now in use for those samples in which both U(1V) and U(V1) determinations are desired. LITERATURE CITED
(1) Boyd, C. M., Menis, O., ANAL. CHEM.33. 1016 (1961). (2) Farrar, L.--G.: Thomason, P. F., Kelley, M. T., Ibid., 30, 1511 (1958). (3) Frankenthal, L., Neuberg, C. N., Exptl. Med. Sura. 1, 386 (1943).
(4)Ginocchio, B." J.; "Uranium, Auto-
matic Potentiometric Ferric Sulfate Method," Method Nos. 1 219224 and 9 00719224 (2-24-58), ORh'L Master Analytical Manual, TID-7015, See. 1. ( 5 ) Kelley, M. T., Jones, H. C., Fisher, D. J., ANAL.CHEW31,488 (1959). (.6.) Kolthoff, I. M.. Lingane. J. 'J.,' J. Am. Chem: SOC.55; 187i-( 1933). (7) Kubota, H., ANAL.CHEM. 32, 610 ( 3 win). \----,-
(8) Lundell, G. E. F., Knowles, H. B., J. Am. Chem. SOC.47,2637 (1925). (9) Shults, W. I)., Thomason, P. F.. ANaL. 6 H E Y . 31, 492 (1959).
(10) Subbaraman, P. R., Joshi, N. R., Gupta, J., And. Chim. Acta 20, 89 il95F)i. \ - - - - ,
(11) Zittel, H. E., Dunlap, L. B., ANAL. CHEJI.34, 1757 (1962). (12) Zittel, H. E., Dunlap, L. B., Thomason, P. F., Ibid., 33,1491 (1961).
RECEIVEDfor review September 26, 1962. Accepted November 21, 1962. The Oak Ridge National Laboratory is operated by Union Carbide Corp. for the U.S.Atomic Energy Commission.
Polarographic Reduction of Steroids in N,N- Dimet hy If o rmamide ALLEN 1. COHEN The Sqvibb lnstifvte for Medical Research, New Brunswick, N. 1.
b
The polarographic half-wave potential of a steroid is characteristic of the reducible group. The reduction of ones, enones, and dienones (crossconjugated and linear) and vicinal halogenated derivatives has been studied and their use in structural elucidations discussed. The reduction potentials of the nonvicinal A4-3-ketosteroids studied were not systematically influenced by structure or molecular weight. The average half-wave potential of the A4-3-ketosteroids was 1.66 =t0.02 volts with an average I d of 1.35 == ! 0.16. Reduction may also occur simultaneously a t two isolated sites without influencing the reduction characteristics of each group.
-
F
studies have been conducted to relate polarographic reduction and structure of steroids. Brezina and Zuman (1 i summarized the reduction of steroids in aqueous-alcoholic solutions, but limited their discussion t o A4-3-ketosteroids and to those steroids which form Girard or hydrazone derivatives. Kabasakalian and McGlotten (10) studied the polarographic reduction of A4-3ketosteroids in well-buffered 5001, aqueous-alcohol media. In a later publication, they discussed the reduction of saturated ketosteroids (7). Cross-conjugated dienones and A4-3-ketosteroids have been determined in the presence of each other by polarography ( 6 , l b ,13). -4number of steroids were examined polarographically in N,N-dimethylformamide. The most significant findings are reported and discussed. It is shown that polarographic data can be used in the characterization of steroids. I n certain cases they are more meaningful than commonly published ultraviolet spectroscopic data. EW
128
ANALYTICAL CHEMISTRY
EXPERIMENTAL
The equipment and procedure have been discussed (2). The steroids investigated have been prepared and characterized by various members of this laboratory. Briefly, 4, 2-, 1-, and 0.5-ml. aliquots of a N,N-dimethylformamide (DMF) solution containing between 5 and 10 mM steroid were pipetted into IO-ml. volumetric flasks, 5 ml. of supporting electrolyte containing 0.2M tetrabutylammonium iodide and 0.02JE tetraethylammonium iodide in DMF was added, and the contents were diluted to mark with DMF. The polarogram were obtained with a dropping mercury cathode and a mercury pool anode. The potential of the mercury pool us. a saturated calomel electrode was -510 mv. The capillary characteristics of the dropping mercury electrode were determined a t various voltages and the appropriate value was used in the calculation of the diffusion current constant, I d (6). The temperature of the solution was 25" i 0.5" C. The
Table 1.
I .. A4 1.66 I1 A1,4 1.44 I11 A4.8 1.21 IV a Volts us. mercury pool anode. = Ba./mM 1712'3 TO -2.3 volts.
b I d
RESULTS AND DISCUSSION
The reduction of the 17a-oxa-D-homoandrostane-3,l'l-diones shown in Table I exemplifies the influence of conjugative unsaturation on the reduction of 3-ketosteroids. While Kabasakalian and Mc. Glotten (7) have polarographically reduced saturated 3-ketosteroids in alcoholic solutions, compound I and other saturated 3-ketosteroids are not reducible in DMF. I n the case of the reduction of the unsaturated 3-ketosteroids, the ease of reduction is determined by resonance, which is greatest in the linearly conjugated dienone(IV), intermediate in the cross-conjugated dienone(", and least in the enone(I1). The rate of electrochemical reduction can be evaluated, qualitatively, from values of the apparent number of electrons transferred (4). The approximate integer value of n indicates that com-
Polarographic Reduction of 17a-Oxa-D-homoandrostane-3,17-diones in N,N-Dimethylformamide
Wave 1 -Em" IC* N o reductionc
C
apparent number of electrons transferred, n, was determined by computer regression program (8).
t116.
1.6 1.5 1.4
Wave 2 n 0.84 0.86 1.10
-Ell,"
2.05 1.89
Idb
n
2.3 1.0
0.59 0.97
pounds 11, 111, and IV are reduced substantially in the first reduction step by a diffusion-controlled (4) process to the mononegative ion. In protonated solvents, generally, one reduction wave is reported for the cross-conjugated dienones (6,12, I S ) . Kabasakalian and McGlotten (9) have reported two reduction waves for prednisone at pH's greater than 6.3 in ethanol-water solutions and one reduction wave for prednisolone in the same pH range. In DMF, 111 reduces to the dinegative ion in a second kinetically controlled ( 4 ) step, since n is substantially less than one. Compound IV aleo reduces to the dinegative ion in a second step, in this case by a diffusion-controlled process. Compounds I1 and 111 differ by approximately 0.2 volt in their most positive reduction steps but only by 3 m p in their ultraviolet maxima ( 5 ) . The A1-3-ketosteroids (Table 11) are reduced a t the same voltage with the same sensitivity coefficients as the A4-3-ketosteroids. The average half-wave potential of 17 (not all tabulated) A4-3-ketosteroids was - 1.66 -I: 0.02 volts Kith an average I d of 1.35 + 0.16. The constancy of I,l indicates there is little difference in thr diffusion coefficient of steroids of molecular Keight between 300 and 450 in DMF; thus the sensitivity coefficient ('an be considered as a function of thr reducible group only. The A1r4-3-ketcsteroids (XVII, XVIII, and XXIT.') and t h e A4K3-ketosteroid (=HI) (cf. 'Table VI) are reduced a t similar potentials as the 17a-oxa-D-homo-4-androstene-3,17-diones (111 and IT', rcspectively). In the five-membrrtd A3(5)-A-nor-2kctosteroids (Table III), resonance is 1c.s than in the siu-membered A4-3ketosteroids. This causes increased difficulty in reduction, a9 shown by a -0.1s-volt shift and a shift of -7 mp in the ultraviolet maximum. The reduction of various progesterones is shown in Table IV. The molecular extinction coefficients of the a4-3keto group and the A16-20-ketogroup a t 240 mp are additive ( a = 16,600 and 10,500 liter-mole-1 respectively). Similarly, the AI6-20-ketogroup and the A-ring enone of V reduce concurrently, resulting in a total I d of 3.3 for the compound and an I d of approximatrly 2.0 for the former group. A Al6-20-keto derivative without a A4-3-keto group (5P-pregn-l6-en-20-one, 3a, 12a-dihydroxy-, diacetate) is reduced a t -1.64 volts with an I d of 2.0 and n of 0.81. Reduction may therefore occur simultaneously a t several sites in a molecule nithout affecting the individual sensitivity coefficients of each reducible group. The 16a,l7a-oxido-20-keto group of VI reduces concurrently with the A-ring group, which results in a total I d of
4.2 and an approximate I d of 2.9 for the former group. The ultraviolet absorption coefficient of progesterone is not augmented by the introduction of the 16a,l7a-oxido group; the group is therefore classified as an inductive negative substituent. In the reduction of the l&,lia-dihydroxy cyclic ketal with acetophenone (VI1 and VIII), a reduction wave of a t least 3 electrons appeared near -2.0 volts. This reduction stfp is unusual, since acetophenone itself is reduced under the same conditions only to the dinegative ion in two distinct steps (3) (E,lz = -1.45 volts, I d = 2.6, n = 0.90; = -1.95 volts, Id = 2.4, n = 0.50). The group does not give rise to any significant ultraviolet bands. Thus, the acetophenone ketal, too, is an inductive negative group. Ihbasakalian and LlcGlotten (7) observed the polarographic reduction of saturated ketosteroids in alcoholic media as well as of 20-ketosteroids having vicinal (17a and/or 21) hydroxyl or acetoxyl groups. In DMF, the saturated ketosteroids were not reducible. The 20-ketosteroids with vicinal hydroxyl or acetoxyl groups, in DMF, gave poorly defined waves betn een -2.0 and -2.15 volts which were of little use in structural correlations. a-Substituted halogenated keto groups are reducible, as demonstrated by the reduction of I X and X (cf. Table IV) and the 16-haloandrostrne3,17-diones (cf. Table V). The chloro group of XITI is more easily reducible than the fluoro group of the analog (XII). The fluoro group of the 20keto-21-fluoro derivative (X), the 16afluoro-17-keto derivative (XI), and the 16~-fluoro-17-ketoderivative (XII), reduces concurrently with the A-ring enone, possibly a t a slightly more
Table IV.
Table It. Polarographic Reduction of 5a-Androst-1 -en-3-one, 17P-hydroxy1 -methyl Esters in N,N-Dimethylformamide 0 --CR
1.66 1.4 CH, 1.3 (CHt),CHs 1.62 Volts us. mercury pool anode. =
h I d
Table 111.
pa./mM
0.89 0.91
m213 t116.
Polarographic Reduction of
A3(6)-A-nor-2-ketosteroidsin N,N-Dimethylformamide
R
R
n
1.5 0.92 1.85 COCHs 0.84 1.6 02CCHgCHa 1 . 8 4 Volts us. mercury pool anode. h I d = pa./mV 1n2'3 t 1 / 6 .
poeitive potential than the A-ring group. It appears that the reduction of the fluoro group is influenced by conformation. The side chain of X rotates freely and assumes no ronforniation. Because the D-ring of thc 17-keto derivatives (XI anti XII) is nearly planar, the angle formed between the 16a- and 16p-C-F bonds and the> 17-keto groups is equal and approximately 55".
do
Polarographic Reduction of Progesterones in N,N-Dimethylformamide
0 '
Wave 1
V
A16
VI
16a,l7a-Oxido
VI1
16a,17a
0
O)C(CH,)C,H,
VI11 4,5-Dihydro 16a,17a 0 > c ( c H , ) c 6 H 6 0 IX Sa-Fluoro, 11-oxo X Qa,21-Difluoro, 11fl,17a-dihydroxy a
Wave 2
-Ema
Idh
n
1.65 1.65
3.3 4.2
0.84 0.79
1.66
1.3
0.99 2.03 6 . 8 0 . 7 4
2.08
6.8
0.62
1.51 1.68
2.6 3.2
0 . 9 1 1.73 1 . 3 1 . 3 1 0.65
-El/tu
Idb
n
Volts us. mercury pool anode. = pa./mM m218 t 1 / 6 .
I d
VOL. 35,
NO. 2, FEBRUARY 1963
129
Table V.
enone. The Ga-fl~oro-A~~~-3-ketosteroid (XXV) and the 6a,9a-difluoroA1,4-3-ketosteroid (XXVI) reduce in four steps. One possible mechanism would have the 6a-fluor0 group reduced and cleaved in the first step, followed by the reduction of the cross-conjugated dienone in the next two steps. Polarographic data therefore prove to be more significant for the characterization of &fluorinated steroids than the published ultraviolet spectra (21). The 6a-chloro, 9a-fl~oro-A~~~-3-ketosteroid (XXVII) reduces in four steps at similar potentials as the 60-fluor0 analog (XXVI), although it was expected that the chloro derivative would have its first reduction step at a more positive potential. As expected, fluorine substituents] which are nonvicinal to a keto group or conjugated unsaturation do not reduce or affect reduction (cf. compounds XVII and XVIII),
Polarographic Reduction of 1 6-Haloandrostene-3,17-diones in N,NDimethylformamide
0
Wave 1 XI XI1 XI11
16a-F 168-F
-E11za
Idb
1.59 1.59 1.23
2.3 2.4 2.3
16/3-C1 Volts us. mercury pool anode. I d = pa./mM mz/$ t1/*.
The Sa-fluoro group of I X is axial and approximately at right angle to the 11-keto group. In the 6p-fluoro-A4-3-ketoderivative (XIX) (cf. Table VI), the 6p position is axia! and perpendicular to the carbonyl group, while the 6a-fluor0 epimer (XXI) is equatorial and in the plane. Thus, the first reduction step of the 6p-fluor0 epimer (XIX) is more easily reduced than the 6a-fluor0 epimer (XXI) by 0.13'volt. The 6a-fluoro-A4-3-ket osteroids (XVI, XX, XXI, and XXII) and the -6$-fluoro derivative (XIX) reduce in a number of steps. The reduction
Table VI.
Wave 2 n 1.00 0.71 0.55
-E~IP
Idh
n
1.72 1.72 1.71
1.6 1.5 1.8
0.97
...
1.00
wave near -2.0 volts of the corticosteroids is due, a t least in part, to the reduction of the hydroxy group vicinal to the 20-ketone. The average halfwave potential of the first wave of four 6a-fluoro derivatives is -1.315 0.025 volts with an average I d of 2.1 k 0.1. The second wave occurs between -1.7 and -1.9 volts with an average I d of approximately 2.0. A possible reduction mechanism might involve the reduction and cleavage of the 6-fluor0 group in the first step, followed by the reduction of the enone at a potential close to that of the nonfluorinated
*
ADDENDUM
Subsequent to the submission of this paper for review, Kabasakalian and McGlotten (8) reported the polarographic reduction of epimeric a-halosteroids and their vinylogs. While a-fluoroketosteroids are reduced between -1.5 and -1.7 volts in DMF close to the half-wave potential of the
Polarographic Reduction of Corticosteroids in N,N-Dimethylforrnamide
CH2OR
0
Substitution 6
XIV
xv
XVI XVII XVIII
CY-F A1
A1
9
11
16
Wave 1 R
a-F 8-OH a-F 8-OH a-OH
-H -H a-OH -H 8-OH -H a-F @-OH OI-OZCCHB --COCH,
-E,/p 1.66 1.63 1.36 1.45 1.43
Wave 2
Wave 3
Idh
n
-Ell2"
Idb
n
1.4 1.3 2.2 1.3 1.4
0.63 0.55 0.48 0.77 0.79
2.10 2.11 1.94 2.15 2.11
1.% l.lc 3.4 2.7 5.9'
0.69 0.67 0.54 0.34 0.30
2.5 2.3 1.9 1.8 3.1 5.6 0.7 0.7 0.7
0.60 0.58 0.60 0.62 0.40 0.40 0.45 1.6 1.09
Idb
Wave 4 n
-El/z"
Idh
72
16a,l7~~-4cetonides CH20R
XIX
8-F LY-F CY-F
xx
XXI XXII XXIII XXIV
xxv
a-F A0 A1
A' A'
a-F
... ...
8-OH a-F @-OH a-F @-OH
... ...
...
...
...
-COCHa -H -COCH3 -COCH3 -COCHz -H -COCHs -COCH, -COCHa
... a-F a-F 8-OH XXVI ... XXVII A' OI-C~a-F &OH Volts us. mercury pool anode. b I d = wa./mM m2lat116. 0 I d = decreases with concentration; at 1mM. d Prewave; Ell2 = -1.08, I d = 0.4, n = 0.68. 0
130
ANALYTICAL CHEMISTRY
1.18 1.31d 1.31 1.28 1.26 1.44 1.16 1.20 1.17
2.7 2.0 2.1 2.2 1.4 1.3 2.2 2.4 1.9
0.57 0.68 0.72 0.92 1.05 0.92 1.00 0.98 0.76
1.81 1.86 1.72 1.72 1.85 2.00 1.52 1.43 1.52
2.13 1.4 0 . 5
... 2.03 0 . 5 ... 2.06 0 . 7 2.16 1.SC 1.02 1 . 7 7 1.5 1 . 7 6 1.8 1.83 1 . 4
0 . 6 5 2.06 0 . 7 1.10 0.59 2.08 1 . 2 1.10 0.66 2.17 1.0 1 . 0 7
enone group (about -1.65 volts), the fluoro group is reduced at more negative potentials (about -2.0 to -2.2 volts) than the A4-3-keto group (about -1.7 volts) in ethanol-aqueous media. The epimeric 6-fluoro-A4-3-ketosteroids reduce with the same relative ease in ethanol-water and DMF-Le., the ,%isomer preceding the a-isomer. In DMF, a second wave appears a t a more negative potential because of the reduction of the enone. This second reduction step is not reported in the protonated solvent.
ment throughout the investigation and E. J. Becker, B. Berk, P. A. Diassi, J. Fried, L. B. High, and F. L. Weisenborn for the donation of steroids.
(6) Kabasakalian, P., DeLorenzo, S., McGlotten. J.. ANAL. CHEM.28. 1669
LITERATURE CITED
(1) Brezina, M., Zuman, P., “Polar-
ography in Medicine, Biochemistry, and Pharmacy,” Interscience, New York,
1958. (2) Cohen, A. I., Keeler, B. T., Coy, N. H., Yale, H. L., ANAL.CHEM.34, 216 (1962). (3) Cohen, A. I., Snyder, R. E., un-
published results. P., “New Instrumental Methods in Electrochemistry,” Interscience, New York, 1954. (5) Dorfman, L., Chem. Reus. 53, 47 (4) Delahay,
ACKNOWLEDGMENT
The author thanks If,H. Coy and K. Florey for their help and encourage-
(1953).
RECEIVEDfor review Julv 20, 1962. Accepted November 30, 1962.
Amperometric Titrations with Very Dilute Solutions of Permanganate H.
P. SILVERMAN’
and
D. A. SKOOG
Department o f Chemistry, Stanford University, Stanford, Calif.
b A satisfactory amperometric end point can b e obtained with permanganate solutions as dilute as 1 X 10-+le Titrations of very dilute solutions of Fe(ll), U(IV), and oxalate ion revealed the presence of a constant error of approximately 2 X lo-‘ meq. of KMn04. This error appears to arise from an induced reaction of the oxidizing agent with the solvent. A blank titration will not permit correction for the error and with very dilute permanganate solutions an empirical calibration curve must b e employed.
T
permanganate ion is sufficiently intense in color so that solutions of this reagent as dilute as 0.01 to 0.02N can be employed for titrations without the necessity of an indicator material. Knop and Kubelkova ( 2 ) have shown that milligram quantities of iron can be titrated accurately with 0.005N permanganate solutions, if certain triarylmethane dyes are employed as indicators. Kirk and Tompkins ( 1 ) have used the o-phenanthroline-ferrous complex as an indicator for oxalate titrations with permanganate solutions of similar strengths. The amperometric method appeared to offer a means by which end points could be determined with permanganate solutions more dilute than those heretofore employed. Kolthoff and Jordan HE
Present address, Magna Corp., 1001 South East St., Anaheim, Calif.
(4) have mentioned briefly that currentvoltage waves for permanganate ion are obtainable with a rotating platinum electrode from 131 sulfuric acid, and have described the use of 0.002N solutions as a reagent for the determination of micro quantities of iodide ion. V e have undertaken the further investigation of the amperometric end point for titrations with very dilute solutions of permanganate ion. These studies have shown that n ell-defined and reproducible end points are obtained n-ith reagent concentrations appreciably lower than those required to give a satisfactory end point by other means. Titration of ferrous iron and other reducing agents has revealed, however, the existence of a constant positive error which is large enough to have serious effects on the outcome of analysis based on permanganate solutions more dilute than about 5 x 10-3N. We have made some preliminary inrestigations of the source of the error, but have only been able to come to some tentative conclusions regarding its cause. Unfortunately, the error is not revealed by a blank titration and satisfactory analyses with very dilute permanganate solutions require the use of an empirical calibration. REAGENTS AND PROCEDURES
Apparatus and Techniques.
Current-voltage curves were obtained with a Sargent Model XXI polarograph. The electrolysis cell included a platinum wire microelectrode 3 mm.
long and 0.05 mm. in diameter. It was rotated at a rate of 600 r.p.m. The reference electrode was a saturated calomel half cell coupled to the electrolysis compartment by means of a sodium sulfate bridge. The cell had a resistance of 300 ohms when filled with 1Jf potassium nitrate. A rate of voltage change of 0.074 volt per minute was employed in obtaining currentvoltage curves. The microelectrode was generally stored in a dilute nitric acid solution. When current-voltage curves were t o be obtained, it was covered with concentrated hydrochloric acid, rinsed with distilled water, and placed in 0.1N potassium permanganate solution for 10 minutes. Following this treatment, the electrode was thoroughly washed with distilled water, Biped dry, and allowed to stand 5 minutes in the solution t o be run. This elaborate treatment was not followed for successive amperometric titrations. Amperometric titrations were carried out a t an applied voltage of $0.4 volt us. the saturated calomel electrode. Generally, 100 to 110 ml. of solution were titrated. A microburet readable to 0.002 ml. was employed and the volume of reagent used was small enough to make unnecessary a current correction for volume change. Reagents and Solutions. Reagent grade chemicals were used throughout. I n some instances the water employed was twice distilled, once from alkaline permanganate solution. Deionized water was found to contain oxidizable impurities, and always had to be distilled before use. Standard permanganate solutions having normalities of I x 10-4 to I X VOL 35, NO. 2, FEBRUARY 1963
131
