Langmuir 1985, 1 , 110-119
110 in the silicon and the pretreatment.
Conclusions The uncatalyzed reaction between Si(100) and CH3C1 to form silanes was studied at 84 kPa and 545-695 K in a differential batch reactor. Kinetic studies and surface analysis show the following: Silicon-containingproducts do not form on clean Si(100). The active surface for chlorosilane and methylchlorosilane product formation is formed by CH3C1decomposition and contains Si, C1, graphitic carbon, and 0. Most of the Si is bound to C1. A long induction period for silane formation occurred, apparently due to the time required to form Si-C1 active sites. The active Si(100) surface reacts with CH3C1at 545-620 K to form HSiC13 and CH3HSiC12plus smaller amounts of SiCl,, CH3SiC1, and (CH&SiC12. Steady-state reaction is fast; 8 X 1015 silane molecules/(cm2 s) form at 620 K.
Between 620 and 670 K, secondary chlorination of silanes occurs, and CH&l desorption may limit silane formation. Methyl chloride and silane decomposition reactions do not occur below 670 K. At 670-695 K, CH3Cldecomposition, silane chlorination, and silane decomposition are significant side reactions. At 695 K, the main silane products are the stable, chlorinated silanes CH3SiC13and SiC1,. The Si(LVV) Auger peak at 84 eV is due to Si bound to c1. Acknowledgment. We gratefully acknowledge support of this work under National Science Foundation Grant CPE 80-24236. We also thank Union Carbide Corp. Silicones and Urethane Intermediates Division, and the University of Colorado for partial support of this work. We appreciate the valuable discussions with Drs. Kenrick M. Lewis and Keith B. Kester. Registry No. CH,Cl, 74-87-3; Si, 7440-21-3.
Systematic Examination of Oxidation and Surface Processes for Acetylenic and Related Steroid Hormones at Mercury Electrodes A. M.
Bond,*l
I. D.
Heritage,l
and M. H. Briggs2
Diuisions of Chemical and Physical Sciences and Biological and Health Sciences, Deakin Uniuersity, Victoria 321 7, Australia Received July 13, 1984 Acetylene-substituted steroid hormones exhibit oxidative responses at mercury electrodes associated with the formation of mercury acetylide compounds. Reactions are also accompanied by strong and specific reactant and product adsorption. A simplified reaction scheme in which S is a steroid and mercury and protons are not shown is as follows: S'
11 S
Sads
* s ' ~ t~ e~
11 S"ads t e
11 S"
The faradaic and nonfaradaic components of the electrochemistry have been studied by use of a range of acetylenic and closely related non-acetylenic steroids. Techniques used include dc and ac polarography, cyclic voltammetry, and controlled potential electrolysis. The faradaic responses of individual steroids are characterizedby behavior reflected in structural differences associated with regions of the steroid molecule not participating in the mercury acetylide formation directly. The structural differences of the compounds examined can be grouped into two general categories, non-phenolic and phenolic, and are associated with irreversible and reversible faradaic responses, respectively. All steroids examined, including the nonacetylenic ones, are adsorbed at the dropping mercury electrode. An investigation into the nature of the adsorption of these compounds also leads to the conclusion that the mechanism of adsorption, as was the case for the faradaic response, is dependent upon structure and solvent conditions. The differences observed for the electrode processes at mercury electrodes of phenolic and non-phenolic compounds are interpreted as being the result of different orientation and adsorption of these compounds. Introduction Investigation of the electrochemistry of steroid hormones has been concerned primarily with the reduction process (1)Division of Chemical and Physical Sciences. (2) Division of Biological and Health Sciences.
associated with the conjugated ketone functional group that many of the compounds c ~ n t a i n . ~ - ~ (3) Kabasakalian, P.; McGlotten, J. J. Am. Chem. SOC.1956, 78, 5032-5036. (4) Cohen, A. Anal. Chem. 1963,35 (2), 128-131.
0743-7463/85/2401-0110$01.50/0 0 1985 American Chemical Society
Langmuir, Vol. 1, No. 1, 1985 111
Oxidation and Surface Processes for Steroid Hormones
OH
(0)
Ethynyl Estradiol
(c)
IlOrgestrel
(bl
Mestranol
(d)
Chloronorgestrel
T
ob
fl
OA
03
0.2
0.1
0.0
- E , Volts
Figure 2. Current-sampled dc polarogram of 0.8 X lo4 M ethynylestradiol a t "pH" 12.2.
H
(e) P-Estradiol
(f) a-Estradiol
a@
*GCH
(h) Lmestronol
(9)
Testosterone
Figure 1. Structures and trivial names of steroid hormones used in this study. More recently the oxidation of "phenolic type" steroid hormones has been studied at the glassy carbon electrode? For acetylenic steroids, work in this laboratory has indicated that oxidation reactions can be observed at mercury electrodes.lOJ1 Acetylenic steroids are widely used in oral contraceptives (oc).12Figure l a gives the structural representation of an acetylenic steroid commonly used in oc, ethnylestradiol. The presence of the acetylenic group at carbon-17 distinguishes this class from other structurally related steroids. The acidic terminal acetylene is capable of replacement by mercury and as a result redox process may be observed at mercury electrodes.lOJ1 13C NMR experiment^'^ show that these compounds are sterically rigid; furthermore the structure is predominantly planar.12 The nature of the steroid backbone and substituents indicates that steroids should be surface active.'* The surface activity has been ~
(5)Zuman,P. Collect. Czech. Chem. Commun. 1968,33,2548-2552. (6)De Boer, H. S.; Den Hartigh, J.; Ploegmakers, H.; Van Oort,W. J. Anal. Chim. Acta 1978,102,141-155. (7)De Boer, H. S.;Van Oort,W. J.; Zuman,P. Anal. Chim.Acta 1981, 130,111-132. (8)Bond, A. M.;Heritage, I. D.; Briggs, M. H. Anal. Chin.Acta 1981, 127,135-145. (9)Shihabi, Z.K.;Scaro, J.; Thomas, B. F. J. Chromatogr. 1981,224, 99-104. Heritage, I. D.; Briggs, M. H. Anal. Chim.Acta 1982, (10)Bond, A. M.; 138,35-45. (11)Bond, A. M.;Heritage, I. D.; Briggs, M. H. Anal. Chem. 1984,56, 1222-1226. (12)Gar@, S.;Szaoz, G. 'Analysis of Steroid Hormone Drugs"; Akademia: Kiado, Budapest, 1978. (13)Blunt, J. W.;Stothers, J. B. Org. Magn. Reson. 1977, 9 (8), 439-464 and references cited therein. (14)Damaskin, B.; Petril, 0.; Batrakov, V. "Adsorption of Organic Compounds on Electrodes";Plenum Press: New York-London, 1971;and references cited therein.
noted in passing on studies of individual steroids; however, no detailed studies on either the oxidation process or surface phenomena at mercury have been reported. The acetylenic and related steroids are ideal for systematic investigations due to the availability of a wide range of closely related compounds. Figure 1 gives the structural information on the compounds studied in this work. Important variations available include substituted terminal acetylenic protons by chloro groups (d), nonacetylenic analogues (e,g), stereoisomers (e,f), and functional variability (h). The steroids available should allow the faradaic (i.e., oxidation-reduction) and nonfaradaic (double-layer charging) components of the current to be rationalized. Experimental Section Reagents. Samples of pure steroids were obtained from Wyeth International, Schering E. G. Berlin, or Sigma Chemicals. Acetylenic steroid purity was confirmed by HPLC. Other reagents were of analytical quality. Instrumentation and Procedures. All polarographic procedures were performed with an E.G. and G. Princeton Applied Research Corporation (Princeton, NJ) PARC Model 174A Polarographic Analyser in conjunction with a standard three-electrode system. This system was comprised of a dropping mercury electrode (DME), flow rate 0.98 mg s-l, together with a Ag/AgCl (3 M KCl) reference e l d e and platinum wire counter electrode. Direct current (dc) experiments were performed with a 0.5-9 controlled drop time and using the sampled dc mode (sdc) with a scan rate of 5 mV s-'. Experiments at a hanging mercury drop electrode (HMDE) were conducted with a PARC Model 303 static mercury drop electrode (SMDE) with an electrode area of 0.16 cm2. Phase-selective alternating current (ac) polarographic experiments were performed by interfacing a PARC Model 129 lock-in amplifier to a PARC Model 174A via a PARC Model 174/50 interface. An external sine wave oscillator was used for both input and reference signals. Cyclic voltammetry (CV) at the hmde was accomplished by interfacing a PARC Model 175 Universal Programmer to the PARC Model 174 instrument. All polarographic and CV experiments were recorded on a Houston Omnigraph 2000 X/Y recorder. Controlled potential coulometry was conducted with PARC Model 173potentiostat with a PARC Model 179 digital coulometer interface. DC polarograms of electrolysis solutions were recorded in the coulometry vessel directly. Both reference electrode and platinum counter electrodes were individually isolated from the electrolysis solution by a reference bridge filled with supporting electrolyte. Electrocapillary measurements were made manually by averaging the drop time over five successive drop falls for each po-
112 Langmuir, Vol. 1, No. 1, 1985
Bond, Heritage, and Briggs
tential measurement. All electrochemical experiments were performed upon test solutions sparged with high-purity nitrogen. Mass spectral analysis was performed with a Finnigan Series 3200 mass spectrometer coupled to a Computer Automation Data System. Samples were introduced via a solid probe a t 70 eV. Test solutions for electrochemicalanalysis were methanol/water mixtures to provide sufficient solubility of the steroid hormones. Apparent pH measurements, “pH”,of these solutions were made at a glass pH electrode standardized in aqueous buffers.
Results and Discussion The electrochemistry at mercury electrodes of each of the steroids in Figure 1was examined with a wide range of techniques. Ethynylestradiol is described in greatest detail. The behavior of other compounds is described relative to the redox and surface activity of this compound. (A) Ethynylestradiol. Dc Polarography. Figure 2 shows a dc polarogram of ethynylestradiol at a dropping mercury electrode (DME). Two well-defined oxidation processes, I and 11, with equal limiting currents were observed for a 0.8 X lo4 M solution with Ellzvalues of -0.325 and -0.080 V, respectively (50% v/v methanol/water, 0.1 M Na2C03,“pH” 12.2). Furthermore, it is apparent that the presence of the steroid significantly modifies the background current (obtained in the absence of steroid). Clearly the electrochemistry is characterized by both faradaic and nonfaradaic processes. The origin of the functional group giving rise to the oxidation response was examined by recording dc polarograms of @-estradiol(Figure le). This steroid is structurally identical with ethynylestradiol but lacks the acetylene functional group. No faradaic response was observed in a wide range of solvent mixture or “pH” within the range 4-12. However, the nonfaradaic modification of the background current is observed as in the case of ethynylestradiol. Clearly the acetylenic group is not required for adsorption and the comparative electrochemistry of these two steroids allows the separate study of faradaic and nonfaradaic responses. The mercury electrode is implicated in the faradaic behavior since no oxidation response was observed at glassy carbon, gold, or platinum electrodes at potentials less than +1.0 V. Oxidation at very positive potentials on glassy carbon has been reported for such compounds and is associated with the phenolic A ring.g The above data indicate that the faradaic component of the electrode processes observed for ethynylestradiol at the DME are associated with its acetylene functional group and the mercury electrode. Results are consistent with mercury acetylide compound formation. Indeed the oxidation of mercury in the presence of acetylene itself results in a polarographic response reported by Geyer and Geibler.15 In that work the oxidation of mercury in the presence of acetylene dissolved in alkaline solution was proposed as in eq 1. HCXH
+ 2Hg + 20H-
-
Hg2Cz+ 2H20 + 2e-
(1)
In a related fashion, the least positive polarographic response of ethynylestradiol might be described by one of the following overall electrode reaction mechanisms where S denotes the bulk of the steroid structure:
+ Hg 3 SC=CHg + H+ + e+ Hg Hg(SCrC-)2 + 2H+ + 2e-
SC=CH 2SC=CH
i -
(2) (3)
~
(15)Geyer, R.; Geibler, L. 2. Chem. 1966,6,438-439. (16)Stricks, W.;Kolthoff, I. M. J. Am. Chem. SOC. 1952, 74, 4646-4653. (17)Stankovich, M. T.;Bard, A. J. J. Electroanal. Chem. 1977,75, 487-505.
1;
1
6
I
I
10
0
I
12
1
14
“PH”
Figure 3. Dependence of Ellz for response I (a) and I1 (b) on “pH”.
Analogous reaction schemes to (2) and (3) have been used to describe polarographic reactions of thiol- (SH) containing amino acids.16J7 Reaction mechanisms of this kind predict a dependence of the response upon the hydrogen ion concentration. (i) pH Dependence of E l l z and iL. Direct current polarograms of 0.8 X lo4 M solutions of ethynylestradiol in 50% v/v methanol-buffer mixtures were recorded between the “pH” limits 4.0 and 13.2. This concentration was chosen due to the comparatively well-defined polarographic waves observed. The half-wave potential, El12, and the limiting current, iL, of each response I and I1 (Figure 2) were measured and plotted as a function of “pH”. Due to poor resolution of I and I1 below “pH” 8.0 some values of Ell2 and iL are not presented. The Ellz dependence upon pH is shown in Figure 3. Both responses exhibit a linear dependence of EIl2upon “pH” between the range 6 and approximately 12. At higher “pH” values the slope of both plots decreases and presumably would ultimately become independent of “pH”. These results can be explained by consideration of the acidity function associated with proposed mechanisms (2) or (3). The initial step in either mechanism must be the deprotonation of the terminal acetylene, e.g., SCECH
& SC=C- + H+
(4) the conjugate base, SC=C-, being the reacting species. The K, for this reaction is approximately 10-12.18 If the rate of attainment of the equilibrium conditions expressed in eq 4 is sufficiently fast and the electron-transfer process is reversible, then the following expression relating the El12 to the pH of the solution can be derived,
RT [H’l” Ellz = Eo‘- - In nF K, where the value of K, is derived from eq 4 and E”’ = EoHg+lHg - R T / ( n F ) In K 1and K, is equal to the equilibrium constant of the following reaction SC=C-
K1 + Hg+ e HgCECS
(6)
for reaction scheme 2. A similar treatment can be applied t o reaction scheme 3. Equation 5 predicts that at pH values less than (pK, - 1)the slope of a E,,, vs. pH plot (18)Morrison, R.T.;Boyd, R. N. “Organic Chemistry”;3rd ed., Allyn and Bacon Inc.: Boston, 1976.
Langmuir, Vol. I , No. 1, 1985 113
Oxidation and Surface Processes for Steroid Hormones
b
as
, 0.4
I
I
0.2 -E ,Volts
0.3
0.1
ao
Figure 4. Current-sam led dc polarograms of (a) 0.4 X lo4, (b) M ethynylestradiol (c) 1.2 X 102,and (d) 1.6 X 0.8 x at “pH” 12.2. will have a value of -mRT/(nF) where m is equal to the number of protons accompanying the electron-transfer step. This theoretical treatment also predicts that, at pH values greater than (pK, + l), El will become pH independent. The slope of the plots /or waves I and I1 are 63 and 55 mV, respectively, for low pH values, suggesting that a one-electron one-proton transfer process is occurring (assuming that the electron-transfer process is reversible). If the latter assumption is incorrect, other combinations involving the electron-transfer coefficient are possible. As predicted, at pH values greater than pK, + 1 the El12 becomes pH independent (Figure 3). The limiting current iL for wave I appears to be independent of pH in the measurable range although the values of iL a t “pH” 50 ms. Under these conditions diffusion control and the same number of electrons for both processes is indicated and is in agreement with dc polarographic data. At concentration values less than 1.6 X lo4 M under the conditions described, the electrode process may be described by the Nernst and Ilkovic equations to give the expression (7)
for reaction scheme 2, where Eo” is equal to EoHg+H g R T / ( n F )In (Kl + K, - [H+])and id is equal to the diffu; sion-controlled limiting current. If eq 7 is valid a plot of -logl, (id- i ) / i vs. E should be linear with a slope of 58 mV/n at 20 OC. The logarithmic analysis of 0.8 X 1.6 X lo4, and 2.4 X lo4 M solutions of ethynylestradiol was undertaken for wave I. For the 0.8 X lo-“ M solution the plot is linear with a slope equal to 53 mV thereby adequately satisfying eq 7. At higher concentrations, the slope of the plot decreases particularly in potential regions positive of El12,as would be expected if adsorption rather than diffusion control becomes rate determining. This explanation would account for the concentration dependence of Ellz, iL,and nonlinear “log plot” at high concentration levels. In the limiting case the activity of the adsorbed product may approach unity and eq 7 would be reduced to the expression involving insoluble film formation, e.g., E = Eo’’ -RT/(nF) In (id- i ) (8) (iii) Cyclic Voltammetry. Cyclic voltammetry (CV) experiments at a hanging mercury drop electrode (HMDE) were conducted in similar solutions to that used for the dc polarographic study. The CV of a 1.6 X lo+ M solution of ethynylestradiol in 50% v/v methanollwater, 0.1 M buffer “pH” 12.2, is presented in Figure 6. An oxidation wave (a) and corresponding reduction wave (a’) are observed at -0.320 and -0.370 V, respectively, due to process I. Oxidative and reductive waves (b) and (b’) are observed
114 Langmuir, Vol. 1, No. 1, 1985
Bond, Heritage, and Briggs
red. ox.
T
0.5
r
r 4
0.3
0.2
0.1
Figure 7. C clic voltammograms of (1)0.8 X (2) 1.6 X (3) 2.4 x 10-K, and (4) 3.2 x M ethynylestradiol. Scan rate, 100 mV s-l; “pH” 12.2.
1 I
0.5
0.4
-E, Volts
-0.2rA
L
0.4
0.3
0.2
0.l
0.0
-E ,Volts
Figure 6. Cyclic voltammcgram of 1.6 X Scan rate, 100 mV s-l, ’pH” 12.2.
lod M ethynylestradiol.
for process I1 at approximately -0.100 V. The redox couple (a-a’) displays characteristic electrochemical stripping behavior. If the applied potential is scanned from -0.5 to -0.2 V and held at this potential for a period of time ( t ) then the peak height and the peak potential of (a’) on the reverse scan are proportional to the duration (t). Similar, although not identical, behavior is observed for the second redox couple (b-b’) when appropriate potentials and delay times are applied. If on the other hand the potential is scanned from -0.5 to -0.2 V and held for a similar period of time ( t )then scanned still further positive to -0.010 and reversed, the peak currents and potentials of the second redox couple (b-b’) are altered in a manner proportional to t. These observations can be interpreted as the product of oxidation process I being accumulated at the electrode surface, presumably by an adsorption process. The second oxidation process I1 is due to the oxidation of the product of I to form another adsorbed product. Both electrode processes I and I1 are electrochemically reversible at low scan rates. The shape of the second redox couple (b-b’) is indicative of a reversible process controlled by reactant and product adsorption. The sharp symmetrical peaks of (a’), (b), and (b’) clearly indicate the importance of adsorption. The scan-rate (V) dependence for the redox couple (a-a’) was examined for values up to 500 mV s-l. The peak current of a is proportional to the square root of the scan rate (W2)but for values >lo0 mV s-l the peak potential shifts toward more positive potentials. This latter result could be interpreted as being due to a quasi-reversible electron transfer or RC characteristics of the electrode process. The reverse-scan reduction peak current and peak potential of (a’) display similar behavior at faster scan rates (>lo0 mV 8-l) and lower concentrations (2.4 X lower concentrations but slower scan rates (C50 mV s-l), Le., at high surface coverages of product, a small sharp current spike is observed on oxidation wave a (Figure 7). The methanol concentration determines the exact nature of the behavior. Due to the observed scan rate and concentration dependence the spike is presumed to be related to the extent of the surface coverage by the product. Essentially identical observations have been made con-
Table I. Peak-to-Peak Separation of Cyclic Voltammograms (mV) for Process I of Ethynylestradiol as a Function of Scan Rate and “pH” in 50% v/v MethanoVWater scan rate, mV s-l “DH” 50 100 200 Fjon 13.2 12.2 10.0 8.0 6.0 4.0
30 30 35 60 150 150
55 50 50 75 170 160
a5 65 60 130 220 200
165 110 100
cerning “spikes” on the CV wave for the oxidation of cysteine, a thiol-containing amino acid at mercury.17 With cysteine the occurrence of the current spikes was attributed to the attainment of a tightly packed monolayer of adsorbed product allowing lateral interaction^.'^ The same explanation would appear to apply in the present case. The integrated reduction current of (a) at concentrations greater than 2.4 X M with a scan rate of 100 mV s-l limits to a value of 2.5 pC cm-2. Under these conditions the electrode surface is presumably saturated and monolayer coverage is attained. The limiting value of the integrated peak current also coincides with the appearance of the current spike on the oxidation response (a) providing confirmatory evidence that monolayer coverage has been achieved and is the origin of the spike. The lower value of the limiting integrated current for ethynylestradiol of 2.5 p C cm-2 compared to 80 pC cmw2for cysteine17 is anticipated because of its substantially larger size. The peak-to-peak separation for process I (E,(a) -E,(a’)) depends upon scan rate and concentration as well as pH (Table I). However, the trend of increasing peak-topeak separations with decreasing “pH” for similar scan rates and at the same concentration indicates that the process is becoming less reversible with lower “pH”. As the “pH” is lowered from 12.2 to 8.0 the second oxidation process is obscured by the normal oxidation of mercury limit as in the dc polarographic example. (iv) Coulometry. Controlled potential electrolysis (CPE) experiments were conducted upon solutions of ethynylestradiolin 50% v/v methanol, 0.1 M Na2C03“pH” 12.2, at a mercury pool electrode. The test solution was monitored by dc polarography. From these experiments an estimate of n, the number of electrons involved per molecule of ethynylestradiol, can be calculated by measuring the net charge transferred during the electrolysis with the aid of a coulometer. To a 0.8 x lo-‘ M solution of ethynylestradiola potential of -0.150 V was applied, corresponding to the limiting-
Langmuir, Vol. 1, No. 1, 1985 115
Oxidation and Surface Processes for Steroid Hormones
fortunately no other moleculer ion above 296 was observed indicating that either the compound is very unstable or the molecular ion is out of the mass range, Le., >700. Similar grey precipitates can be obtained by adding excess mercurous nitrate to a methanolic solution of ethynylestradiol or by adding water to a stoichiometric solution of each. This indicates that the compound is precipitating probably after diproportionation to form finely divided mercury in the precipitate. Similar precipitation experiments can be performed with mercuric salts and ethynylestradiol; however, in this instance once solubility is exceeded the precipitate is white without free mercury. The electrolysis results can be rationalized by equations such as 2Hg
0.6 A
os
OA 0.3 -E, Volts
a2
0.1
current region of oxidation process I. A net charge of 0.15 f 0.01 C was measured, which corresponds to a calculated n value of 1.0 f 0.15 from three separate determinations. The dc polarograms of the original preelectrolysis solution and the solution after electrolysis at -0.150 V are shown in Figure 8, parts a and b, respectively. The Ell2value of the reduced form is equal to -0.325 V, and the corresponding El12value for the oxidized form is equal to -0.320 V. The limiting currents, are, however, nqt exactly the same. The above results are clearly indicative of a oneelectron reversible process for oxidation reaction I. After exhaustive electrolysis of the test solution at -0.150 V, the potential was adjusted to -0.050 V and the electrolysis continued. A further 0.10 C was measured to give a total of 0.25 C. If, however, the original test solution was electrolyzed immediately a t -0.050 V without the intermediate step, then 0.28 C was measured giving n values of 2.0 f 0.2. The E l I z values for the reduction of the second process, 11, Figure &, compare favorably with the original solution oxidation response. These results also infer that the oxidation process I1 is reversible with 1 electron per molecule of ethynylestradiol transferred. The dc polarographic background current is significantly modified by each product of electrolysis, consistent with differing degrees of adsorption of the steroid and mercury complexes. Furthermore, this experiment proves that the mercury complexes are soluble in solution at these concentration levels. When the electrolysis of ethynylestradiol solutions of much higher concentrations was undertaken (i.e., 1 X M), the 'iR drop" was significant and the applied potential was not determinate. However, when a very positive potential was selected, before the oxidation limit of mercury, the electrolysis at the second oxidation limit could be performed with reasonable certainty. In these instances n values of 2 f 0.1 were realized. Concomitant with electrolysis of high concentration was the formation of a grey precipitate, which after washing and placing in a solid probe of a mass spectrometer yielded the parent ion of ethynylestradiol m l e 296 and a mercury cluster about mle 200 at probe temperatures greater than 100 OC. At lower temperatures the total ion spectrogram showed another compound coming off the probe with the characteristic isotope cluster about m l e 200 of mercury. This is indicative of free elemental mercury in the precipitate. Un-
2HgS
+ 2H+ + 2e
3Hg
(94
2HgS + HgzSz
(9b)
+
(9c)
HgzS2 Hg + Hg3Szz++ 2e
QO
Figure 8. Current-sampled dc polarograms of (a) preelectrolyzed solution, (b) electrolyzed solution at -0.15 V, and (c) electrolyzed solution at -0.05 V, "pH" 12.2.
+ 2SH
+ 2SH
-
Hg3Sz2++ 2H+ + 4e
(10)
The reactions Hg3S?+ + Hg
+ Hg2++ HgS2
and HgzS2 + Hg
+ HgS
(11)
presumably are also important. At higher concentrations, precipitation of HgS,, Hg2Sz,or basic mercury salt will influence the reactions. Chemical reactions producing similar products may occur as follows:
-
+ 2SH Hg + HgSz + 2H+ Hg2++ 2SH HgS2 + 2H+
Hg?+
-
(12) (13)
After simplification of equations by elimination of the proton and elemental mercury term, but incorporation of surface reactions, the equations appear to be best represented by the scheme S' K
S e Sad,
11.'
e SIodr t
e
(14)
11 11.'
S'bdr
t e
S"
The surface phenomena will be considered subsequently. (B) Norgestrel. The dc polarographic behavior of norgestrel (Figure IC)and the other acetylenic steroids not containing the phenolic group differs from that of ethynylestradiol significantly, particularly with respect to reversibility of the faradaic response. Only one well-defined oxidation response (I) was observed for norgestrel in alkaline methanolic solution and at a potential significantly different than for ethynylestradiol,-0.175 V (Figure 9, scan 1). However, a t higher concentrations (>2.4 X M), there was a small peak, suggesting that another process occurred at approximately Ell2 = -0.050 V, although it was almost obscured by the normal mercury oxidation limit. As with ethynylestradiol, norgestrel structural analogues without the acetylene functional group do not give an electrochemical response at any solid electrode. As before
Bond, Heritage, and Briggs
116 Langmuir, Vol. 1, No. 1, 1985
T
-02pA
-0.1PA
1
a
1
I
0.6
0.6
0.4
a3
02
0.1
- E , Volt8
Figure 11. Cyclic voltammogram of 1.6 X rate, 100 mV s-l; “pH” 12.2. X
lod M norgestrel. Scan
lo4 and 3.2 X lo4 M. Some curves are shown in Figure
9. I 0.5
1
0.4
0.3 0.2 -E, Volts
0.1
0.0
Figure 9. Current-aam led dc polarograma of (1) 0.8 X lo4, (2) 1.6 X lo4, (3) 2.4 X 10-Pand (4) 3.2 X lo4 M norgestrel at “pH” 12.2.
I
-a5
1
I
-0.4
-0.3
-0.2 -0.1 E,Vdtr
01)
+01
*42
Figure 10. Current-sampled polarograms of (1)0.8 X lo4, (2) 1.6 x lP,(3) 2.4 X lP,and (4) 3.2 X 10-4M norgestrel in solution, “pH” 4.0. these results infer that the oxidative response is intimately associated with the acetylene functional group of the steroids in question. A significant structural analogue of norgestrel, chloroethynylnorgestrel (Figure Id), which has a chlorine atom substituted in place of the terminal hydrogen of the acetylene group, was also investigated for oxidative polarographic behavior. The lack of an oxidation response in a wide range of solvent mixtures for this compound gives additional support to the mechanisms proposed in eq 2 and 3 of mercury acetylide formation. (i) pH Dependence of E l and i p The El and i~ values from a 0.8 X M soiution of norgestrei in 50% v/v methanol/H20, 0.1 M buffer, were measured as a function of “pH” in the range 4.0-13.2. This concentration was chosen due to the comparatively well-defined polarographic waves obtained a t this level. The Ell2 results showed that at “pH” values in the range 10-13 the plot was linear and obeyed the dependence predicted by eq 5 with a slope of 60 mV/“pH” unit. However, in this instance no pH-independent region was observed possibly due to the pK, for norgestrel being larger than for ethynylestradiol. Below “pH” 10 surface phenomena became dominant culminating at “pH” 4 (Figure 10) where no faradaic process was observed, only depression of the background for subsequent additions of norgestrel. Mixed faradaic and nonfaradaic terms occurred a t intermediate pH. (ii) ConcentrationDependence of Ellaand iL. Direct current polarograms were recorded for norgestrel in 50 % v/v methanol/H,O, 0.1 M Na2C03“pH” 12.2, between 0.4
The value of i L is shown to be linearly dependent upon concentration up until approximately 0.8 X lo4 M where pronounced curvature began, reaching a limiting value at approximately 3.2 X 10“ M under the conditions of the experimental procedure. These results are similar to those obtained for ethynylestradiol as the limited surface area of the mercury electrode can only allow a finite surface coverage and current produced as a result. The current per unit concentration is also similar to that of ethynylestradiol suggesting the same number of electrons transferred per molecule of steroid. At higher concentrations, >1.6 X lo4 M, a maximum was observed at approximately -0,050 V, which may correspond to the unresolved second oxidation process that was observed for ethynylestradiol (Figure 9). The ElI2data as a function of concentration show that the El12values become more positive with increased concentration up until approximately 3.2 X M when it, as does the limiting current, remains constant. This is in direct contrast to the case of ethynylestradiol. The shift in El12in the lower concentration region is indicative of reactant adsorption. Accurate wave-shape analysis is impossible. An interpretation similar to the case of ethynylestradiol of a one-electron, one-proton oxidation of mercury to form a mercury steroid compound (eq 2 and 3) can be drawn from these results. However, particularly at low pH, the overall rate of the reaction is significantly slower than for ethynylestradiol and is presumably dominated by surface phenomena. (iii) Cyclic Voltammetry. Cyclic voltammetry experiments were conducted upon the same solutions used in the dc study at a HMDE. The CV of 1.6 X M norgestrel in 50% v/v methanol/H20, 0.1 M Na2C03”pH” 12.2, is presented in Figure 11. An oxidation wave (a) and corresponding reduction wave (a’) were observed at -0.125 and -0.525 V, respectively. The redox couple (a-a’) displayed characteristic “stripping” behavior as was the case for ethynylestradiol. When the potential was scanned from -0.6 to -0.1 V and held at this potential for a period of time t, then the reverse peak current i, (a’) increased in magnitude, and peak position E, (a’) was shifted in a manner proportional to (t).No second response was discernible from the oxidation limit of mercury. However, in contrast to ethynylestradiol of approximately -400 the peak-to-peak separation (a,) mV was very large, indicating that the electrode process was not reversible. The overall shape of the CV waves was similar to those described by Laviron20for the irreversible electron transfer of strongly adsorbed product and reactant (20) Laviron, E. In “Electroanalytical Chemistry“; Bard, A. J.; Ed.; Marcel Dekker: New York, 1982; Vol. 12.
Langmuir, Vol. I, No. 1, 1985 117
Oxidation and Surface Processes for Steroid Hormones
1 0.0 -0.3
-0.2
-0.1
0.0 E , Volts
+0.1
a8
0.4
1.2
-E, V d t ~
~~~
+0.2
Figure 12. Cyclic voltammograms of 1.6 X loa M norgestrel in “pH” 4 solution at (1)50, (2) 100, and (3) 200 mV s-l. in CV. The peak potential of the oxidative response E,(a) is a linear function of scan rate, which also indicates that the process is not reversible. Similar results are obtained for the other non-phenolic acetylenic steroids mestranol, (chloroethynyl)norgestrel, and lynestrenol. The AE, value for norgestrel increased as the “pH” of the solution decreases from -400 mV at “pH” 13.2 to -560 mV at “pH” 10.0. In solutions below “pH” 10 the “stripping” peak E,(a’) became much smaller in size than for comparable experiments at higher ,pH”. As the “pH” was lowered further to values less than 6.0 the stripping peak disappeared to be replaced by another peak E,(a”) at potentials very similar to the forward or oxidative scan peak E,(a). The resulting highly symmetrical response (Figure 12) did not display any of the characteristic stripping features of the former couple (a-a’) (Figure 11). Both peaks at this “pH” increased with concentration until M under the experimental a limiting value, at 4.8 X conditions of (Figure 12), was reached. The scan-rate dependence of both i,(a) and i,(a”) was indicative of adsorption processes as both peaks are linearly dependent upon the scan rate between 50 and 200 mV s-l (Figure 12, scans 1-3). These results are consistent with the dc results and with the interpretation that in low-“pH”solutions the electrochemistry is essentially nonfaradaic. Similar results with respect to the nonfaradaic component were obtained for the nonterminal acetylene steroid hormone analogue of norgestrel, (chloroethynyl)norgestrel, and the steroid methyltestosterone. In both instances the symmetrical peaks were only observed in low-pH solutions. The peaks were proportional to scan rate and the peak potentials were almost identical with those of norgestrel at low “pH”. The results reinforce the idea that in low “pH” norgestrel and all other non-phenolic acetylenic steroid hormones produce nonfaradaic peaks. (iv) Coulometry. Controlled potential electrolysis experiments were conducted upon solutions of norgestrel in 50% v/v methanol/H20, 0.1 M Na2C03“pH” 12.2, at a mercury pool electrode. The test solution was monitored as before by dc polarography. A 0.8 X M solution of ethynylestradiol was electrolyzed at a potential of -0.050 V corresponding to the limiting current region of the oxidation process of norgestrel (Figure 9). A net charge of 0.14 f 0.02 C was measured, which corresponds to a calculated n value of 0.9 f 0.15 from three separate determinations. The EIlzvalue of the dc polarogram before electrolysis due to oxidation norgestrel was equal to -0.175 V, as before. After electrolysis a reduction dc wave was observed at -0.500 V,
Figure 13. Electrocapillary curves at “pH” 12.2 of (m) background, @) ethynylestradiol,).( p-estradiol, (A)mestranol, and ( 6 ) norgestrel. which presumably corresponded to the reduction of the mercury-steroid compound formed by the oxidation process. The large difference in Ellz values for reduction and oxidation that the electrode process described by either eq 2 or 3 for norgestrel, and indeed all non-phenolic acetylenic steroids, is irreversible. In contrast, El values before and after controlled potential electro ysis are identical within experimental error for ethynylestradiol. After controlled potential electrolysis of the test solution, significant turbidity was observed similar to the highconcentration CPE of ethynylestradiol. In a similar manner, methanolic solutions of norgestrel when subjected to mercury(1) or mercury(II) solution produced precipitates that obviously contained elemental mercury for the Hg(1) solution and a creamy white precipitate for the Hg(II) case. Reactions below would be consistent with the data.
i
Hg
+ SH 2HgS Hg&
HgS
+ H+ + e
(154
Hg2S2
(15b)
HgS2 + Hg
(154
Chemical reactions with mercury(1) and mercury(I1) salts were as for ethynylestradiol. (C) Adsorptive Behavior of Steroid Hormones. The preceding experimental results leave little doubt that adsorption processes play a significant role in the faradaic and nonfaradaic electrochemistry of acetylenic steroid hormones. As a consequence of this, the adsorption of those and other steroid hormones were studied by various methods in an attempt to elucidate the fundamental differences observed between the electrochemistry of phenolic and non-phenolic acetylenic steroid hormones. (i) Electrocapillary Studies. Electrocapillary measurements were made of various steroid containing solutions in 50% v/v methanol/water, 0.1 M buffer “pH” 12.2 and “pH” 8.0, over the potential range -1.2-0.05 V (Figures 13 and 14). The results are presented for the phenolic steroid hormones ethynylestradiol, @-estradiol,and the non-phenolic steroid hormones mestranol and norgestrel. Both ethynylestradiol and @-estradioldecreased the surface tension (drop time) of the DME compared to the background in a relatively specific potential region from approximately -0.6 to 0.00 V in buffered methanol solutions of “pH” 12.2. These results indicate that both of these steroid hormones adsorb onto the DME in potential regions near to and positive of the point of net zero charge, the electrocapillary maximum (ecm). Of specific interest here is that even though 0-estradiol does not have the acetylenic group its adsorptive behavior was very similar
118 Langmuir, Vol. 1, No. 1, 1985
Bond, Heritage, and Briggs
:-=----I
I -Q2rA
B ‘
Q
o
OIO
0.8
C
?
6
12
-E, Vat6
Figure 14. Electrocapillary curves at ”pH”8 of (m) background, ( 0 )ethynylestradiol, (A)@-estradiol, and (+) norgestrel.
YY
i
-1,OpA
I
i il
..4
IC b
6
1.2
1.0
0.8 0.8 - E , Volts
0.4
0.2
0.0
Figure 16. Cyclic voltammograms of lo4 M (A) @-estradioland (B)a-estradiol. Scan Rate,100 mV s-l; (- - -) background; “pH” 12.2.
I
1.0
a8
0.6
0.4
a2
-E, V a s
Figure 15. Ac polarogram of (a)ethynylestradiol, (b) norgestrel, (c) @-estradiol,and (d) mestranol. Appliced AC signal 10 mV p p at 196 Hz. to that of ethynylestradiol. For the non-phenolic steroid hormones norgestrel and mestranol the drop time was decreased even further via adsorption and they adsorb over a much wider potential range particularly in negative regions of the ecm (Figure 13). That is, these two compounds adsorbed onto the DME more strongly than did either of the two phenolic steroids, especially negative of the ecm. At lower “pH” values, the adsorption of the phenolic steroids ethynylestradiol and @-estradiolwas less distinguishable from the non-phenolic group than at “pH” 12.2. Both steroids adsorbed over a larger negative region to approximately -1.0 V and increased the strength of adsorption around the ecm. The “pH” dependence of adsorption of the phenolic steroids coincided with formation of the protonated form of the phenolic A ring. At high “pH”, 12.2, the phenol was essentially in its anionic form (pK, 10) and there was strong specific adsorption relative to non-phenolic steroids. At ‘pH” lo, the non-phenolic acetylenic steroids do not exhibit specific electrode adsorption behavior and are much more strongly adsorbed than the corresponding phenolic steroids. The preferred orientation and strong adsorption of this class of steroid is believed to induce the observed irreversible electrode reaction behavior. The orientation of adsorbed intermediates is known to exert profound effects on electrochemical pro~esses.~~ Registry No. Ethynylestradiol, 57-63-6;mestranol, 72-33-3; norgestrel, 6533-00-2;chloronorgestrel, 14115-33-4;p-estradiol, 50-28-2;a-estradiol,57-91-0;testosterone,58-22-0; lynestranol, 52-76-6. (21) Soriaga, M. P.; Hubbard, A. T. J . Am. Chem. SOC.1982, 104, 2735-2742. (22) Soriaga, M. P.; Hubbard A. T. J. Am. Chem. SOC.1982, 104, 2742-2147. (23) Soriaga, M. P.; Hubbard, A. T. J. Am. Chem. SOC.1982, 104, 3937-3943. (24) Soriaga, M. P.; Stickney,J. L.; Hubbard, A. T. J. Mol. Catal. 1983, 21,211-221.
Crystal Growth of Calcium Phosphates in the Presence of Magnesium Ions M. H. Salimi, J. C. Heughebaert,*t and G . H. Nancollas Chemistry Department, State Uniuersity of New York at Buffalo, Buffalo, New York 14214 Receiued August 28, 1984 The influence of magnesium ions upon the crystallization rates of dicalcium phosphate dihydrate and octacalcium phosphate has been investigated at constant supersaturation. While having no detectable effect on the growth of dicalcium phosphate dihydrate, magnesium ions appreciably retard the rate of octacalcium phosphate growth, probably by adsorption at active growth sites. The mineralization of the thermodynamically most stable hydroxyapatite is much more strongly inhibited by the presence of these added ions, which may therefore mediate in the precipitation process by selectively stabilizing the more acidic precursor phases. Introduction The precipitation of calcium phosphates is important both in biological mineralization and in natural water systems.’S2 Traces of magnesium ion have been shown to reduce the overall rate of seeded calcium phosphate crystallization3 and markedly delay the transformation of amorphous calcium phosphates to more stable apatitic phase^.^ In natural water systems, magnesium ion conmol L-1,2while in centrations may be as high as 5 X biological calcification magnesium concentrations ranging from 0.5% in outer tooth enamel layers to 2% in the innermost dentines is likely to have important consequences on the rates of remineralization. Numerous studies have
been made of the influence of magnesium ions on calcium phosphate formati~n.~?’ The results of spontaneous precipitation experiments have suggested that magnesium ions kinetically hinder the nucleation and subsequent growth of hydroxyapatite (Ca5(P04)30H,hereafter HAP) by competing for lattice sites with the chemically similar but
On leave from Institut National Polytechnique de Toulouse,
621. (7) Ferguson, J.; McCarty, P. L. Enuiron. Sci. Technol. 1971,5, 534.
31400 Toulouse, France.
(1)‘Fological Mineralization and Demineralization”;Nancollas, G. H., Ed., Springer-Verlag: Heidelberg, 1982. (2) S t u “ , W.; Morgan, J. J. “Aquatic Chemistry”;Wiley Interscience: New York, 1981. (3) Nancollas, G. H.; Tomazic, B.; Tomson, M. B. Croat. Chim. Acta 1976, 48,431. (4) Eanes, E. D.; Ratner, S. L. J. Dental Res. 1981, 60, 1719. (5) Shaw, J. H.; Yen, P. K. J. J. Dental Res. 1972, 51, 95. (6) Martens, C. S.: Harriss, R. C. Geochim. Cosmochim. Acta 1970,34,
0743-7463/85/2401-0119$01.50/0 0 1985 American Chemical Society
