424 (1968).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 2, FEBRUARY 1970
(9, IO), potentiostats apparently can effect accurate potential control in very high resistance solvent systems-e.g., 1,2dimethoxyethane-0.11 tetrabutylammonium perchlorate (TBAP) (II), methylene chloride-0.1M TBAP (IO), acetonitrile with millimolar supporting electrolyte (IO), acetonitrile in absence of supporting electrolyte (12), etc. The only important restriction imposed by such high resistance media is a limitation on the potentiostat bandpass (9) which ensues at frequencies well beyond those of significance in most analytical applications. These instrumental developments played a decisive role in stimulating the expansion of interest in aprotic solvent electrochemistry. The enhanced activity has led to the present state where much is known about mechanistic schemes and numerous solvents have been shown useful for study. In addition to those mentioned above, benzonitrile (13), dimethylformamide (13-13, dimethylsulfoxide (13), propylene carbonate (16), nitromethane (17), pyridine ( l a ) , and 96z dioxane-water (14) are representative of other aprotic solvents which have been accorded sufficient study to reveal the purification schemes and general handling procedures necessary for voltammetric work. The relevant mechanistic information which has evolved in recent years is too vast and varied in scope to summarize at this point and we refer the reader to appropriate reviews (13,14,19) or symposia publications (20, 21). However, electrolytic mechanisms revealed for organic compounds in aprotic solvents feature a number of important common denominators whose existence is worth noting. For example, studies on reductive processes have established that facile one-electron reduction processes leading to radical anions of at least transient stability are characteristic for neLitral molecules possessing r-electron conjugation. The stability of the radical anion, which determines the overall reversibility of the electrode reaction, has been found to increase with increasing dimension of the conjugated a-electron system and with increasing electrophilicity of substituent groups. Special substituent groups sharply reduce radical anion lifetimes--e.g., certain halogens or pseudo-halogens which facilitate radical anion decomposition by halide ion ejection, or acidic protons on the parent electroactive molecule which may lead to protonation of the radical anion product by the unreacted parent species. It also has been found that chemical decomposition of the radical anion is (11) J. Q. Chambers, A. D. Norman, M. R. Bickell, and S. H. Cadlz, J . Amer. Chem. Soc., 90, 6056 (1968). (12) D. A. Hall and P. J. Elving, Electrochim. Acta, 12, 1363 (1967). (1 3) R. N. Adams, “Electrochemistry at Solid Electrodes,” Marcel Dekker, Inc., New York, N. Y.,1969. (14) M. E. Peover in “Electroanalytical Chemistry,” Vol. 2, A. J. Bard, Ed., Marcel Dekker, Inc., New York, N. Y., 1967, Chapter 1.
(15) S. Wawzonek and J. H. Wagenknecht in “Polarography 1964,” Vol. 2. G. J. Hills,. Ed.,. Interscience, New York, N. Y.. 1966. 1035-io41. (16) R. F. Nelson and R. N. Adams. J. Electroanal. Chem.. 13. i84 (1967). (17) J. D. Voorhies and E. J. Schurdak, ANAL.CHEM.,34, 939 (1962). (18) P. J. Elving and R. F. Michielli, J. Elecrrochem. SOC.,114, 68C (1967). (19) I. M. Kolthoff in “Polarography, 1964,” Vol. 1 , G. J. Hills, Ed., Interscience, New York, N. Y., 1966, pp. 1-23. (20) Disc. Faraday SOC.,45 (1968), all papers. (21) “Symposium on the Synthetic and Mechanistic Aspects of Electroorganic Chemistry,” G. R. Ghirardelli, Ed., U. S. Army Research Office-Durham, Durham, N. C., October 1968, all
papers.
usually accompanied by uptake of additional electrons by the decomposition product. Finally, the dc polarographic halfwave potential is often estimable on the basis of well-established correlations between this observable and the quantum mechanical parameter related to the energy difference between the highest occupied and lowest unoccupied molecular orbitals of the parent molecule. Using the foregoing precedents as guidelines, one can often predict with reasonable accuracy the type of voltammetric behavior which a previously untested compound will exhibit. The latter concept is particularly important for most pharmaceuticals whose aprotic solvent electrochemical behavior has not been studied explicitly by electrochemists and, at best, only the behavior of related model compounds is known. We report here the results of a survey of dc and ac polarographic behavior of a variety of pharmaceutically-important molecules in a representative aprotic solvent system: acetonitrile-tetrabutylammonium perchlorate. Of primary concern is the presentation of data which demonstrate in an effective manner the sensitivity and scope of aprotic solvent electrochemistry for the analysis of pharmaceuticals. The consideration of analytical routines for specific sample preparations is not of paramount interest in this presentation. However, it should be mentioned that the data presented here have been applied as guidelines to the successful development of assay procedures for certain commercial pharmaceutical preparations (22,23). EXPERIMENTAL
All polarographic measurements were effected with instrumentation based on operational amplifiers, an approach which has been widely discussed (3-10). Philbrick Model P25AU and SP2AU operational amplifiers were housed in two Philbrick Model R P Operational Manifolds on which all circuits were constructed with the aid of Pornona Electronics, Inc., Electronic Test Accessories. The manifolds were rewired slightly to accommodate simultaneous operation of seven amplifiers per manifold, rather than the five-amplifier operation provided by the unmodified unit. The potentiostat was constructed in the current-follower mode (4, 9) utilizing P25AU amplifiers. Direct current signal sources consisted of a precision ( = t O . l % ) initial voltage source and a voltage ramp generator (scan rate = 50 mV per minute) constructed from conventional operational amplifier circuits (8) using P25AU and SP2AU amplifiers, respectively. A Wein Bridge Oscillator (24) constructed from a P25AU amplifier provided a stable 16-Hz sinusoidal signal source. Voltage division of this source provided a 20-mV peak-to-peak amplitude which was applied to the electrochemical cell in ac polarographic measurements. Conditioning of the potentiostat output signal (cell current signal) in ac polarographic measurements consisted of tuned amplification of the harmonic of interest (8), precision fullwave rectification (8, 25) of the tuned amplifier output, and filtering of the rectifier output with a low-pass filter of secondorder Butterworth response (26), whose dc output was suitable for recording. Model P25AU operational amplifiers were (22) A. L. Woodson, L. L. Alber, J. Assoc. Oft: Anal. Chem., 52, 847 (1969). (23) A:L.-Woodson, unpublished work, Food and Drug Adrninistration, Chicago District Laboratory, Chicago, Ill., 1969. (24) Burr-Brown Research Corp., “Handbook of Operational
Amplifier Applications,” Burr-Brown Research Corp., Tucson, Arizona, p 66. (25) Zbid.,p 73. (26) Philbrick Researches, Inc., “The Lightening Empiricist,” Vol, 13, Nos. 1 and 2, Philbrick Researches, Inc., Dedham, Mass., 1965.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 2, FEBRUARY 1970
243
Table I. Compilation of dc and ac Polarographic Responses of Pharmaceuticals in Acetonitrile-O.1M Tetrabutylammonium Perchlorate Approximate detection limits -Z(at)* Z(2wt)' Idon Compound ndod nwte n d EPb EminC Em" 0.44 0.45 0.40 -1.76 -1.87 5 x 10-5 1 x 10-4 1. Acetylsalicylic acid" -1.64 1 x 10-5 2. Atropine" -2.14 N.R.i N.R. 0.60 N.R. N.R. 5 x 10-5 N.R. N.R. 3. Atropine methyl nitraten -2.04 -2.10 -2.13 0.50 ... 0.60 1 x 10-4 5 x 10-4 5 x 10-5 4. Colchiceine" -0.96 -1.00 -1.02 0.80 0.58 0.55 2 x 10-5 3 x 10-5 2 x IO5. Colchicine" -1.47 -1.47 -1.47 1.00 1.00 1.00 3 x 10-5 1 x 10-5 5 x 10-7 6. Deserpidine" -2.14 -2.14 -2.12 1.00 1.00 1.00 3 x 10-5 1 x 10-5 5 x 10-7 N.R. N.R. N.R. N.R. N.R. N.R. N.R. N.R. N.R. 7. Diethystilbestrolp N.R. N.R. 3 x 10-5 N.R. 0.30 N.R. N.R. 8. Estradiolp -0.64 N.R. N.R. N.R. N.R. N.R. N.R. N.R. N.R. N.R. N.R. 9. Estronep 10. Hydrochlorothiazidep -1.56 -1.65 -1.m 0.80 0.70 ... I x 10-5 3 x 10-5 3 x 10-6 11. Hydrocortisone" -1.58 -1.63 -1.68 0.90 0.56 0.54 1 X loW5 3 X 10-5 2 X 10-6 12. Methyclothiazideq -1.58 -1.60 --1.69k 0.58 0.45 3X 3X 6 x 10-6 N.R. N.R. 2 x 10-5 N.R. N.R. 13. Nitroglycerine' -0.59 N.R. N.R. 0.50 14. Phenobarbital" -2.07 -2.14 -2.w 0.83 0.60 . .. 5 x 10-5 I x 10-4 2 x 10-5 -1.85 0.50 -1.64 -1.80 0.93 0.38 5X 8 X 8 X 10-6 15. Prednisolone" 0.32 0.35 0.26 -1.70 -1.80 5X 5 X IO-' 5 X 10-5 16. Prednisone" -1.65 -1.91 -1.90 0.93 0.84 17. Progesteronem -1.85 0.86 5X 1X 5 x 10-6 18. Reserpine" -2.14 -2.15 -2.14 1.00 1.00 1.00 5 X lo-' 5X 1 X 10-6 19. Salicylic Acid" -1.54 -1.70 -1.66 0.50 0.60 0.60 8X 2X 2 x 10-5 20. Sulfacetimide" -1.85 -1.98 0.35 0.27 ... 5 x 10-5 5 x 10-5 I x 10-5 21. Sdfadiazine" -1.581 -1.31 -1.40 -,1.66 0.75l 0.69' 0.75l 5 X 10-51 5 x 10-5' 5 x 1Q-61
4::;l
")
-1.49k) -1.62) -1.68&} 1.00 ... ... -2.05) -2.OokJ 23. Sulfamethazine"' -1.45 -1.57 -1.51k 0.33 0.37 ... -2.02 1.10 0.95 1.00 24. Testosteronem -2.02 -2.03 a El,>= dc polarographic half-wave potential (volts us. silver reference). b Ep = fundamental harmonic ac polarographic peak potential (volts is. silver reference). c Emin= potential of second harmonic ac polarographic minimum (volts us. silver reference). 22. Sulfamerazine"
-1.56 -1.95
apparent n-value calculated from dc polarographic data using the relationship (28) n potential and E314= three-quarter-wave potential in millivolts. d lido
6
=
rzwt =
=
I
x 10-41 5 x 10-5'
5X 3 x 10-5
3X 2 x 10-5
56 Esia
- Eli4 where
apparent rz-value calculated from fundamental harmonic ac polarograms using the relationship (8);n
of wave at half height in millivolts. f nZwt= apparent n-value calculated from second harmonic ac polarograms using the relationship (8); n
=
90
--
W1,Z
68 --
AEP
E114
5 x 10-6' 5 X 10-8 2 x 10-6
=
quarter-wave
where Wl/2 = full-width
where AEp = separation of
second harmonic peak potentials in millivolts. 0 Zdc = detection limit based on the dc polarographic current (molesiliter). * Z(wt) = detection !hit based on fundamental harmonic ac polarographic current (moles/liter). i Z(2wt) = detection limit based on second harmonic ac polarographic current (moles/liter). i N.R. designates no detectable response-Le., no wave. . . . designates either that measurements were not performed or nature of data precluded essential calculation. k designates that second harmonic ac polarogram does not exhibit a minima; number represents peak potential. 1 designates that number is based on first (most anodic) wave of polarogram. m compound source: USP Standard. n compound source: K and K Laboratory Standard. p compound source: NF Standard. q compound source: Abbott Laboratories (Enduron). compound source: Atlas Powder Standard Lactose-Nitroglycerine mixture (9.6 nitroglycerine). 7
employed in this signal conditioning train. A single-stage tuned amplifier sufficed in fundamental harmonic measurements, whereas a two-stage unit was required for second harmonic measurements. In dc polarographic measurements, the potentiostat output was suitable for recording without further signal conditioning. In both dc and ac polarography, the sample-and-hold readout technique (27) was employed in most instances. The necessary circuitry for this operation, including the timing circuit for electromechanical synchronization of mercury drop life with the current sampling operation, were constructed in a manner identical to that described in the literature (27), except that Model P25AU amplifiers were used in place of the Model P45AU amplifiers previously employed (27) E. R. Brown, T. G. McCord, D. E. Smith, and D. D. DeFord, ANAL. CHEM., 38,1119 (1966). 244
a
in the timing circuit (27). The sample-and-hold circuit output (cell current) was recorded with the aid of a Hewlett-Packard Model 7000A X-Y recorder whose x-axis was driven by the dc ramp generator output. A Hewlett-Packard Model 141A oscilloscope with Model 1402A and 1420A plug-ins aided in signal monitoring, ac amplitude measurements, etc. A Metrohm Model ELA unthermostated polarographic cell was employed. A dropping mercury working electrode, a platinum wire auxiliary electrode and a silver wire reference electrode in 0.1M tetrabutylammonium iodide comprised the three electrodes. Oxygen dissolved in the polarographic solution was removed by bubbling with nitrogen gas which had been presaturated with acetonitrile vapors in a gas bubbler. Eastman Spectrograde acetonitrile and Matheson Coleman and Bell Polarographic Grade tetrabutylammonium perchlorate (TBAP) were employed as solvent and supporting electrolyte, respectively, without further purification.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 2, FEBRUARY 1970
Figure 1. Polarogram of 2.79 X lO-dM testosterone in acetonitrile-0.10M TBAP A--dc polarogram B-Fundamental harmonic ac polarogram C-Second harmonic ac polarogram (Ordinates uncalibrated in all cases)
Figure 2. Polarograms of 1.73 X lO-4M reserpine in acetonitrile-0.10M TBAP Legend same as Figure 1
Table I lists the compounds investigated in this work, together with their sources (see footnote of table). All compounds were used as obtained from the sources without further purification. RESULTS AND DISCUSSION
Important features of the dc and ac polarographic behavior exhibited by the various pharmaceuticals in acetonitrile-0.1M TBAP are compiled in Table I. Some representative polarograms are shown in Figures 1-4. Although the figures provide full details of the response characteristics of only four of the 24 compounds studied, the general quality of the polarographic response for analytical applications is adequately
represented by the data of Table I. These data, combined with appropriate polarographic principles, enable a variety of conclusions regarding the analytical utility of the polarographic responses. For example, the dc polarographic halfpeak potential (El,*),fundamental harmonic ac polarographic peak potential (Ep),and second harmonic minimum potential (&in) not only locate the relative positions of the waves for the various compounds, but also allow conclusions regarding the reversibility of the relevant electrode reactions. It is known that for a strictly diffusion-controlled or reversible electrode reaction (8)
EIIZ = Ep
= Emin
ANALYTICAL CHEMISTRY, VOL. 42, NO. 2, FEBRUARY 1970
(1) 0
245
oc
FOTlh-AL
D L POTEYTldL (VOLTS,
l“0Liii
Di
PC-lhT#AL IVOL-S,
Figure 3. Polarograms of 3.48 X lO-4M phenobarbital in acetonitrile-0.10M TBAP Legend same as Figure 1
! A
i P
f
i D C
P0TEN-Io.L
‘YOL-SI
DC
POTENTIAL
‘“o.-il
Figure 4. Polarograms of 3.27 X 10-4M colchicine in acetonitrile-0.10M TBAP Legend same as Figure 1
Thus, electrode reactions of compounds whose polarographic data obey Equation 1--e.g., colchicine, deserpidine, reserpine, testosterone-are characterizable as diffusion-controlled (or at least nearly diffusion-controlled) on both the dc and ac polarographic time scales. Appreciable deviations from diffusion-controlled behavior of the ac and/or dc polarographic response are indicated. when data fail to obey Equation l. Similarly, the apparent n-values, ndo, nul,and nhl provide information regarding the reversibility of the electrode reaction. They also provide a measure of the width or sharpness of the waves in question as is apparent from the equations used for their calculation (see legend of Table I). Because the expressions used to calculate ndo, nut and n h l are all based on theory for diffusion-controlled reactions, only integral or near-integral n-values are mechanistically meaningful in a strict quantitative sense. The unity or near-unity n-values of Table I (note that integral n-values greater than unity are not found) suggest a one-electron diffusion-controlled response for the technique in question. Although a unity ndo value can be misleading in this regard (24, if one finds that
246
holds, together with Equation 1, a diffusion-controlled response is definitely confirmed. The only significance one should apply to non-unity n-values is that they indicate rate control by processes other than, or in addition to diffusion, that their deviations from unity roughly indicate the magnitude of the departure from reversible behavior, and that these values provide a measure of wave-width. The approximate detection limits stated in Table I represent our assessment of the lowest concentration at which a wave is definitely detectable and an approximate (i30P;: relative error) quantitative analysis can be performed. They provide a comparison of the wave magnitudes, relative to the background current, for the various techniques and compounds. We consider these detection limits as definitely on the conservative side. In some cases they are influenced by background current due to electroactive solvent impurities, a consequence of employing the solvent as received. A significant improvement on these limits is expected to accompany the use of some preliminary solvent purification steps (10). This was not attempted here because the sensitivity limits obtained are more than adequate for most analyses of interest to us. We would also expect at least an order-of-magnitude improvement in the fundamental harmonic ac polarographic
ANALYTICAL CHEMISTRY, VOL. 42, NO. 2, FEBRUARY 1970
detection limits if phase-sensitive detection (8) were employed in place of the total current measurement scheme used in these studies. The reduction in the background signal associated with the double-layer charging current, which is realized through phase-sensitive detection of the fundamental harmonic current, should yield sensitivities more comparable to those observed for second harmonic measurements (Table I), the latter being the only technique employed in the present work which provided active discrimination against doublelayer charging current (8). The importance of this suppression of charging current becomes particularly evident when one notes that, with a few exceptions, the second harmonic technique provided superior sensitivity to the other methods. This sensitivity advantage of the second harmonic method often applies even when the overall ac polarographic response is grossly attenuated by relatively irreversible electrode reactions (8)--e.g., acetylsalicylic acid, atropine methyl nitrate, phenobarbital, and all sulfa drugs-a situation which tends to swing the advantage to the dc polarographic method. However, only three compounds, atropine, estradiol, and nitroglycerine, exhibited sufficiently irreversible responses that the dc polarographic method yielded significantly better sensitivity than the second harmonic ac polarographic measurement. One important feature of the polarographic behavior for analytical applications is the form of the current-concentration profile. Data on this polarographic characteristic were not included in Table I simply because of the redundancy of the results obtained. Current-concentration profiles of the dc polarographic limiting current and ac polarographic peak currents were carefully determined for colchicine, deserpidine, phenobarbital, reserpine, and testosterone in acetonitrile0.1M TBAP. The dc profile was determined for nitroglycerine. The concentrations of electroactive compound employed ranged from near the detection limit to approximately millimolar, with five to eight data points used to determine the form of the profile. In all cases linear responses were observed. For the case of the second harmonic ac polarogram of colchicine, the smaller (most cathodic) of the two peaks exhibited a noticeably nonlinear peak current-concentration response. However, at the larger peak, which is the one normally preferred as a basis for analysis, the corresponding response was essentially linear. Although current-concentration profiles were obtained for only a small fraction of the twenty-four compounds involved in this work, the results from this sampling strongly suggest that a linear or nearlinear response will be obtained in most instances. The ability of the type of instrumentation employed in this work to eliminate effects of very large ohmic resistances was mentioned earlier. This feature enormously increases the versatility of voltammetric methods, not only because of the much wider choice of solvents it affords, but also because requirements on supporting electrolyte concentrations become much less restrictive. Analytically useful voltammetric responses already have been reported using acetonitrile with very low supporting electrolyte concentrations (10, 12). Although migration (28) and unusual double-layer effects (29-31) are anticipated under these conditions, the analytical utility of the polarograms is not expected to be seriously affected by these perturbations. The possibility of effecting dc and ac polarographic analyses of the compounds in ques(28) J. Heyrovsky and J. Kuta, “Principles of Polarography,” Academic Press, New York, N. Y., 1966. (29) F.C.Anson, J . Phys. Chem., 71,3605 (1967). (30) P. Delahay and G. G. Susbielles, ibid., 70, 3150 (1966). (31) G.C.Barker, J . Elecrroanal. Chem., 12,495(1966).
1
A
I l
i
I C * E n ‘
1
-
2 -
I
,
--
-
> c1 c
I
*I‘
s
Figure 5. Polarograms of 1.00 X 10-3M testosterone in acetonitrile-1.0 X lOP3MTBAP A-Fundamental harmonic ac polarogram B-Second harmonic ac polarogram (Ordinates uncalibrated)
tion using abnormally low supporting electrolyte concentrations is not simply of academic interest. When one must employ the relatively expensive tetraalkylammonium salts, the pragmatic overtones associated with the possibility of consuming 10 or 100 times less supporting electrolyte than normal are not insignificant, particularly in a laboratory where large numbers of analyses are contemplated. Accordingly, the dc and ac polarographic behavior in a~etonitrile-lO-~M TBAP was examined for most of the compounds listed in Table I (acetylsalicylic acid, atropine methyl nitrate, colchicine, deserpidine, hydrochlorothiazide, methylclothiazide, phenobarbital, prednisolone, prednisone, progesterone, reserpine, sulfacetamide, sulfadiazine, sulfamerazine, sulfamethazine, testosterone). With the exceptions of sulfadiazine and sulfamerazine, the polarographic waves observed with 10-3M TBAP were remarkably similar to those obtained using acetonitrile0.1M TBAP. Normally, slight changes in dc and ac polarographic wave position, magnitude, or shape attend the two order-of-magnitude change in TBAP concentration. While perhaps of some kinetic-mechanistic significance, the small changes are essentially inconsequential as far as analytical applications are concerned. The two anomalies, sulfadiazine and sulfamerazine, were the only compounds examined which yielded closely-spaced multiple waves (see Table I). The significant changes in the response of the latter compounds with reduction in TBAP concentration appear to be due in a large part to an attendant shift in the relative positions of the multiple waves. In no case was the analytical utility of a polarogram deteriorated by the reduction of TBAP concentration to millimolar. On the contrary, the one significant and general change attending the hundred-fold decrease in TBAP concentration is a noticeable enhancement in sensitivity (2-3 fold) due to a reduced double-layer charging current (not enhanced faradaic currents) which is a consequence (expected) of the lower supporting electrolyte concentration. Figures 5 and 6 show fundamental and second harmonic ac polarograms of reserpine and testosterone in acetonitrile-10-3M TBAP. Comparison of these polarograms with the corresponding data for 0.1M TBAP (Figures 1 and 2) provides an illustration of the foregoing remarks. The data of Table I are rich in mechanistic implications which could, in principal, he discussed compound by compound. This will not be attempted because such a discussion is out of context with the main concern of this report. Fur-
ANALYTICAL CHEMISTRY, VOL. 42, NO. 2, FEBRUARY 1970
*
247
'
B
_>,*
I"0
c
POTENTI
i"LT5
223
-200
c i
POTLY.
A.
VOLTS
Figure 6. Polarogram of 3.76 X lW5M reserpine in acetonitrile-1.00 X lO+MTRAP Legend same as Figure 5 ther, with the exception of those compounds exhibiting simple diffusion-controlled behavior, the conclusions would be speculative because the data, while adequate to provide the basis for tentative conclusions, is inadequate to serve as a basis for firm, quantitative mechanistic conclusions. However, one general qualitative observation is worth noting. Namely, the electrochemical behavior of the pharmaceuticals investigated here is quite consistent with the precedents established by the studies of simpler organic compounds in aprotic media, which were discussed above. A small, but significant fraction of the compounds investigated yield polarogram characteristic of simple, diffusion-controlled one-electron reductions leading to relatively stable anion radicals of the parent compound. The majority of compounds which do not exhibit strictly diffusion-controlled responses also appear to undergo an initial rapid one-electron reduction. However, in the latter cases the radical anions decompose irreversibly with associate? half-lives comparable to, or less than the polarographic time scale, thus destroying the reversible character of the electrode reactions. Subsequent electron uptake by the decomposition products i s also indicated-Le., ECE or extended ECE type mechanisms (13, 32) appear to be ope1ative when the radical anion decomposes. The reversible one-electron character of the initial reduction step was confirmed for colchicine, colchiceine,hydrocortisone, prednisolone, prednisone, and progesterone in a separate triangular wave cyclic voltammetric study (33) where it was found possible to raise the triangular wave frequency to the point where the follow-up chemical reaction could not significantly influence the voltammogram. The result was a cyclic voltammogram characteristic of a rapid, reversible one-electron process. For the majority of compounds not subjected to cyclic voltammetric confirmation of the oneelectron character of the primary step, circumstantial evidence in favor of this pathway is abundant. Second harmonic ac polarograms whose shapes are very characteristic of oneelectron reversible charge transfer followed by chemical decomposition of the reduced species (34) and the complete (32) R. S. Nicholson, J. M. Wilson, and M. L. Olmstead, ANAL. CHEM., 38, 542 (1966). (33) H. L. Hung and D. E. Smith, unpublished work, Northwestern
University, Evanston, Ill., 1968. (34) T. G. McCord and D. E. Smith, ANAL.CHEM., 41,1423 (1969). 248
ANALYTICAL CHEMISTRY, VOL. 42,
absence of apparent n-values exceeding unity (Table I) are among the more convincing forms of this circumstantial evidence. The ECE character of these nondiffusion-controlled processes is suggested primarily by the fact that the dc polarographic limiting current magnitudes for processes which deviate significantly from diffusion control exceed the values expected for one-electron transfer. Also, the relatively broad shape of the dc polarograms exhibited for the type of compounds in question can be interpreted on the basis of a simple EC mechanism (13) only by invoking very slow charge transfer, a suggestion contrary to all reliable precedent (13,14). On the other hand, the broad waves are readily interpretable in terms of an ECE type mechanism (35) without invoking slow charge transfer. The foregoing mechanistic conclusions may be incorrect for particular compounds involved in this study. However, the bulk of evidence strongly suggests that the favored pathway involves an initial, rapid oneelectron reduction with the stability of the anion radical product determining the overall reversibility of the electrode reaction and total number of electrons ultimately transferred under dc conditions. The aspects of the polarographic results considered above, the simple facts that 22 of the 24 compounds yielded a dc response and 19 of 24 exhibited analytically useful ac polarograms, and the excellent sensitivities obtainable when charging current discrimination is operative (second harmonic measurements) are among the observations leading to the inescapable and expected conclusion that acetonitrile represents an eminently useful media for polarographic analysis of pharmaceuticals and related organic compounds. Because acetonitrile is a reasonably representative aprotic solvent, a similar conclusion is justified regarding other aprotic solvents which have been used for electrochemical studies. For several decades a vast methodology involving dc polarographic analysis of pharmaceuticals using aqueous media has been developed. The monograph of Zuman and Brezina (36) provides an excellent compilation of this progress. We do not view the work described here, including the precedents quoted, as a keynote to abandonment of this methodology in favor of one based on aprotic organic solvent polarography. It is true that for polarographic analysis of the class of compounds in question, one can quote advantages of aprotic organic media such as: normally enhanced solubilities, a wider range of dc potentials available for the observation of both oxidative and reductive processes, minimization of troublesome adsorption effects, and more numerous reversible electrode reactions making feasible the use of highly-sensitive ac polarographic methods. However, for many compounds some of these advantages may not be realized and/or equally convincing arguments in favor of aqueous media might be forwarded. Thus, a biased view is not recommended. Rather, we hold that the results reported here support, not a revolution, but a considerable broadening of the analysts outlook with regard to choice of solvent, supporting electrolyte concentration and measurement technique for polarographic analysis of pharmaceuticals and other classes of organic compounds. RECEIVED for review August 18, 1969. Accepted December 5,1969. (35) H.R.Sobcl, Ph.D. Thesis, Northwestern University, Evatiston,
Ill., 1969.
(36) P. Zuman and M. Brezina, "Polarography in Medicine, Biochemistry and Pharmacy," Interscience, New York, N. Y., 1958.
NO, 2, FEBRUARY 1970
