Polarographic behavior of benzylpenicillenic acid - American

50,NO. 13, NOVEMBER 1978 ·. 1917 these fractions are obtained, they could be separated further by normal-phase chromatography on. µ-Bondapak-CN...
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978

these fractions are obtained, they could be separated further by normal-phase chromatography on p-Bondapak-CN (chromatographic system 1) or p-Bondapak-NH, (chromatographic system 2). At this stage, phenolic compounds with more than one aromatic ring could be separated from phenol and alkylphenols. Fractions could then be collected, and injected onto a reversed-phase column such as p-Bondapak C18 (chromatographic system 4). The separation on chromatographic system 4 is mainly by aliphatic carbon number, assuming the same number of aromatic double bonds for the compounds to be separated. By using the correlation factors in these latter two separation steps and information as to the number of ortho substituents obtained from the first separation step, several possible structures can be assigned to an isolated phenolic compound. By combining chromatographic systems, a t least a partial separation of several isomeric phenols with the same number of ortho substituents and aliphatic carbon number should be possible. The concepts developed in this work are presently being used for the separation of phenols from coal liquefaction solvents.

LITERATURE CITED (1) (2) (3) (4) (5) (6) (7)

Mobil, R . & D Corp., EPRI Project RP 410-1 (1976). R. Amos, Talanta, 20, 1231 (1973). V. Das Gupta, J . Pharm. Sci., 65, 1706 (1976). A. F. Cunningham and D. E. Hillman, J . Chromatogr., 148, 528 (1978). A. N. Crabtree and A. E. J. McGill, Mikrochim. Acta, 1, 85 (1967). J . J. Kirkland, J . Chromatogr. Sci., 9, 206 (1971). B. L. Karger, K. Conroe, and H. Engelhardt, J. Chromatogr. Sci., 8, 242 (1970).

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(6) K. Karch, I.Sebestian, I. Halisz, and H. Engelhardt, J . Chromatogr.,

122, 171 (1976). (9) A. W. Wolkoff and R. H. Larose, J . Chromatogr., 99, 731 (1974). (10) S. Husain, P. Kunzelmann, and H. Schildknecht, J . Chromatogr., 137, 53 (1977). (11) J. C. Kraak and J. F. K. Huber, J . Chromatogr., 102. 333 (1974). (12) C. P. Terweij-Groen and J. C. Kraak, J . Chromatogr., 138, 245 (1977). (13) K. Callmer, L.-E. Edholm, and 8. E. F. Smith, J . Chromatogr., 138, 45, (1977). (14) L. S. Bark and J. T. Graham, J . Chromatogr., 23, 120 (1966). (15) L. S. Bark and J. T. Graham, J . Chromatogr., 23, 417 (1966). (16) L. S. Bark and J. T. Graham, J . Chromatogr., 27, 116 (1967). (17) L. S. Bark and J. T. Graham, Manta, 13, 1281 (1966). (18) R. V. Vivilecchia, R. L. Cotter, R. J. Limpert, N. Z.Thimot, and J. N. Little, J . Chromatogr., 99, 407 (1974). (19) P. K. C. Tseng and L. B. Rogers, 29th Pittsburgh Conference Abstr. on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, Feb. 27-Mar. 3, 1978, No. 197. (20) L. R. Snyder, "Principles of Adsorption Chromatography". Marcel Dekker, New York, N.Y., 1966. (21) J. March, "Advanced Organic Chemistry: Reactions, Mechanisms, and Structure", 2nd ed., McGraw-Hill Book Company, New York, N.Y., 1977. (22) H. W. Kohlschutter and K. Unger, "Thin Layer Chromatography, A Laboratory Handbook", E. Stahl, Ed. Springer-Verlag, New York, N.Y., 1969. (23) R. T. Morrison, and R. N. Boyd, "Organic chemistry", 2nd ed.,Allyn and Bacon, Boston, Mass., 1966. (24) R. S. Drago and B. B. Wayland, J . Am. Chem. Soc., 87, 3571 (1965). (25) J. F. Schabron, R . J. Hurtubise, and H. F. Silver, Anal. Chem., 49 2253 (1977). (26) R. P. W. Scott and P. Kucera, J . Chromatogr., 142, 213 (1977). (27) F. Dietz. J. Traud, P. Koppe. and Ch. Rubelt, Chromatographia, 9, 380 (1976).

RECEIVED for review July 27, 1978. Accepted September 5 , 1978. Financial support was provided by Department of Energy, Washington, D.C., Contract No. EX-76-S-01-2367.

Polarographic Behavior of Benzylpenicillenic Acid Mohammed Jemal' and Adelbert M. Knevel" Deparlment of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907

The direct current and differential pulse polarographic behavior of benzylpenicillenic acid based on the sulfhydryl group has been investigated in 0.056 M citrate buffer of pH 6.45. Benzylpenicillenic acid gives a single wave at low concentrations and two waves at high concentrations. The single wave is adsorption-controlled and the double wave is diffusion-controlled. Triton X-100 shifts the first wave to a more positive value. It also increases the peak current and the increase varies from 2-fold to 14-fold, depending on the concentration of benzylpenicillenic acid. The i d / C value is independent of concentration and presence of Triton X-100 whereas the iJCvalue changes with concentration and in the presence of Triton X-100. Constant i,/C is obtained for M in the presence of Triton concentrations below 1.0 X X-100 at 0.004% and above. The iJCvalue calculated from the idCvalue was higher or lower than the experimental iJC value depending on the absence or presence of Triton X-100, respectively.

Benzylpencillenic acid (BPA) has bepn reported to be the prominent acylating agent in the formation of antigenic determinants in penicillin allergy (1). Since BPA is a deg'Present address, Squibb Institute for Medical Research, New Brunswick, N.J. 08903. 0003-2700/78/0350-1917$01.00/0

radation product produced in parenteral solutions of benzylpenicillin (BPN) under normal conditions of storage ( I ) , parenterally administered solutions (of BPN serve as a potential source of BPA for pencillin allergy in the body. Analysis of BPA is based on its UV absorbance a t 322 nm (2, 3). The chief drawback to this method of analysis is that other substances related to BPA also absorb a t this wavelength. Depending upon the absence or presence of oxygen and concentrations of buffer, BPA may be converted to such products as benzylpenicillenic acid disulfide (and possibly sulfinic and sulfonic acids) and benzylpenicilloylpenicillenic acid ( 3 ) . These substances all absorb radiant energy at 322 nm and, hence, interfere with the UV analytical method for BPA. We have shown in a previous study ( 4 ) that differential pulse (DP) polarography is capable of resolving the sulfhydryl containing compounds, benzylpenicillenic acid and penicillamine, known degradation products (of BPN. Hence it was decided to study the polarographic behavior of BPA based on the electrochemical reactivity of the :sulfhydryl group. With the exception of one reference ( 5 ) which attributes the presence of catalytic waves in Brdicka solutions containing BPN to the sulfhydryl group of' WPA formed as a degradation product of BPN, no study has been reported in the literature on the polarographic behavior of BPA on this functional group. This paper describes the systematic study of the DP polarographic behavior of BPA in 0.056 M citrate buffer at pH 6.45 based on the electrochemical reactivity of the sulfhydryl C 1978 American Chemical Society

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group. Direct current (dc) polarographic studies were conducted in a n attempt to explain the interesting effects observed when various surfactants were added to solutions of BPA. The results reported in this paper serve as a basis for the assay of BPA in the presence of BPN, its degradation products, and derivatives of BPA which absorb a t 322 nm. EXPERIMENTAL Apparatus. A Polarographic Analyzer Model 174A in conjunction with a Model 174/70 Drop Timer was used (Princeton Applied Research Corporation, Princeton, N.J.) along with the PAR Model 9300 and 9301 top and bottom polarographic cell. A three-electrode system was employed with platinum wire serving as the counter (auxiliary) electrode, calomel electrode model 3-712 (Coleman Instruments, Oak Brook, Ill.) as the reference electrode and dropping mercury as the working electrode. The capillary had the following characteristics. In 0.056 M citrate buffer of pH 6.45 (with 5% ethanol) at open circuit, the mercury flow rate was 2.57 mg/s and the natural drop time was 3.0 s at hUnCOrI of 78.6 cm. Except when the effect of mercury pressure on wave height was studied, h,,, at 78.6 cm was used. Unless otherwise indicated, a drop time of 2 s and a pulse amplitude of 25 mV were employed in the DP mode and a natural drop time was used in the dc mode. The scan rate was 2 mV for both DP and dc studies. Materials. BPA was obtained commercially from Sigma Chemical Company, St. Louis, Mo. Since this substance is unstable even in solid form, it was necessary to estimate its purity using the technique as reported (6). Scintillation grade Triton X-100 was obtained from Eastman Kodak Co. Benzylpenicilloic acid was prepared from BPN according to a literature procedure ( 7 ) . Other chemicals were obtained commercially and used as purchased. Selection of Buffer. A 0.056 M citrate buffer of pH 6.45 (containing 14.03 mg of trisodium citrate and 0.47 mg of citric acid per mL) was used in this study because it is one of the buffers in which Benzylpenicillin U.S.P. is stored. Also this system was selected because it provides maximum stability of both BPN and BPA in solution (8, 9). The buffer concentration was found to be dilute enough not to cause the dimerization of BPA t o such products as benzylpenicilloylpenicillenic acid ( 3 ) . Procedure. All experiments were carried out at 22 "C h 1. Since BPA is unstable in aqueous media ( 3 , 8 ) ,all stock solutions of this compound were prepared in absolute ethanol. Polarographic solutions were prepared as follows. A volume of absolute ethanol which gave a final concentration of 5% upon dilution, was introduced into a 10-mL volumetric flask. A pre-determined amount of BPA stock solution was then added followed by the buffer (containing a surfactant when required) to bring the volume to the 10-mL mark. The solution was then shaken and immediately transferred to the polarographic cell, where pre-purified nitrogen was bubbled through it. When bubbling was started, about 100 s had elapsed since the time mixing began. Bubbling was continued for about 200 s and then the nitrogen flow was directed so it formed a blanket over the solution. Recording was started when the solution was exactly 400 s old. The initial potential for all recordings was 4 . 8 V and the potential scan rate was 2 mV/s. It was essential to maintain uniform timing, and initial potential and scan rate settings so that the age of all analytical solutions at their peak potential (for DP polarography) or a t their plateau (for dc polarography) was the same. In this way the relation between concentration and polarographic output could be compared. The concentrations of BPA reported here are for time zero, unless otherwise stated. When desired, the ratio between initial concentration and the concentration at a given time was determined using UV absorbance. The absorbance at time zero for an aqueous solution was taken to be equal to the absorbance in absolute ethanol.

RESULTS A N D D I S C U S S I O N To determine if the concentration of BPA affected the rate of degradation, absorbance-vs.-time scans of different concentrations were run for a period of over 1h in pH 6.45 citrate buffer. The ratio of the absorbance a t t = 0 and t = x after solution preparation was found to be independent of con-

Table I. Wave Height vs. Concentration of Benzylpenicillenic Acid Giving Single and Double Waves no. of wave height,a concn, M waves WA 0.96 x 10.~ 1 0.023 1.91 x l o - 5 1 0.048 0.098 3.82 x 10-5 1 0.150 5.73 x 10-j 1 7.64 x 10-5 0.200 2 9.55 x 0.254 2 0.346 13.37 x 10-j 2 17.19 x lo-' 0.438 2 19.1 x 10-5 2 0.486 63.0 X lo-' 2 1.62 126 X 2.85 2 For concentrations giving two waves, wave height is the total wave height as measured from the residual current to the plateau of the second wave. centration. Hence, the relationship between the initial concentration of BPA in the polarographic solution and the concentration at the time the polarographic signal is measured remained linear. Direct Current Polarographic Behavior. Concentration Effects. BPA exhibited a well-defined wave with a half-wave potential of -0.54 V a t low concentrations. This wave height increased up to a concentration of 7.0 X lV5M. At this point, a second wave with a half-wave potential of 4 . 3 4 V began to appear. When the concentration was further increased, the height of the second wave steadily increased and the height of the first wave remained practically the same. Table I lists concentrations of BPA giving a single wave and double wave along with respective wave heights. As can be seen from Table I, there was a direct relationship between concentration and wave height. Diffusion and Adsorption Control. To determine whether the wave heights are limited by diffusion, adsorption, kinetic, or catalytic phenomena, the effect of mercury pressure on the heights of the first and double wave was studied. First, a concentration of BPA (3.90 X M) known to give only one wave, was investigated at different pressures of mercury. The wave height varied in direct proportion to hii,', indicating that the limiting current of the single wave was diffusion controlled. A concentration of BPA (1.22 X 10 M) known to give two waves a t all pressures of mercury investigated, was then studied. The limiting current of the first wave exhibited the behavior of an adsorption controlled wave as shown by the direct proportion relationship of the wave height to h,,,. On the other hand, the limiting height of the double wave was found to vary in direct proportion to h&(:,indicating that the total limiting current was diffusion controlled. M BPA solution gave two waves a t h,,,, of A 7.13 X 31.85 cm but only one wave a t h,,,, of 91.75 cm confirming the fact that the height of the first wave of a double wave increases a t a larger rate than the total wave when mercury pressure is increased. Diffusion Coefficient. The diffusion coefficient was calculated using the Ilkovic equation. A solution concentration which gave a double wave was employed and the total wave height was measured at -0.2 V. Because of degradation of BPA during the polarographic procedure, the concentration used in the Ilkovic equation was 82.7% of the concentration at t = 0. The diffusion coefficient determined in this manner was found to be 5.32 X 10F cm2/s. Electrocapillarj Curups. Electrocapillary curves in 0.056 hl citrate buffer of p H 6.45 with 5% ethanol, the buffer with 5% ethanol and 0.00870 Triton X-100, and the buffer with 5% ethanol and 1.50 X M BPA are shown in Figure 1. A comparison of curves a and c shows that the 1.50 X LO M

ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978

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BPA solution markedly decreases the drop times indicating that the interfacial tension between the mercury drop and the solution has been lowered. The decrease of drop times is seen in the potential range between 4 . 7 \' and +0.2 V. The course of drop time depression indicates that there is adsorption (or film formation) on the electrode surface starting at the potential where BPA does not undergo electrode reaction and t h a t the adsorption continues in the potential region where all of the BPA has been converted to its product (note that M BPA solution gives a double wave which starts a 1.50 X a t -0.6 V and reaches its second plateau at -0.2 V). Curve b shows that Triton X-100 in the medium is adsorbed over a long range of potential including the region where BPA or its product is adsorbed. Log Plot Analysis. Direct current polarograms exhibiting only single waves were analyzed. The log plots for a 6.50 x M solution are shown in Figure 2. The results show that the electrode reaction is of the type RSH + Hg + RSHg H+ + e ( 1 0 , I I ) . The slope of plot a is 0.05 V, which is smaller than the theoretically expected value of 0.059 V at 22 "C. The smaller slope value is probably due to complications of adsorption. The reliability of plot a is evidenced by the fact that the half-wave potential obtained from the plot is equal t o that obtained from the polarogram. Another indication of the reliability of the plot is seen when comparing the slope of the plot with the value of E3,4 - E114 obtained from the polarogram. The measured value of E3,4 - E114 was 0.046 V, as compared t o its calculated value of 0.048 V. E f f e c t s o f Surfactants. First, the effect of different concentrations of Triton X-100 (0,002-0.020%) on a given concentration of BPA in citrate buffer of pH 6.45 was studied. For a concentration of BPA which gave a single wave, the

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half-wave potential was shifted to a more positive value by the presence of Triton X-100. As the concentration of Triton X-100 was increased, the half-wave potential continued to shift accordingly. At a concentration of O.008%, the half-wave potential reached a value of -0.39 V where it remained constant even though the Triton X-100 concentrations were further increased. On the other hand, the wave height was not affected. For a concentration which gave two waves, the half-wave potential of the second wave was not shifted at all, whereas the first wave was shifted in the manner of the single wave described above. Thus, as the Triton X-100 concentration was increased, the two waves came closer together until a t sufficiently high concentrations the two waves merged for all practical purposes. The effects of gelatin and camphor were also studied. Gelatin generally exhibited an effect similar to that of Triton X-100. However, none of the concentrations of gelatin investigated (0.0054.050%~moved the wave to the same extent as that of Triton X-100. Whereas gelatin a t concentrations of 0.025% and above caused a maximum shift of 0.08 V in the wave of BPA solution (6.39 X M), a saturated solution of camphor caused a shift of only O.Of! V. The height of the wave was not affected in either case. Differential Pulse Polarographic Behavior. Concentration Effects. As in the case of dc polarography, low concentrations of BPA exhibited a single peak whereas high concentrations showed two peaks. Once the second peak started to appear, the first peak remained at practically the same height. The peak potential of the first peak changed only slightly with change in concentration (e.g., 4.51 V at 2.54 X lo4 M). Until the appearance of the second peak, the height of the first peak increased with concentration. .4s shown in Figure 3, the relationship between concentration and peak height did not show a proportionality. The ratio of i,/C continued to increase with concentration up to the point where the second wave was about to appear. It was believed that some of the impurities found in the commercial products of BPA used in this study caused the variation of i,/C with concentration. One such impurity, benzylpenicilloic acid (BPO),is known not to give a DP peak in the potential region of BPA; but it was thought that it might affect the height of the BPA peak indirectly. When mixtures of BPA and BPO of different proportions were investigated, there was little or no change in the heights of the BPA peaks due to presence of BPO. Hence, BPO did not cause the

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variation of the ratio i p / C . Another substance in the BPA product that was suspected as causing variation of i,/C was an impurity of protein origin. Ordinary BPN preparations are known to contain high molecular weight compounds, proteins, and sulfur-containing polymers (12). Since the BPA product used was prepared from BPN, it was possible that it contained some of the impurities. Since the concentration of the impurities in the polarographic solutions would change in proportion to the volume of the BPA stock solution taken, the polarograms of the different concentrations of BPA would be affected by different concentrations of the impurities. If a given concentration of BPA is followed with time, this in effect gives different concentrations of BPA but with the same concentration of impurity. Accordingly the peak height of a BPA solution a t selected ages of the solution was compared with the UV absorbance at 322 nm of an identical solution. The ratio of peak height to absorbance was found to decrease with the age of solution which was in agreement with the decreases of i,/C with decrease in concentration. Thus it was concluded that the inconstancy of the i,/C ratio was not caused by an impurity in the BPA preparation. In an attempt to find a medium in which a proportionality exists between concentration and peak height, the following buffer systems were investigated: tetraethylammonium phosphate (pH 6.0), sodium potassium phosphate (pH 6.80), acetate (pH 5.18), borate (pH 8.0),and McIlvaine buffer (pH 6.65). BPA in all buffers investigated, behaved in the same manner as in citrate buffer of p H 6.45 in that the ratio i,/C increased with concentration. It is not clear why i,/C should increase with concentration whereas id/ C remains unchanged. Literature survey shows t h a t in the few studies reported most of the sulfhydryl compounds show a linear relationship between concentration and peak height (13, 14). I t is conceivable that i,/C could change with concentration if a change in concentration is accompanied by a change in reversibility. However, it was found that the EBI1- Ell4values for concentrations covering the range in which i,/C increased were found to be the same (46 mV). Effects of Surfactants. First the effect of Triton X-100 (0.0024.020%) on BPA in pH 6.45 citrate buffer which give single peaks was studied. The peak potential was shifted to a more positive value with increase in Triton X-100 through 0.008% after which there was little change. For example, for a 3.74 X lo4 M solution the shift in the presence of 0.008% Triton X-100 was from -0.52 V to 4 . 4 0 V. A very interesting and analytically useful effect of Triton X-100 was that the peak height increased significantly. The peak height increased as the concentration of Triton X-100 was increased through 0.004% after which it showed little or no change when concentrations as high as 0.020% were used. The effect of Triton X-100 on solutions (e.g., 2.54 X M) which gave two peaks was also studied. In the absence of Triton X-100, the two peaks were well resolved, but as Triton X-100 was added the resolution between the two peaks became poorer. This was because the first peak was shifted toward the second peak while the latter remained unaffected. None of the concentrations of Triton X-100 included in this study caused a complete merger of the two peaks, and only a slight effect was observed on the peak heights. The effects of gelatin and a saturated solution of camphor were also studied to determine if the effects observed with Triton X-100, particularly the increase in peak height, were also obtained with other surfactants. Gelatin (0.005-0.050%) in pH 6.45 citrate buffer exhibited qualitatively a similar effect both on peak potential and peak height. For example, the peak potential of a 3.74 X lo4 M solution moved from -0.52 V to -0.46 V as gelatin concentration was increased through

PENlClLLENlC ACID, M x IO6 Height of the first peak of benzylpenicillenic acid vs. concentration in the presence of 0.004% Triton X-100

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0.035% after which there was no change. The peak height increased with gelatin concentration until 0.025% and then remained unaffected as gelatin concentrations were increased. The peak height obtained with 0.025% gelatin was about half the peak height obtained with 0.008% Triton X-100 (0.120 FA as compared to 0.228 PA). The peak in the presence of Triton X-100 was sharper than that obtained in the presence of gelatin for the same concentration of BPA and experimental conditions. To investigate the effects of camphor, a saturated solution of camphor in citrate buffer of pH 6.45 was used. The M solution was shifted to a peak potential of a 3.74 X more positive value by only 0.01 V. Height of the peak was increased but it was only about the height obtained in the presence of 0.008% Triton X-100 and the peak was not as sharp as in the presence of Triton X-100. Because of the larger peak height and narrower peak width, Triton X-100 would be the surfactant of choice as an analytical aid for the analysis of BPA. I t is not clear why Triton X-100 brings about an increase values in the absence and in peak height. When ESl4presence of Triton X-100 were compared, the value obtained in the presence of Triton X-100 was higher; e.g. for a 2.17 X M solution, it was 46 mV without Triton X-100 but 61 mV with 0.004% Triton X-100. According to these results, the electrode reaction became less reversible in the presence of Triton X-100 and, therefore, the peak height was expected to decrease. This, however did not occur. The use of surfactants with D P polarography is not common. In the few cases where Triton X-100 was used, it caused depression of peak currents (15,16). In a recent report on the effects of surfactants in D P polarography, Jacobsen and Lindseth (17)mention that the cationic surfactant decylamine increased DP peak current of folic acid by 50% whereas Triton X-100 completely inhibited the electrode reaction. They suggest that the cationic surfactant makes the electron transfer more reversible. Concentration Effects in Presence of Triton X-100. Relationships between peak height and concentration of BPA in the presence of Triton X-100 at concentrations of 0.00270, 0.004%, 0.006%, 0.008%, 0.010%, 0.012%, 0.014%, 0.016%, and 0.020% were investigated. In the 0.002% Triton X-100 solution, i,/C increased with an increase in the BPA concentration. The relationship between concentration and peak height in BPA solution containing 0.004% Triton X-100 is shown in Figure 4, and this is typical of Triton X-100 at concentrations ranging through 0.020%. A linear relationship in the concentration region below the 1.0 X M level was obtained. A comparison of Figures 3 and 4 shows that in the concentration region where ip/ C is decreasing with concentration in the presence of Triton X-100 (0.004% and above). i,/C is increasing with concentration in the absence of Triton X-100. This indicates that the ratio of peak height in the presence of Triton X-100 to that in the absence of Triton X-100 is not

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constant but decreases with concentration of BPA. A plot of such a ratio vs. concentration is shown in Figure 5 . The increase in peak height due to the presence of Triton X-100 varies from 14-fold for a 2.5 X l o 4 M solution to 2-fold for a 9.0 X M solution. Comparison of Experimental a n d Theoretical Values of Peak Current. Since i,/C values did not remain constant, it was necessary in reporting these values to give the concentrations of BPA and state whether or not Triton X-100 was or was not used in the determinations. This was not necessary when reporting id/ C values, because they remained constant for all concentrations of BPA in the absence or presence of Triton X-100. Noting that i,/C varies from about 4 to 18 kA/mM in the absence of Triton X-100 and from 61 to 26 pA/mM in the presence of Triton X-100, it was of interest to compare the experimental values of i,/C with the theoretically expected (15, 18) values. The i d / C value obtained under the same conditions (e.g., same drop time of 2 s) as the i,/C values was 2.45 pA/mM. T h e calculated i,/C value is 2.47 kA/mM which is much lower than the experimental values. Much of the difference is due to instrumentation. In the polarographic analyzer PAR model 174A, the differential pulse mode has a tenfold gain in the output not present in the other modes. The peak currents reported here are the values as read from the instrument. T o obtain the "true" values, they must be divided by 10. Therefore, the "true" experimental i,/C varied from 0.4 to 1.8 in the absence of Triton X-100 and from 6.1 to 2.6 in the presence of Triton X-100. Thus the experimentally obtained i,/C in the absence of Triton X-100 is lower than the calculated i,jC at all concentrations of BPA, but the converse is true in the presence of Triton X-100. While there has been a report where the experimental i, is lower than calculated i, due to irreversibility and instrumental artifacts ( 1 5 ) , we have not come across a case where experimental i, is higher t,han calculated i,. Selection of the Optimum Concentration of Triton X-100 for Quantitative Analysis of BPA. It was noticed, when using a high sensitivity and a large pulse amplitude, that there was a small inflection just before the start of the main peak for BPA solutions containing Triton X-100. The source of the inflection was investigated to determine whether it was due to Triton X-100, BPA or one of its impurities, or ethanol. By studying the effect on the inflection of change of concentration

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of the suspected source while keeping the concentration of the others the same, it was concluded that the inflection was due to the Triton X-100. When solutions of different concentrations of Triton X-100 containing no BPA were examined, a small peak a t about -0.6 V was seen. The size of the peak decreased as the concentration was increased from 0.002% through 0.008% and then increased slightly as the concentration was further increased. In the presence of BPA, the peak due to Triton X-100 became a shoulder preceding the BPA peak and appeared larger. The resolution between the Triton X-100 peak and the BPA peak improved as the concentration of Triton X-100 was increased from 0.002% through 0.008% but there was no improvement above 0.00870. Therefore, the medium containing 0.008% Triton X-100 seemed to be the optimum concentration for BPA analysis a t low concentrations. In this medium, a direct relationship between concentration and peak height was observed from M down to 4.0 X lo-' M after which measurement 1.0 X of the peak was difficult.

CONCLUSION The finding that Triton X-100 causes a dramatic increase in peak current is quite interesting because previous studies (15-1 7 ) of other compounds showed that this surfactant caused a decrease. The increase in peak current of BPA was shown not to be due to a change in reversibility. Experimental values of zp/C in the presence of Triton X-100 were found to be higher than theoretical values calculated using the method reported by Parry and Osteryoung (18). T o our knowledge, this is the first example of its kind to be reported in the literature. The relationship between peak current and concentration seems to be quite unique in that the ratio of i,/C increases with concentration in the absence of Triton X-100 but remains either constant or decreases (depending on the concentration region) with increased concentration i n the presence of Triton x-100. Although some of the results observed in this study could not be readily explained, Triton X-100 was shown to be a useful analytical aid in the analysis of benzylpenicillenic acid using D P polarography. The applications of D P polarography for the simultaneous analysis of benzylpenicillenic acid and penicillamine in parenteral benzylpenicillin preparations is planned for future work.

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RECEIVED for review May 10, 1977. Resubmitted May 15, 1978. Accepted August 24, 1978. Supported in part by the General Research Support Grant 5SOlRRO5586-07 and the African-American Institute.