Spectrophotometric assay of ampicillin (. alpha.-aminobenzylpenicillin

of alkaline hydrolysis of ampicillin when compared to a titrimetric method. A novel use for this assay is sug- gested, involving analysis of mixtures ...
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Spectrophotometric Assay of Ampicillin (a=AminobenzylpeniciIlin) Involving Initial Benzoylation of the Side Chain a-Amino Group David E. Tutt‘ and Michael A. SchwarW Department of Pharmaceutics, SchooE of Pharmacy, Srate University of New York at Buffalo, Buffalo, N . Y . 14214 The rate of formation of a-aminobenzylpenicillenic acid, 2, from ampicillin (a-aminobenzylpenicillin, 1) under strongly acidic conditions in the presence of mercuric chloride at 50 “C is very slow owing to stabilization of the penicillin by the side chain a-amino group. Consequently, ampicillin may not be readily assayed spectrophotometrically under these conditions. However, treatment of an aqueous solution of sodium ampicillin with benzoyl chloride in dioxane under mildly alkaline conditions produces a new, acid-unstable penicillin, a-benzamidobenzylpenicillin, 3, which on treatment with mercuric chloride in acid solution rearranges rapidly to form a-benzamidobenzylpenicillenic acid, 4, which may be assayed spectrophotometrically at 322 nm after only 48 minutes. Optimum conditions are reported for the benzoylation procedure and the subsequent spectrophotometric analysis. The overall assay, which may be used for concentrations as low as 4 X 1O-W ampicillin, i s shown to be very reproducible and to yield accurate results for the rate of alkaline hydrolysis of ampicillin when compared to a titrimetric method. A novel use for this assay is suggested, involving analysis of mixtures of ampicillin and other penicillins.

SEVERAL PROCEDURES have been described for the assay of penicillins, involving either of two general chemical modifications of the penicillin nucleus. Initial nucleophilic cleavage of the p-lactam of penicillins under alkaline conditions is the basis of titrimetric ( I ) , iodometric ( I ) , and hydroxamic acid (2) assays. Treatment of penicillin under acidic conditions (pH 1-5) in the presence of heavy metal ions (mercuric or cupric) produces the corresponding penicillenic acid (3, 4 ) which contains a chromophore (oxazolone) with an absorption maximum at 320-360 nm, depending on the side chain. Since intact penicillin is required for oxazolone formation and the presence of penicilloyl derivatives does not interfere with the procedure, the penicillenic acid assay is specific for penicillin and has been used extensively for the spectrometric analysis of penicillins. The method described originally by Herriott (5) involved treatment of penicillin solutions at pH 4.6 and 100 “C. This was modified by Brandriss and coworkers (3) for analysis of aqueous and protein-containing solutions of penicillins at ambient temperature using mercuric chloride and pH 1-3. The procedure described by Brandriss et al. has been used extensively in our laboratory for analysis of aqueous solutions of a variety of penicillins. However, acid-stable penicillins, such as ampicillin (a-aminobenzylpenicillin, l), cannot be Present address, Dept. of Chemistry, University of Sussex, Sussex, England. To whom correspondence should be addressed. (1) J. V. Scudi and H. B. Woodruff, “The Chemistry of Penicillin,” H. T. Clarke, J. R. Johnson, and R. Robinson, Ed., Princeton University Press, Princeton, N. J., 1949, p 1025. (2) J. H. Ford, IND. ENG.CHEM., ANAL.ED.,19,1004 (1947). (3) M. W. Brandriss, E. L. Denny, M. A. Huber, and H. G . Steinman, “Antimicrobial Agents, and Chemotherapy-1962,” American Society for Microbiology, Ann Arbor, Mich., p 626. (4) A. Holbrook, J. Pharm. Pharmacol, 10,762 (1958). ( 5 ) R . M. Herriott, J. Biol. Chem., 164,725 (1946). 338

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analyzed readily by this method since the rate of formation of penicillenic acid, 2, is extremely slow at ambient temperature. Smith and coworkers (6) used a modification (7) of the original assay procedure, in which solutions of sodium ampicillin, 1, containing copper sulfate were heated at 75 OC and pH 5.2 for 30 minutes. Attempts to use their procedure in our laboratory for the assay of ampicillin in kinetic reaction solutions yielded unsatisfactory results, and another assay procedure was sought. It has been recognized (8) that the nature of the side chain is important in determining acid stability of penicillins. Thus, a-methoxy, a-chloro-, and a-aminobenzylpenicillin are increasingly more acid-stable than benzylpenicillin (9), and this effect has been rationalized in terms of the inductive effect of the a-substituent decreasing the nucleophilicity of the side chain amide carbonyl oxygen, which is involved in oxazolone formation. It seemed likely that removal of this inductive effect by suitable chemical modification of the side chain amino group would yield a new, acid-unstable penicillin which could be assayed readily at low pH. One possible procedure involves benzoylation of the amino group under very mildly alkaline conditions (ea. pH 9) using benzoyl chloride to give a-benzamidobenzylpenicillin, 3. (A review points out that although it is reasonable to assume that only benzoylation is taking place we have not isolated and identified the reaction product.) Under such conditions, alkaline hydrolysis of the P-lactam would be negligible, although benzoylation would be fairly rapid. We now describe an analytical procedure for ampicillin involving initial benzoylation of the side chain amino group and subsequent spectrophotometric assay for penicillin of the resulting solution using a modification of the method of Brandriss and coworkers (3). EXPERIMENTAL Materials. Sodium ampicillin was provided by Bristol Laboratories. Benzoyl chloride was purified by distillation [lit. (10) bp 197 “C at 760 mm Hg]. Commercially available 95 ethanol, n-propanol, and 1,Cdioxane were used without further purification. Water was deionized and distilled under nitrogen in all-glass apparatus. Benzoylation Procedure. One milliliter of a solution of benzoyl chloride in dioxane was added from a syringe to 40 ml of the appropriate solution of sodium ampicillin in water at pH 9.0 stirred in a reaction cell thermostated at 31.5 O C . pH was maintained at 9.0 i 0.5 during this procedure by the addition of titrant (1.ONNaOH) by the pH-stat. The mixture (6) . , J. W. G. Smith, G . E. DeGrey, and V. J. Patel, Analyst (London), 92,247 (1967). (7) F. G. Stock. ibid.. 79.662 (1954). and reference (5). (8j F. P. Doyleand J, H:C. Nayler, “Advances in Drug Research,” Vol. 1, N. J. Harper and A. B. Simmonds, Ed., Academic Press, London, England, 1964, p 26. (9) F. P. Doyle, J. H. C. Nayler, H. Smith, and E. R. Stove, Nature, 191, 1091 (1961). (10) “Dictionary of Organic Compounds,” Vol. I, 1. Heilbron, A. H. Cook, H. M. Bunbury, and D. H. Hey, Ed.,Eyre and Spottiswoode Ltd., London, England, 4th ed., 1965, p 358.

was stirred for a further period at pH 9.0 and 31.5 OC until liberation of benzoic acid in the system was complete (determined by the addition of titrant). It was important to maintain rapid mixing throughout this procedure because addition of dioxane solution to the aqueous solution of ampicillin produced a dispersion of fine droplets of benzoyl chloride which dissolved completely within about 5 minutes. An aliquot of the reaction solution was then removed and analyzed for penicillin (a-benzamidobenzylpenicillin, 3) by the niethod of Brandriss and coworkers (3). The appropriate concentrations of benzoyl chloride (0.80M) and ampicillin (2.0 x IO-") solutions, the rate of addition of benzoyl chloride (1 ml, 0.5 ml/min), and time of mixing of the reaction solution (12 min) were kept constant except in studies to determine a particular effect upon the maximum absorbance (Amax)in the penicillenic acid assay. The absorbance values reported have been corrected for the effect of dilution of the initial ampicillin solution by the dioxane and titrant except the spectra in Figure 1 and curves in Figure 2. Assay for Penicillin. The basic procedures for this assay have been described previously (3),and only the specific conditions employed for the spectrophotometric assay for abenzamidobenzylpenicillin are given here. An aliquot of the benzoylated ampicillin solution was diluted (20-fold) with the appropriate buffer solution preequilibrated at 50 O C and assayed spectrophotometrically by conversion of the abenzamidobenzylpenicillin to the corresponding penicillenic acid in the presence of mercuric chloride at pH 1.8 (preliminary investigation), pH 0.9-2.3 (assay pH dependence), and pH 1.71 f 0.03 (all other studies) in 19% ethanol-water at 50 "C. A glycine-hydrochloric acid buffer (0.2M) was used for pH 2.3 and hydrochloric acid was used for pH 0.9-2.0. Apparatus. A Radiometer pH-stat system, consisting of a TTT-lc pH-stat, SBR 2c titrigraph, and SBU I C syringe buret fitted with a jacketed reaction cell, was used for maintenance of pH during benzoylation of ampicillin, for measurement of pH at 31.5 "C, and for maintenance and measurement of pH at 31.9 "C in a study to compare the benzoylation assay procedure with a titrimetric method used to obtain kinetic data for alkaline hydrolysis of ampicillin. Ultraviolet spectrometric measurements were made using a Beckman-Gilford recording quartz spectrophotometer fitted with a thermostated block (50 " C )and a Beckman DB-G double-beam spectrophotometer fitted with a Sargent SR recorder. Since the DB-G spectrophotometer was not equipped with a thermostated block, the absorption spectra shown in Figure 1 were obtained by transferring an aliquot of penicillin assay solution (stored at 50 " C ) to a silica cell and immediately measuring the absorption spectrum in the region 290-355 nm. Fresh aliquots were taken for each spectrum recorded. Stoppered silica cells (1 cm) were used for all spectral measurements. A Radiometer pH meter 26 was used for measurement of pH values of assay solutions at 50 O C . Constant temperatures (31.5 f 0.1, 31.9 i 0.1, or 50 & 0.5 " C ) were maintained using Bronwill circulating water pumps. RESULTS

Preliminary Investigation. A solution of sodium ampicillin (40 ml, 2.0 X lO-3M) was treated with a solution of benzoyl chloride in dioxane (1 ml, 0.8OM; concentration after dilution with ampicillin solution, 20 X 10-aM) under the reaction conditions described in the Experimental section. An aliquot of this solution was assayed spectrophotometrically for penicillin. The presence of a new, acid-unstable penicillin was revealed by the development of a chromophore in the region 320-330 nm [characteristic of an oxazolone structure (3)which attained a maximum absorbance of 0.7 at 322 nm after 50 minutes under the assay conditions (See Experimental section)]. In contrast, treatment of a solution of sodium ampicillin with dioxane alone and subsequent spectrophotometric

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assay of the solution under the same conditions revealed the much slower development of a chromophore (corresponding to 2) in the region 320-330 nm. The absorbance of this solution was only 0.05 at 322 nm after 54 minutes and was still rising after 7 hr ( A = 0.34 at 322 nm). It should be emphasized that, since the penicillenic acid ( 2 or 4) is an intermediate in a series first-order reaction, only the maximum absorbance of the chromophore corresponding to the intermediate (2 or 4) is proportional to the initial concentration of penicillin (1 or 3, respectively) assayed by this method ANALYTICAL CHEMISTRY, VOL. 43, NO. 3, M A R C H 1971

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Figure 3. Absorbance as a function of benzoyl chloride concentration (see text for details) (4, 11). Thus, a considerably longer time would be required to reach the maximum absorbance for a-aminobenzylpenicillenic acid, 2, than for the benzoylated penicillenic acid, 4 (see Scheme). Spectrometric analysis of a sample of water treated with benzoyl chloride under the reaction conditions showed a negligible absorbance at 322 nm even after 3 hr, demonstrating that excess benzoic acid (formed by hydrolysis of benzoyl chloride) does not interfere with the assay procedure. We concluded that sodium ampicillin, an acid-stable penicillin, had been benzoylated under mildly alkaline conditions to yield a-benzamidobenzylpenicillin, 3, an acid-unstable penicillin, which was readily converted into the corresponding penicillenic acid on treatment with acid (see Scheme). Subsequent investigations were concerned primarily with the determination of a suitable solvent and the optimum pH and wavelength for the penicillin assay; and the optimum molar excess and rate of addition of benzoyl chloride solution, and time of sampling from the benzoylation mixture. Penicillin Assay. The method of Brandriss and coworkers (3) was used to assay for a-benzamidobenzylpenicillin by its conversion to a-benzamidobenzylpenicillenic acid. As solvent, 19% ethanol-water was used rather than water (deter(11) A. A. Frost and R. G. Pearson, “Kinetics and Mechanism,” J. Wiley and Sons, Inc., New York, N. Y.,2nd ed., 1961, p 166. 340

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mined by the solubility of 4). The variation of absorbance as a function of wavelength in the region 280-360 nm for a benzoylated solution of ampicillin (assayed at pH 1.74) showed an absorption maximum at 322 nm after about 48 minutes (Figure 1). The curves in Figure 1 represent the experimentally measured spectra of the assay solution during the formation of a-benzamidobenzylpenicillenic acid after benzoylation with 1 ml of 0.80M benzoyl chloride and treatment at pH 1.8 at 50 “C. Sodium ampicillin was treated with a constant molar excess of benzoyl chloride (see Experimental section for conditions) and the mixture was assayed for penicillin at a variety of pH. The variation of absorbance at 322 nm as a function of time for pH 0.9-2.3 is shown in Figure 2. The choice of a suitable pH value for the penicillin assay rests on three considerations: the time (tmnx) taken to attain maximum absorbance, the absorbance (Amax)at tmax, and the period of time ( A t ) during which A,,, is maintained. Using these criteria ca. pH 1.7 was considered suitable (t,,, = 48 min, A,,, = 0.7, and At = 9 min) for the a-benzamidobenzylpenicillin assay. The effect of variation of mercuric chloride concentration upon A,,, was constant (0.715 & 0.005) within the reproducibility of the overall procedure for the range 4.0-12.1 X 10-aM HgC12,and as a result, a constant concentration of mercuric chloride (8.0 X 10-3M)was used for subsequent assays. Benzoylation Procedure. A solution of sodium ampicillin in water was treated with increasing concentrations of benzoyl chloride in dioxane (1 ml, 0-l.6M; 0-40 X 10-3M after addition to ampicillin solution under the benzoylation conditions, see Experimental) and assayed spectrophotometrically at 322 nm in pH 1.7 buffer at 50 “C. Figure 3 shows the variation of absorbance (AmaX)as a function of initial concentration of benzoyl chloride after addition to the ampicillin solution. The full circles represent experimentally determined absorbance (corrected for dilution of the benzoylation solution by dioxane and titrant) and the intercept on the absorbance axis (square) represents the absorbance measured at 322 nm after 48 minutes (corresponding to rmax for the a-benzamidobenzylpenicillin assay at pH 1.7) of a solution of sodium ampicillin treated under the reaction conditions in the absence of benzoyl chloride. An essentially constant absorbance was observed for assay solutions which were treated with benzoyl chloride at initial concentrations (after addition to the aqueous

solution of ampicillin) 7.4-10 times that of ampicillin (2.0 X ~o-~M and, ) in subsequent procedures, a constant initial concentration of benzoyl chloride (20 X 10-3M) was employed. Variation of the rate of addition of benzoyl chloride solution (1 ml, 0.1-1.0 mlimin) to sodium ampicillin solution under the reaction condition produced a negligible change in absorbance (A,,, = 0.716 i 0.004) at 322 nm in the spectrophotometric assay for penicillin. In subsequent assays, the benzoyl chloride solution was added to the sodium ampicillin solution during a period of 2 min (0.5 ml/min). The addition of titrant (NaOH) was usually complete after about 10 min, corresponding to complete hydrolysis of excess benzoyl chloride. In experiments to investigate the effect of the sampling time on A,,,, aliquots were removed at subsequent times (12-20 min after addition of benzoyl chloride) and assayed spectrophotometrically at 322 nm. The results 0.008) was independent of samindicated that A,,, (0.716 pling time within the period studied, and, for convenience, aliquots were removed for assay 12 minutes after the addition of benzoyl chloride. In order to estimate the reproducibility of the overall benzoylation and penicillin assay procedures, two series of four solutions of sodium ampicillin (1.0 X 10-3M and 2.0 x 10-3M) were benzoylated under optimum conditions and assayed spectrophotometrically at 322 nm. The absorbances (Amny)for each series of experiments (1.0 X 10-3M: A,,, = 0.356 i 0.005; 2.0 X 10-3M: A,,, = 0.715 i 0.005) are reported as mean i standard deviation, and demonstrate the method to be very reproducible. A series, of sodium ampicillin solutions of increasing concentration was benzoylated under optimum conditions and assayed spectrophotometrically. The variation of A,,, as a function of initial sodium ampicillin concentration is shown in Figure 4, and demonstrates that Beer's law is obeyed by a-benzamidobenzylpenicillenic acid in the concentration range studied. Comparison with Titrimetric Assay. A solution of sodium ampicillin in water was treated under mildly alkaline conditions (4.0 X 10-lMampicillin, pH 10.66, p = 0.2M, 31.9 "C) and aliquots assayed periodically using the benzoylation procedure and subsequent spectrophotometric analysis at pH 1.7. The apparent first-order rate constant for loss of penicillin P-lactam ( k o b s = 1.03 x min-l; kOb,/(OH-) = 12.7 1. mol-' min-I) was in agreement with the rate constant for formation of penicilloic acid under the same conditions ( k o b s = 1.10 X min-I; kOb,/(OH-) = 13.2 1. mol-') measured titrimetrically by the pH-stat system. Moreover, the secondorder rate constant for alkaline hydrolysis of ampicillin was in good agreement with the previously reported (12) rate constant ( k = 12.3 1. mol-' min-I) determined titrimetrically at 31.5 "C.

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Treatment of a solution of sodium ampicillin under acidic conditions (e.g., 1.0 X 10-4M ampicillin, 8.0 x 10-3M HgC12, pH 1.7, 50 "C 19 ethanol-water) and spectrophotometric analysis at 322 nm using the method of Brandriss and coworkers ( 3 ) revealed the slow development of a chromophore (oxazolone) corresponding to a-aminobenzylpenicillenic acid ( A = 0.34 and rising after 7 hr). Since only the absorbance (Amax)corresponding to the maximum concentration of (12) R. D. Kinget and M. A. Schwartz, J . Pharm. Sci., 58, 1102 (1969).

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Figure 4. Absorbance as a function of ampicillin concentration, assayed under optimum conditions penicillenic acid is directly proportioned to the initial concentration of penicillin (3, I I ) , this method is unsatisfactory for the analysis of ampicillin and other acid-stable penicillins. However, when a solution of benzoyl chloride in dioxane was added to an aqueous solution of sodium ampicillin under very mildly alkaline conditions ( e g . , 2.0 X 10-3M ampicillin, 20 x 10-3M benzoyl chloride, pH 9.0, 31.5 "C) a new penicillin, a-benzamidobenzylpenicillin was produced within 10 minutes. Spectrophotometric observation of the resultant solution under acidic conditions revealed the rapid formation of a chromophore ,,A(, = 322 nm), corresponding to abenzamidobenzylpenicillenic acid, which attained a maximum absorbance (A,,, = 0.7) after only 50 minutes. Thus, benzoylation of the side chain amino group of ampicillin, which had conferred acid stability on the penicillin, afforded a new, acid-unstable penicillin, (a-benzamidobenzylpenicillin) which could be assayed readily under acidic conditions. The benzoylation procedure was shown to be insensitive to large changes in either the rate of addition of benzoyl chloride solution (0.1-1.0 mlimin) to the ampicillin solution or the time of sampling (12-20 min) for the spectrophotometric analysis. An initial concentration of benzoyl chloride in the assay solution of 7-10 times the initial concentration of ampicillin (2.0 X 10-3M) produced the highest A,,, values (see Figure 3) corresponding to the maximum conversion of ampicillin to a-benzamidobenzylpenicillin in the benzoylation procedure. The assay of ampicillin using this modified procedure was shown to be very reproducible (i1 %) and in good agreement with the titrimetric analysis for penicillin in the alkaline hydrolysis of ampicillin. Although ampicillin concentrations as high as 2.0 X 10-3M were used to investigate the dependence of A,,, in the spectrophotometric assay upon the initial concentration of ampicillin in the benzoylation solution (Beer's law, see Figure 4), solutions containing as low as 4 X 10-5M ampicillin may be assayed using the benzoylation procedure by employing suitable variations in the experimental conditions (spectrophotometer absorbance scale, assay dilutions, cell path length). Thus, treatment of a solution of ampicillin (4 X lO-5M) with a 10-fold molar excess of benzoyl chloride at pH 9.0 and 31.5 "C and subsequent spectrophotometric analysis at 322 nm of an aliquot of the reaction mixture

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(diluted 5-fold with p H 1.7 buffer containing HgCL a t 50 “C) would produce a n absorbance (A,,, = 0.06 after 48 minutes) which could be measured accurately using the Beckman-Gilford spectrophotometer. Since benzoylation of the side chain amino group and subsequent rearrangement of a-benzamidobenzylpenicillin to form a-benzamidobenzylpenicillenic acid is specific for intact ampicillin (6),the overall procedure could be employed for the spectrophotometric assay of ampicillin both in kinetic reaction solutions and presumably in solutions containing a mixture of ampicillin and another penicillin. For example, treatment of an aliquot of an aqueous solution of benzylpenicillin and ampicillin at pH 2.1 in water at ambient temperature would yield benzylpenicillenic acid only after ca. 30 minutes (3) (measured spectrophotometrically at 322 nm) since the rate of degradation of ampicillin under such condi-

tions is negligible (9). Benzoylation of another aliquot of the mixture and treatment of an aliquot of the resultant solution a t p H 1.7 and 50 O C in 19% ethanol-water would yield both benzylpenicillenic acid and a-benzamidobenzylpenicillenic acid (from ampicillin). However, an absorbance measured a t 322 nm after 48 minutes would correspond almost entirely to a-benzamidobenzylpenicillenic acid since the benzylpenicillenic would have decomposed completely under these conditions. ACKNOWLEDGMEhT

The authors thank Bristol Laboratories for the gift of sodium ampicillin.

RECEIVED for review August 3, 1970. Accepted December 2, 1970.

Application of a Computerized Electrochemical System to Pulse Polarography at a Hanging Mercury Drop Electrode H. E. Keller’ and R. A. Osteryoung Department of‘ Chemistry, Colorado State Unicersity, Fort Collins, Colo. 80521

Application of computerized pulse polarography on a hanging drop to analysis of extremely dilute solutions i s demonstrated. An approximate theory is developed which shows that for reversible systems functionally identical behavior can be expected on the dropping and hanging drop mercury electrodes. A decrease in sensitivity for irreversible reactions would be observed under otherwise identical conditions with the stationary electrode. Ensemble averaging and digital smoothing are described and their effect on signalto-noise ratio is demonstrated. Variations of pulse height, pulse width, and time between pulses are briefly discussed. Response obtained on 4 X 10-8M Cd2+solution indicates that usable data can be obtained at this level while a precision of 10% i s indicated on 4 x lO-’M Cd2+.

DERIVATIVE MODE PULSE POLAROGRAPHY has been shown to be avery sensitive analytical technique (Z-4). When performed on a stationary electrode, additional advantages may accrue such as increased electrode area (5), increased speed of analysis, and ensemble-averaging (6, 7) undisturbed by drop area uncertainty. Computerization of chemical analysis is becoming very popular today, a fact occasioned by utility and by novelty. In electrochemical analysis, several workers have been enPresent address, Department of Chemistry, Northeastern University. Boston, Mass. 02115 (1) E. Temmerman and F. Verbeek, J . Electroanal. Chem., 12, 158 (1966).

( 2 ) A. Lagrou and F. Verbeek, ibid., 19, 413 (1968). (3) E. P. Parry and R. A. Osteryoung, ANAL.CHEM.,36, 1366

gaged in demonstrating the utility of an on-line computer system (6, 8-10). By employing a computer to take measurements, control the experiment, and analyze the resulting data, maximum use is made of the advantages of derivative pulse polarography at a stationary electrode. Other capabilities such as convolution of the current response to increase the signal-to-noise ratio and automatic determination of peak positions and heights by a real-time successive approximation technique can be developed readily on a computer system. In this paper the characteristics of a computerized electrochemical system are described and demonstrated in an application to pulse polarography a t a stationary electrode. Some of the advantages of the computer system over conventional systems are developed; some further potential advantages are mentioned. The realizable sensitivity of the system as an analytical tool seems, at present, to be limited by background. Instrumental artifacts and oxygen appear to be the primary contributors. With the problems, however, measurable response is obtained with 4 x 10-SM Cd2+. Reducing the concentration of supporting electrolyte to below 10d3Mis an important factor in this achievement. This sensitivity compares with stripping analysis where sensitivities as low as 6 X 10-11M (ZZ) have been reported. More normally, values of 10-9M are seen with respect to this technique. It does require that the species determined be concentrated into another phase, a fact which limits its general utility. Potential sweep voltammetry has a reported sensitivity of -~10-~M( 1 2 )

(1964).

(4) C. Peker, M. Herlem, and J. Badoz-Lambling, Freseiiius’ Z. Anal. Chem., 224,204 (1967). ( 5 ) G. D. Christian, J . Electroanal. Chem., 22, 333 (1969). (6) S. P. Perone, J. E. Harrar, F. B. Stevens, and R. E. Anderson, ANAL.CHEM., 40, 899 (1968). (7) V. W. Lee, T. P. Cheatham, Jr., and J. B. Wiesner, Proc. I.R.E., 38 1165 (1950).

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(8) G. Lauer and R. A. Osteryoung. ANAL.CHEM.,40 (IO), 30A (1968). (9) G. P. Hicks, A. A. Eggert, and E. C. Toren, Jr., ibid., 42, 729 (1970). (10) G. Lauer, R. Abel, and F. C. Anson, ibid., 39,765 (1967). (11) S. P. Perone and J. R. Birk, ibid., 37, 9 (1965). (12) J. W. Ross, R. D. DeMars, and I. Shain, ibid.. 28, 1768 (1956).