LITERATURE CITED
(1)Allen, L. c . , Johnson, L. F . ~ J. A m . Chem. SOC.85, 2668 (1963). ( 2 ) Anderson, R. L., Houseman, E. F., “Tables of Orthogonal Polynominal Values Extended to K = 104,” Agricultural Experiment Station, Iowa State College of Agriculture and Mechanical Arts, Ames, Iowa, 1942. (3) Cole, LaMont C., Science 125, 874 (1957).
(4) Guest, P. G.,“Numerical Methods of Curve Fitting,” p. 349 ff., University Press, Cambridge, England, 1961, (5) Jones, R. N., Seshadri, K. S., Hopkins, J’ w.J Can’ Jour’ Chem. 40, 334 (lg6’). (6) Kerawala, S. M., Indian J . Phys. 15, 241 (lg41). ( 7 ) SavitzkY, A., A N A L . 33, 25.4 (Dec. 1961). (8) Whitaker, S., Pigford, R. L., Ind. Eng. Chem. 52, 185 (1960).
(9) Whittaker, E.,Robinson, G., “The Calculus of Observations,” p. 291 ff., Blackie and Son, Ltd., London & Glasgow, 1948.
RECEIVED for review February 10, 1964. Accepted April 17, 1964. Presented in part at the 15th Annual Summer Symposium on Analytical Chemistry, University of Maryland, College Park, Md., June 14, 1962.
Controlled -Potential Cou Iometric Ana lysis of N-Substituted Phenothiazine Derivatives F. HENRY MERKLE and CLARENCE A. DISCHER College o f Pharmacy, Rutgers-The
State University, Newark 4, N . 1.
b Controlled-potential electrolysis is suitable for the coulometric determination of several pharmaceutically important N-substituted phenothiazines. The concentration of sulfuric acid, used as the supporting electrolyte, has a differentiating effect on the half-wave potentials of the compounds studied. Polarographic measurements obtained with a rotating platinum microelectrode established current-voltage curves. The compounds could b e quantitatively oxidized to a free radical or to a sulfoxide by selection of suitable acid concentrations and applied potentials. Electroreduction of the free radicals occurs at approximately +0.25 volt vs. S.C.E. on a platinum electrode. In the case of the sulfoxides, a single 2-electron reduction step occurred at ca. -0.95 volt vs. S.C.E. on a mercury pool cathode. The determinations showed good reproducibility and an accuracy of ca. 1% was obtained with sample concentrations of 1 0-3 M or greater.
T
establishment of an oxidation mechanism for the various N amino-substituted phenothiazines is a subject of considerable biological significance ( 2 ) . The character of this oxidation has been investigated by using a variety of analytical methods (1, 4 , 7 , 8). However, because of the transient nature of a free radical intermediate, relatively little has been done to demonstrate the quantitative aspects of this reaction. The application of controlledpotential electrolysis to the analysis of phenothiazine derivatives is an extension of previous work by the authors on the electro-oxidation of chlorpromazine (6). Since these compounds are electrolytically active a t moderate applied potentials, controlled-potential coulometry offers a rapid and absolute approach for their HE
quantitative determination. Moreover, this technique makes possible the direct determination of the individual species involved in the oxidation sequence. The oxidation reactions for phenothiazine derivatives are, in general, represented by:
R:
+ .
R.
+ e-
(1)
and
R.
+ HzO
+
S
+ 2H+ + e -
(2)
in 12N sulfuric acid, and
R: and 2R.
+ H20
+ .
R.
+ e-
--
spontaneous
R:
(3)
+ S + 2H+ (4)
in 1 N sulfuric acid ( 2 , 6 ) , where R: represents the initial reduced form of the compound, R . represents the free radical obtained upon 1-electron oxidation, and S represents the corresponding sulfoxide. EXPERIMENTAL
Instrumentation. Polarograms were obtained on a Sargent Model XXI recording polarograph. An H-type cell was used, with a sintered-glass disk of medium porosity separating t h e two electrode compartments. A rotating platinum microelectrode served as the working electrode us. S.C.E. as reference. T o obtain well defined reproducible S-shaped curves it was necessary to pretreat the microelectrode by anodic polarization for 10 minutes in 1N or 12N sulfuric acid at f1.0 volt vs. S.C.E., followed immediately by a brief 2- to 3minute electrolysis with the platinum microelectrode as the cathode. The controlled-potential electrolyses were performed with an electronic controlled-potential coulometric titrator, Model Q-2005 ORNL ( 5 ) . Readout voltages were measured with a ?JonLinear Systems Model 484 A digital voltmeter.
Electrolysis Cells and Electrodes. Two closed cells with operating capacities of 100 and 20 ml., respectively, were used for oxidations. They were constructed to accommodate large cylindrical, wire mesh, rotating platin u m electrodes, 2.5 cm. in diameter a n d 5 cm. in height for the large cell, and 1 cm. in diameter and 3 cm. in height for the small cell. The reference electrode (S.C.E.) and auxiliary platinum cathode were isolated from the sample compartment by sintered-glass diaphragms and agar plugs. The sample compartment of the reduction cell consisted of a 125-m1. wide-mouthed Erlenmeyer flask with a standard-taper neck and fitted. cover. Approximately 30 ml. of mercury was used as the cathode pool. A glass propeller-type stirrer served to agitate the mercury pool. The reference electrode (S.C.E.) and auxiliary electrode (platinum anode) chambers mere separated from the cathode compartment by glass side-arms fitted with sintered-glass diaphragms and agar plugs. The three cells were constructed so that nitrogen gas could be bubbled through the solution before and during electrolysis. The platinum gauze electrodes and the glass stirrer were rotated a t 600 r.p.m. using a Sargent Synchronous Rotator. Materials. REAGENTS.T h e acid solutions were prepared using sulfuric acid, Baker analyzed reagent, distilled water, and ethanol U.S.P. grade. PHENOTHIAZINE DhRIVATIVES. The compounds studied were provided in powdered form by the suppliers indicated: chlorpromazine hydrochloride, chlorpromazine sulfoxide hydrochloride, prochlorperazine ethanedisulfonate, and trifluoperazine dihydrochloride, Smith, Kline & French Laboratories; promethazine hydrochloride and promazine hydrochloride, Wyeth Laboratories; triflupromazine hydrochloride, E. R . Squibb Laboratories; thioridazine hydrochloride, SandDz Laboratories. The structural formulas and generic names of the compounds used in this work are shown in Figure 1. VOL. 36, NO. 8, JULY 1964
1639
Procedure for Stabilization of Free Radical Intermediates. Polarograms were prepared and studied to determine the effect of acid concentration on current-voltage relationships for each of the compounds. T h e change in the current-voltage curves for 1 x 10-3M solutions of the compounds previously listed as a function of acid concentration is shown in Figure 2. These polarograms clearly indicate the separation of the single anodic wave obtained in LV sulfuric acid (curve 1) into two single 1-electron oxidation waves in 121V sulfuric acid (curve 2). I n each case the half-wave potentials for the two waves are separated by approximately 0.5 volt. Therefore, the selective coulometric oxidation of the phenothiazine compounds to their respective free radicals should be accomplished a t the proper potential by using a 1211: sulfuric acid solution as the supporting electrolyte. The acid-stabilizing effect was further demonstrated by generating the free radical species electrolytically under controlled-potential conditions in 10.4’ and 1211: sulfuric acid. Polarograms were recorded after 3 hours’ standing under an atmosphere of nitrogen. Curve 3, Figure 2, shows the effect of J ~ L Yacid in stabilizing the electrolytically produced radical for a 3-hour
Table I.
Compound oxidized
period. The anodic current (curve 4, Figure 2) indicates that spontaneous disproportionation of the free radical occurred to a significant extent over a 3-hour period when 10N acid was used. I n the case of promethazine, Figure 2, B , it was necessary to increase the concentration of acid to 14N to stabilize the radical intermediate.
General Electrolysis Procedure Using 12,Y Sulfuric Acid. OXIDATION TO A FREE RADICAL. T h e required volume of sulfuric acid supporting electrolyte was placed in the oxidation cell. Sitrogen gas was introduced and bubbled through the solution during the entire titration. The solution was then pre-electrolyzed by oxidizing a t a potential value slightly more positive than that to be used for the determination, until the current fell to a negligibly small value (less than 0.1 ma.). The phenothiazine compound was then introduced into the electrolysis cell, the desired potential set, and the titration initiated. The electrolysis was continued until the current reached a value between 1 and 0.1% of the initially observed current. On the completion of electrolysis the currenttime integral was read as a voltage from the digital voltmeter. At this point all solutions had an intense color characteristic of the particular compound oxidized: chlorpromazine, red; promethazine, red; promazine, orange-
Controlled-Potential Coulometric Oxidation Titrations
H2S0, No. Of Standard Oxidation concn,, runs Taken, mg. Found, mg. deviationc potentiala N Ad BE A B A B A B
Chlorpromazine Promethazine Promazirle Triflupromazine Trifluoperazine Prochlorperazine Thioridazine
f0.60 f0.70 +0.60 f0.65 +0.70 +0.60 +0.55
Chlorpromazineh Promethazineh Promazineh Triflupromazineh Trifluoperazineh Prochlorperazineh Thioridazineh
+1.00 $0.98 +0.95 +1.05 +1.05 +1.05 f1.00
Oxidation to Free Radical/ 12 5 4 35.6 10.0 35.7 9 . 9 0 . 2 0.1 14 12 12 12 12 12n
4 4 4 4 4 3
5 4 5 4 4 2
32.1 10.0 32.4 9 . 8 0 . 2 0 . 1 32.1 1 0 . 0 3 2 . 3 9 . 9 0 . 1 0 . 1 3 8 . 9 10.0 3 8 . 8 9 . 8 0 . 1 0 . 1 48.0 10.0 48.2 9 . 7 0 . 3 0 . 1 56.410.056.59.70.20.1 40.710.040.99.80.20.1
Oxidation to Sulfoxide’ 12 14 12 12 12 12 120
5 5 3 3 3 3
2 3 2 2 2 2
...
, . .
35.6 32.1 32.1 38.9 48.0 56.4 40.7
Oxidation to Sulfoxidei +0.95 1 3 . . . 35 6
10.0 10.0 10.0 10.0 10.0 10.0 10.0
35.7 32.2 32.0 39.1 48.2 56.5
9.9 9.8 9.9 9.7 9.7 9.7
0.3 0.3 0.2 0.6 0.3 0.4
0.1 0.1 0.1 0.1 0.2 0.1
. , .
...
. . .
, . .
, . .
0.4
. .
Chlorpromazine . . . 35.7 Promethazine +0.75 1 , . . , . . 32.1 . . . Promazine +l.O.5 1 3 . . . 32.1 . . . 32.0 Triflupromazine +1.05 1 3 . . . 38.9 . . . 38.8 Trifluoperazine +1.00 1 3 . . . 48.0 . . . 47.8 Prochlorperazine +1.00 1 3 . . . 56.4 . . . 56.1 Thioridazine +0.75 1 3 . . . 40 7 , . . 40.7 a Anode potential given with respect to S.C.E. * Average value for number of runs indicated. c Standard deviations ralculated with respert to averages given. Oxidation titrations performed using 100-nil. cell. e Oxidation titrations performed using 20-ml. cell. 1 Calculated on basis of 1-electron change. Supporting elect,rolyte containing 30% by volume ethanol. Free radical. Calculated on basis of 2-electron change.
1640
ANALYTICAL CHEMISTRY
R
.. ...
... , . .
... . . .
... 012 0.4 0.2 0.2 0.4
. , .
... ...
... , . .
RIFLUOPERAZINE 4 g k o - W a
-C5
-
DROCHLORPERAZINE
-cI
-
rRIFLUPROMAZIEE
I1
I,
-cs
Figure 1 . Structural formulas and generic names of compounds used in study
brown; triflupromazine, orange ; trifluoperazine, orange; prochlorperazine, red ; thioridazine, blue.
OXIDATIONTO SULFOXIDE. The acidstabilized free radical was oxidized to a colorless sulfoxide by increasing the applied potential. Satisfactory quantitative results were obtained by operating a t potentials which fell on the sloping portion of the second polarographic oxidation wave. The potentials reported in Results were found to be sufficiently positive to perform the second oxidation step within reasonable electrolysis times and without initiating complicating secondary oxidations beyond the sulfoxide. REDUCTION OF FREERADICAL. The free radical species was reduced on a platinum electrode a t a potential of +0.25 volt us. S.C.E. -4small correction was necessary because of a conditioning of the platinum electrode surface on changing from oxidation to reduction conditions. This effect was evaluated by performing a preelectrolysis oxidation using supporting electrolyte only and then subjecting the electrode directly to reducing conditions. The magnitude of this effect was found t o be constant for each of the two electrodes used; the corrections comprised approximately 1% of the total readout with the large cerl and up to 10% of the total readout with the small cell. General Electrolysis Procedure Using 1 . O S Sulfuric Acid. OXIDATION TO SULFOXIDE. Oxidation of the compounds directly to the sulfoxide stage was accomplished by using acid concentrations lower than those necessary to stabilize the free radical intermediate: a 1.V acid was a suitable
I
A- CHLORPROMAZINE
I
.-ST
E
- TRIFLUOPERAZINE
30
3
t1.2
tl.l
+IO
r09
E
+O 8 VI.
+0.7
S C.E.
+OB
+0.5
+Oc)
+I2
+0.3
+l,l
+IO
+oQ
+q t w
+qs
+as tap +p
+a6
t05
VOLT.
B- PROMETHAZINE
F - PROCHLORPERAZINE
30
M
+Ii2
I
IC - PROMAZINE
+I!
UO
+0$
+OB
4-07
E
va
S.C.E.
E
VI.
S.C.E.
+Q4
to3
VOLTS
2 -THIORIDAZINE 30
D -TRIFLUPROMAZINE -30
I
6
Figure 2. Polarograms of thiazine compounds studied 7 - i- - - - 1 , Anodic wave with 1 N sulfuric ocid
----
VOLTS
M solutions of pheno-
as supporting electrolyte Anodic wove with 12N sulfuric ocid as supporting electrolyte 3. Anodic wove of electrolytically produced free radical after 3 hours' standing under nitrogen otmosphere, 12N sulfuric acid supporting electrolyte 4. As in 3, but with 10N sulfuric acid as supporting electrolyte
2.
VOL. 36, NO. 8, JULY 1964
1641
12X ACID
TIME
- SECONDS
Figure 3. Current-time curves for 1 electron oxidations of chlorpromazine 1. 2.
+0.6 volt
VI.
Figure 4. Current-time curves for direct oxidation of chlorpromazine to its sulfoxide at 1 .O volt vs. S.C.E.
+
S.C.E.
+ 1 .O volt vs. S.C.E.
1. 2.
dilution. This concentration allowed the quantitative oxidation to proceed to the sulfoxide within practical electrolysis times. The electrolysis procedure employed was identical to that using If the more positive 12N acid. potentials, on the polarographic oxidation plateaus, were used, significantly shorter electrolysis times were required. REDUCTIONOF SULFOXIDES AT A MERCURY CATHODE IN 1N ACID. The electroreduction of the sulfoxide com-
Table
Compound reduced Chlorpromazine Promethazine Promazine Triflupromazine Trifluoperazine Prochlorperazine Thioridazine
II.
Oxidation in 1 N sulfuric acid Oxidation in 12N rulfuric acid
pounds a t a mercury pool cathode required potentials of approximately -0.95 volt us. S.C.E. The sulfoxide compounds were prepared by oxidizing the phenothiazine compounds in 1N acid at a platinum electrode, as described in the previous section. Aliquots were pipetted directly from the oxidation cell into the reduction cell. Solid chlorpromazine sulfoxide was available and was titrated in a similar manner by introducing the weighed
Controlled-Potential Coulometric Reducrion Titrations
Reduction potentiala f0.25 f0.25 +0.25 + O . 25 +0.25 + O , 25 +0.25
Found, mg.* A B Reduction 12 3 14 2 12 3 12 2 12 2 12 2 120 3
of Free Radical’ 2 35.6 10.0 35.4 2 32 1 10 0 32.0 3 321 1 0 0 3 2 0 3 389100385 2 480100478 2 56.4 1 0 . 0 5 6 1 2 40.7 1 0 . 0 4 0 5
Ci C Reduction of Sulfoxideh 1 3 35.6 ... ... 32.1 32.1 1 3 38.9 1 3 1 3 48.0 56.4 1 4 1 3 40.7
10.0 9 9
9 9 9 9 9 C
9 8 7 7 8
Standard deviationc A B 0.1 0.2 0.1 0.1 0.1 0.1 0.2
0.1 0.1
0.1
0.1 0.1 0.1 0.1
C
0.2 Chlorpromazine - 0.95 35.2 ... Promethazine , . . 31.4 0.1 -6.92 Promazine 0.5 38.5 Triflupromazine - 0.95 0.3 47.1 Trifluoperazine -0.86 0.4 55.6 Prochlorperaxine -0.90 0.2 40.0 Thioridazine -0.92 Reduction of Solid Samples of Chlorpromazine Sulfoxide? -0.95 1 3 37.2 34.9 0.4 a Cathode potential given with respect to S.C.E. b Average value for number of runs indicated. c Standard deviations calculated with respect to averages given. d Reduction titrations performed using 100-rnl. cell. c Reduction titrations performed using 20-ml. cell. Calculated on basis of I-electron change. 0 Supporting electrolyte containing 30%-by volume ethanol. Calculations based on 2-electron change for electrolytically produced sulfoxide. Titrations performed in reduction cell using mercury pool as cathode. j Caldations based on 2-electron change using samples obtained from two different lots.
1642
ANALYTICAL CHEMISTRY
sample into the electrolysis cell containing 50 ml. of 1N sulfuric acid. Nitrogen gas was bubbled through the solutions during the entire reduction titration. RESULTS A N D DISCUSSION
Evaluation of Titration Procedure and Data. The results obtained for the controlled-potential oxidation of the N-substituted phenothiazines are compiled in Table I. All were quantitatively oxidized to the free radical form. However, promethazine and thioridazine could not be oxidized quantitatively to their sulfoxides in 1A’ and 12N acid, respectively. The oxidation of the other compounds studied proceeded quantitatively through two steps involving a colored free radical as an intermediate. The results obtained in reductions of the free radicals and the sulfoxides are summarized in Table 11. The reductions of the sulfoxides were accomplished as easily as the oxidations were. I n the case of thioridazine the inclusion of ethanol was required when 12N acid was used as the electrolyte in order to maintain suitable concentrations. The experimental data indicate that the controlled-potential method is of value in the quantitative determination of various A‘-substituted phenothiazine derivatives. The oxidation and reduction procedures presented are analytically useful when performed under the stated conditions. The controlledpotential coulometric titration of phenothiazine compounds offers two advantages: the compounds may be titrated directly without preliminary treatment; and the different oxidation species of a given phenothiazine may be determined individually in the presence of one another because of the selectivity ~
of oxidation imposed by control of the working potential. The polarographic data proved to be a valuable aid in the select,ion of electrolysis potentials. There were no significant differences in t,he currentvoltage relationships obtained with the microelectrode and with the macroelectrode. Current-Time Curves. T h e general charact.er of t,he current-time curves for the acid concentrations used is shown in Figures 3 and 4. Characteristic curves for the 1-electron oxidations occurring in 1 2 5 ac,id, illustrative of typical initial currents and electrolysis times, are shown in Figure 3. T h e current-time curves for the two-step oxidations (Figure 4) in both 1 S and 1212' acid exhibit two linear segnients with different slopes, representative of the t,wo consecut'ive oxidations. In the early part of the electrolysis the reaction expressed by Equat,ion 1 (or 3) predominates. This is indicated by the first linear portion. As the concentration of radical builds up, the react,ion expressed by Equat'ion 2 is of primary significance and is the main determinant of the slope of the second line segment. Electrode Oxidation Mechanism.
Some observations were made regarding the nature of t h e electrode oxidat,ion mechanism. Pretreatment of the platinum macroelectrode result,ed in the open circuit oxidation of the reduced phenothiazine compounds. When the plat,inum electrode was polarized anodically for a few minutes a t normal working potentials and then
the circuit opened and the sample to be oxidized introduced, any material that came in direct contact with the electrode surface was immediately oxidized. This was indicated by a color changeviz., the reduced colorless compound was oxidized to the colored radical intermediate. This effect could be repeated only when the electrode was again subjected to brief anodic polarization. The amount of material transformed under open circuit conditions was found t o be negligible when compared to the quantities that were taken for analysis. The work of Feldberg, Enke, and Bricker ( 3 ) dealing with the formation and dissolution of platinum oxide films offers an explanation for t,his observation. These workers demonstrated anodic film formation on platinum through two single-electron steps-a very slow step, followed by a fast reversible step. The latter results in an active electrode state. This work leads us to propose that the act,ive electrode state is responsible for the oxidation of phenothiazine compounds observed under the open circuit conditions described. -1 similar situation may exist under closed circuit conditions; the active electrode state is maintained with consequent oxidation of the electroactive material. hdditional evidence supporting this oxidation mechanism was observed during the polarographic studies. Erratic oxidation waves were obtained with an untreated platinum microelectrode. However, when the electrode was first, oxidized and then reduced under controlled-potential conditions in 1.V or
12'T acid, reproducible polarographic waves were obtained. The electrode produced reproducible waves during its entire period of use after one such treatment. ACKNOWLEDGMENT
The authors thank Smith, Kline & French Laboratories, Wyeth Laboratories, Squibb Laboratories, and Sandoz Laboratories for the generous supplies of phenothiazine cornpounds made available for this work. The authors give credit' to the Research Council of Rutgers-The State University for financial assistance which enabled the constructmionand purchase of much of the instrumentation used for the coulometric work. F. H . Merkle thanks the Johnson & Johnson Laboratories for support in the form of a research fellowship for the 1963-64 academic year. LITERATURE CITED
( 1 ) Borg, D. C., Cotzias, G. C., Proc. iVatl. A c a d . Sci. 48, 623 (1962). ( 2 ) Zbid., p. 643. ( 3 ) Feldberg, S. W., Enke, C. G., Bricker, C. E., J . Electrochem. Soc. 110, 826 (1963). ( 4 ) Kabasakalian, P., McGlotten, J., ANAL.CHEM.31,431 (1959).
( 5 ) Kelly, M. T., Jones, H. C., Fisher, D. J., l b i d . , 31, 488 (1959). ( 6 ) Merkle, F. H., Discher, C. A., J. Pharm. Sci., in press. ( 7 ) Piette, L. H., Ludwig, P., Adams, R. N., ANAL.CHEM.34, 916 (1962) (8) Ryan, J. A , , J . A m . Pharm. Assoc. 48,240 (1959).
RECEIVED for review February 14, 1964. Accepted April 23, 1964.
Catalytic Determination of Vanadium in Water M. J. FISHMAN and M. W. SKOUGSTAD U. S. Geological Survey, Denver, Colo.
b A rapid, accurate, and sensitive spectrophotometric method for the quantitative determination of trace amounts of vanadium in water is based on the catalytic effect of vanadium on the rate of oxidation of gallic acid by persulfate in acid solution. Under given conditions of concentrations of reactants, temperature, and reaction time, the extent of oxidation of gallic acid is proportional to the concentration of vanadium present. Vanadium is determined by measuring the absorbance of the sample at 415 mp and comparison with standard solutions treated in an identical manner. Concentrations in the range of from 0.1 to 8.0 pg. per liter may be determined with a standard deviation of 0.2 or
less. By reducing the reaction time, the method may b e extended to cover the range from 1 to 100 pg. with a standard deviation of 0.8 or less. Several substances interfere, including chloride above 100 p.p.m., and bromide and iodide in much lower concentrations. Interference from the halides is eliminated or minimized by the addition of mercuric nitrate solution. Most other substances do not interfere at the concentration levels at which they commonly occur in natural waters.
S
and spectrophotometric techniques are the principal means currently used to determine vanadium in natural waters. Either PECTROGRAPHIC
technique requires concentration of vanadium by precipitation, evaporation, or extraction, to detect trace quantities of t'he element. Haffty ( 1 ) and Silvey and Brennan (4) described general spectrographic procedures suitable for determining vanadium and other minor elements in water. Spectrophotometric methods have been reported by Xaito and Sugawara ( 3 ) and by Sugawara, Tanaka, and S a i t o ( 5 ) . A rapid, qualit'ative test for trace amounts of vanadium was recently reported by Jarabin and Szarvas ( 2 ) . Their test is based on the fact that the oxidation of gallic acid by acid-persulfate is catalyzed by minute amounts of vanadium. In the presence of vanadium the reaction proceeds rapidly, VOL. 36, NO. 8, JULY 1964
1643
