finally returns to the base line when blank is passing through both electrodes. If each sample is not separated by a blank, samples could be run easily at a rate of 15 to 20 samples per hour. Assuming a flow rate of about 2 ml/min and 5 minutes per sample using a 1 plasma sample, a measurement could be conveniently made with 0.1 ml of plasma. In conclusion, the method developed for the assay of M A 0 activity was found to have good sensitivity but, more important, to be very free from interferences because the measurement can be made fortuitously in a voltage pocket that exists between the reduction of oxygen and the oxidation of biological amines, where the background noise is small and constant, and because the differential measurement effectively cancels out faradic interferences which do not change during the time interval of passage between the two electrodes, Initial studies were begun using tubular platinum electrodes, but it was the authors' experience that superior results were obtained at the tubular carbon electrodes.
Although the data are the subject of subsequent publications, kinetic constants for the enzyme reaction of the crude preparations are in excellent agreement with data reported on crystallized enzyme. Also, measurements can be made on other crude biological preparations-for example, measurements have been made on diluted rat brain and rat liver homogenates. In addition, extensive studies of M A 0 inhibitors in the crude preparations are being made. In all cases, excellent results, typical of those presented in the present paper, indicate the general utility of the method.
RECEIVED for review September 30, 1969. Accepted January 29, 1970. This study was supported in part by Grant GM15821, from the National Institutes of Health, U.S. Public Health Service, Bethesda, Md. William D. Mason was a fellow of the American Foundation for Pharmaceutical Education.
Ferrocene as a Primary Standard for Oxidation-Red uction Titrations in Acetonitrile Byron Kratochvil and Peter F. Quirk Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada Ferrocene is a suitable primary standard for solutions of hydrated copper(l1) perchlorate in acetonitrile. Commercial ferrocene can be purified readily by recrystallization and sublimation, and is stable and nonhygroscopic on storage. Concentrations of copper(ll) solutions, determined by weight titrations of ferrocene and aqueous EDTA, agree within 0.2 ppt.
IT HAS RECENTLY been shown that copper(I1) perchlorate can be used as an analytical oxidant in acetonitrile (I, 2). The reduction potential in acetonitrile of the copper(I1)-(I) couple vs. a silver-0.01M silver nitrate reference is 0.801 V (3), 1.27 V greater than that of the copper(I)-(O) couple. Although copper(I1) solutions in acetonitrile are prepared easily and are stable ( I ) , a method for determining their redox titer to a part per thousand or better has not been established. A preliminary investigation showed that copper(I1) perchlorate was too hygroscopic to be used directly. Thiourea, potassium iodide, hydroquinone, and quinhydrone gave drawn out potential breaks and concentration-dependent equivalence points. Other standards used in water-such as arsenic trioxide, sodium oxalate, potassium ferrocyanide, and iron(I1) ethylenediammonium sulfate-are not sufficiently soluble in acetonitrile. Oxalic acid dihydrate, though soluble, is not oxidized by copper(I1). Ferrocene is oxidized readily to the ferricenium ion by copper(I1) in acetonitrile. Its availability and ease of oxidation prompted a study as a primary standard for acetonitrile solutions of hydrated copper(I1) perchlorate. (1) B. Kratochvil, D. A. Zatko, and R. Markuszewski, ANAL. CHEM., 38, 770 (1966).
(2) H. C . Mruthyunjaya and A. R. V. Murthy, Indian J. Chem., 7, 403 (1969). (3) E. Lorah and B. Kratochvil, University of Alberta, unpublished
work, 1969. 492
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EXPERIMENTAL
Reagents. Technical grade acetonitrile (Matheson, Coleman and Bell) was purified according to the method of O'Donnell, Ayers, and Mann (4), except that the decantation following addition of sulfuric acid was replaced by a vacuum distillation below 55 "C. The pure solvent had an absorbance of less than 0.2 at 200 mp. Technical grade heptane (Fisher Scientific Co.) was used as received. Deionized water was used throughout. Crystallization and sublimation were used to purify commercial ferrocene (Arapahoe Chemicals). Recrystallization was done from heptane; the product was dried under vacuum for at least 12 hr. Commercial acetonitrile was not satisfactory for recrystallization. Sublimations were performed under vacuum at 60 to 80 "C; the product was collected on a water-cooled cold finger. The melting point of ferrocene recrystallized twice, then sublimed, was obtained from DTA curves recorded on a DuPont Model 900 differential thermal analyzer; a range of 175.4 to 175.9 "C was found. The solubility of ferrocene in acetonitrile was determined by equilibration under nitrogen with pure solvent at 25 f 0.05 "C; equilibrium was reached within 4 hr. The solutions were analyzed by careful evaporation of the solvent and weighing, and by titration of weighed aliquots with copper(I1) perchlorate in acetonitrile. Anal. by evaporation (av. of 4), 3.41 weight %; by titration (av. of 4), 3.37 weight %. Hydrated copper(I1) perchlorate was prepared by adding a slight excess of 72% perchloric acid (J. T. Baker Chemical Co.) to a suspension of copper(I1) carbonate (Fisher Certified Reagent) in water. The solution was boiled to eliminate carbon dioxide, then cooled, and the resulting crystals were collected and dried at room temperature under vacuum. The copper was determined by EDTA titration with murexide (4) J. F. O'Donnell, J. T. Ayres, and C . K. Mann, ANAL.CHBM., 37, 1161 (1965).
600
-
400
-
200
-
0 I
I
1.o
0.5
[Cu”]
Lu
I t
I [Fc] Ratio
Figure 1. Titration of ferrocene with copper(I1) in acetonitrile indicator ( 5 ) and the perchlorate by reduction to chloride followed by titration of the chloride with silver nitrate (6). The acid present was estimated by pH measurement of a concentrated aqueous solution of the product. Qualitative tests for bicarbonate, carbonate, sulfate, nitrate, and the halides were negative. Assuming the acid to be perchloric acid, the analyses correspond to the formula Cu(CIO&.o.6.1 H 2 0.0.01 HC104. This laboratory preparation was used for most of the experimental work, but in a few specified instances commercial material (G. Frederick Smith Co.) was used. The disodium salt of EDTA (The British Drug House Ltd.) was recrystallized according to the method of Blaedel and Knight (7) and dried overnight at 80 “C. Murexide (J. T. Baker Chemical Co.) mixed 1 :1000 by weight with sodium chloride was used as indicator. Titrant Solutions. Hydrated copper(I1) perchlorate solutions, approximately 0.03m, were prepared in pure or technical grade acetonitrile. To suppress hydrolysis of the copper, about 1 ml of 70% perchloric acid was added per liter of hydrated copper(I1) perchlorate solution. The solutions were stored in borosilicate glass bottles with ground-glass stoppers. Approximately 0.03m aqueous EDTA solutions were stored in polyethylene bottles. Standard solutions of copper(I1) in water were prepared by dissolution of 1.9-g portions of 20-gauge copper wire (Canadian Laboratory Supply), weighed to the nearest microgram, in 13 ml of nitric acid and dilution with water to 1000 0.005 g. The concentration of this solution was expressed as millimoles of copper per gram of solution. Procedure for Titrations. Titrations of ferrocene with copper(I1) in acetonitrile were performed in the cell previously described ( I ) . Exclusion of oxygen was found unnecessary. An Orion digital pH meter was used with platinum indicator and silver-0.OlM silver nitrate in acetonitrile reference electrodes. For weight titrations, samples of ferrocene (0.5 to 0.6 mmole) were weighed to the nearest microgram into small glass cups. The cups were covered to minimize loss of ferrocene through volatilization; as an added precaution all samples were titrated within 30 min of weighing. The cups were dropped into 40 to 50 ml of acetonitrile in a titration cell and 0.03m copper(I1) titrant added by means of a 30-ml hypodermic syringe fitted with a glass capillary tip. The syringe plunger was lubricated with silicone grease to prevent evaporation of the solution and provide more
*
( 5 ) G. Schwarzenbach, “Complexometric Titrations,” English Translation, Interscience, New York, N. Y., 1957, pp. 80-82.
( 6 ) D. A. Zatko and B. Kratochvil, ANAL.CHEM., 37, 1560 (1965). (7) W. J. Blaedel and H. T. Knight, ibid., 26, 741 (1954).
I
I
1.000
0.999
[Cu”]
1.001
1
1.002
/ [ Fc] Ratio
Figure 2. End point region for titration of ferrocene with copper(I1)in acetonitrile positive operation. When a potential of 260 to 280 mV us. the silver reference electrode was reached, indicating that the titration was about 99.9 complete, 0.003m copper(I1) solution (prepared by accurate dilution by weight of the stock solution) was delivered from a 5-ml syringe to complete the titration. The weight of titrant was found by weighing the syringes on an analytical balance. After each titration of ferrocene, an aliquot of the 0.03m copper(I1) stock solution of approximately the same weight as used in the titration of ferrocene was weighed for EDTA standardization. This alternation procedure was necessary to eliminate determinate error by evaporation of acetonitrile from the stock solution. The aliquots were evaporated to dryness and 95z of the stoichiometric amount of 0.03~1 EDTA was added by syringe. The titration solution was diluted to about 500 ml with deionized water and the pH was adjusted first to 2.0 with dilute nitric acid, then to 7.5 i 0.1 with dilute ammonia. The dilution minimized interference by the blue color of the copper-ammonia complex; the two-step pH adjustment provided the needed buffer. A pH of 7.5 gave the best end points. About 0.2 g of diluted murexide indicator was added and the titration continued until the solution changed from green to blue-green. At this point EDTA diluted tenfold by weight was added from a 5-ml syringe until the final end point, a permanent blue color with no trace of green. The EDTA solution was standardized by the same procedure against a standard solution of aqueous copper(I1) nitrate; alternation was again used. Through careful matching of end-point colors on successive titrations, standard deviations of better than 0.5 ppt were obtained routinely. The purity of the copper wire used to prepare the standard solution was determined by electrodeposition according to the ASTM method (8). Current was supplied by a Model 6206B Hewlett-Packard dc power supply. After each run a check for complete copper deposition was made by titration of the remaining solution with EDTA. The EDTA titrant was standardized against standard copper(I1) nitrate solution, RESULTS AND DISCUSSION A titration of ferrocene with hydrated copper(I1) perchlorate in acetonitrile is shown in Figure 1 . The reaction is Fc Cu2+ s Fc+ Cu+ where Fc is ferrocene and Fc+ is the ferricenium ion. Expansion of the titration plot near the inflection (Figure 2) shows
+
+
(8) “ASTM Standards, Chemical Analysis of Metals,” Part 32, American Society for Testing and Materials, Philadelphia, Pa., 1967, pp. 374-75. ANALYTICAL CHEMISTRY, VOL. 42, NO. 4, APRIL 1970
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Table I. Comparison of Concentrations of Copper(I1) Solutions (in mmoles/g of solution) Found with Ferrocene and with EDTA Set I Ferrocene EDTA 0.028745 0.028780 0.028755 0.028762 0.028766 0.028751 0.028746 Av 0.028755 0.028760 0.5 Std dev 0.4 in ppt
Ferrocene 0.028111 0.028118 0.028125 0.028125 Av 0.028120 Std dev 0.2 in ppt
Set I1 Ferrocene EDTA 0.028781 0.028755 0.02878 1 0.028782 0.028789 0.028778 0.028785 0.028804 0.028776 0.028784 0.028766 0.028727 Av 0.028780 0.028788 Std dev 0.3 0.8 in ppt Set 111 EDTA 0.028117 0.028 140 0.028120 0.028125 0.028125 0.4
Table 11. Comparison of Procedures for Purification of Ferrocene Purity found No. by copper(I1) of Procedure titration, titns Std dev, ppt As received 99.37 3 0.3 1 Recrystallization' 99.56 3 0.2 1 Recrystallization 99.82 3 0.5 2 Recrystallizations 99.89 4 0.1 3 Recrystallizations 99.91 3 0.2 1 Sublimation 99.91 3 0.2 2 Sublimations 99.93 2 0.0 1 Recrystallization and 1 sublimation 99.99 2 0.2 2 Recrystallizations and 1 sublimation 100.02 13 0.3 2 Recrystallizations and 2 sublimations 100.02 4 0.3 From commercial acetonitrile; remainder from heptane.
that the end point can be determined within 0.1 ppt. The reaction is rapid, and potential drift during titration is negligible. The copper(I1) titrant was standardized by weight titrations against aqueous EDTA solutions, which were standardized in turn by weight titrations against standard aqueous copper(I1) nitrate solutions prepared from copper wire. The purity of the copper wire found for three analyses was 99.962, 99.954, and 99.954 %; average 99.957 %. Buoyancy corrections were made for all weighings. Results of three sets of data (Table I) show that weight titrations of ferrocene recrystallized twice, then sublimed, give values that agree with EDTA titration values within 0.3 ppt. Copper(I1) solutions prepared from laboratory synthesized copper(I1) perchlorate gave more-drawn-out titration curves with ferrocene than solutions prepared from commercial hydrated copper(I1) perchlorate. The commercial material contained 2.7 moles of perchlorate per mole of copper(I1); apparently excess perchloric acid is present, which reduces copper(I1) hydrolysis and sharpens the titration breaks. The addition of about 1 ml of 70% perchloric acid per 494
ANALYTICAL CHEMISTRY, VOL. 42, NO. 4, APRIL 1970
liter of copper(I1) titrant solution improved the sharpness of the titration breaks, but affected the stability of the potential near the end point by making the ferricenium ion susceptible t o further oxidation, especially when the water concentration was low. A combination of 0.1M perchloric acid in the titrant plus 0.2M water in the solution being titrated gave sharp potential breaks and stable potentials, and was used in all work where pure acetonitrile was the solvent. Higher concentrations of water gave smaller and more drawnout potential breaks, but did not affect the copper(I1)-ferrocene stoichiometry. Since the above combination of acid and water gave satisfactory results, no further work was carried out on the effects of water and perchloric acid on the titration. Comparison of Purification Procedures for Ferrocene. Titrations with copper(I1) of ferrocene recrystallized twice, then sublimed, agreed well with those of EDTA. The purity of commercial ferrocene treated by various procedures, based on titrations by EDTA-standardized copper(II), is shown in Table 11. Stability and Hygroscopicity. Ferrocene recrystallized twice from heptane was divided into three portions. Samples of the first portion were titrated immediately with copper(I1) in acetonitrile, the second portion was stored in a brown bottle at room temperature, and the third was stored at room temperature in a desiccator over a saturated aqueous solution of NazSOl. 10 H20. The relative humidity in the desiccator ranged from 91 to 93 % as measured by a Taylor Humidiguide hygrometer. Samples of the second and third portions were titrated after six months with copper(I1) in acetonitrile. Three or more titrations of each portion gave relative standard deviations of less than 0.3 ppt. The averages of the three portions had a range of only 0.2 ppt. These results indicate ferrocene is nonhygroscopic and stable on storage. Technical Grade Acetonitrile as Solvent. To assess the effects of impurities present in technical grade acetonitrile, two different shipments were used as received for copper(I1)ferrocene and copper(I1)-EDTA titrations. In one case the concentration of copper(I1) as determined by titrations of ferrocene was 5 ppt, and in the other, 3 ppt, lower than that found by titrations with EDTA. The addition of varying amounts of acetonitrile to the aliquots taken for the titrations with EDTA did not affect the results. Two modes of interference were considered, quantitative oxidation and complexation of contaminants in the solvent by copper(I1). The first requires that copper(1) formed by impurity oxidation be titratable by EDTA. Copper(1) in acetonitrile was found to be quantitatively titrated by aqueous EDTA. The second requires that complexed impurities be displaced by EDTA. Acrylonitrile, ammonia, acetic acid, ammonium acetate, and acetamide have been reported to be contaminants in technical grade acetonitrile. None, at the concentrations typically found in technical grade acetonitrile, interfered in the titration of ferrocene by copper(I1). Cyanide was determined by the method of Murty and Viswanathan (9) to be present at about 1 x lO-4M in the technical grade acetonitrile used in this work. Cyanide added to pure acetonitrile as potassium cyanide at the 1 X 10-4M level resulted in a copper(I1) concentration by ferrocene titration 2 ppt lower than the EDTA titration value. Therefore cyanide, most likely present as hydrogen cyanide, appears to be the contaminant in technical grade acetonitrile that interferes with the copper(I1tferrocene stoichiometry. (9) G. V. L. N. Murty and T. S . Virwanathan, Anal. Chim. Acta, 25, 293 (1961).
Use of Ferrocene as a Primary Standard. This work has shown that ferrocene is easy to purify, is stable and nonhygroscopic in air, reacts stoichiometrically and rapidly with copper(I1) in pure acetonitrile, and has a large potential break at the end point. These factors, in addition to a high equivalent weight, make ferrocene a satisfactory primary standard. Factors to be aware of are the temperature coefficient of expansion of acetonitrile and the volatility of both acetonitrile and ferrocene. None of these is a serious disadvantage at the part per thousand level if appropriate precautions are taken.
ACKNOWLEDGMENT We thank Robert Long for preliminary work on the purification of ferrocene, Glenn Johanson for the solubility measurements of ferrocene in acetonitrile, and the G. Frederick Smith Chemical Co. for a donation of hydrated copper(I1) perchlorate.
RECEIVED for review October 2, 1969. Accepted February 3, 1970. Financial support by the National Research Council of Canada and the University of Alberta is gratefully acknowledged.
Acid-Base Microtitrations Based on Serial Dilution Joseph R. Robinson, Honore Stelmach,’ and Stuart P. EriksenZ School of Pharmacy, University of Wisconsin, Madison, Wis. 53706 An acid-base microtitration procedure, based on a serial dilution technique, is presented together with the necessary mathematical description of the system as well as equations to calculate initial concentrations of the sample solution. The procedure, compared with conventional (buret) acid-base titrimetric techniques, has several advantages, namely, rapidity of operation, small sample size requirements, necessity for only one initial standard solution irrespective of the concentration of unknown acid (or base), and end point accuracy. It is shown that, although small volumes are involved, normal environmental conditions of air velocity, room temperature, and humidity have a negligible effect of the technique because the titration can be performed rapidly. Modifications of the basic proposal can be made to enlarge the scope and potential utility of this titrimetric procedure.
ACID-BASE titrations based on buret addition of titrant are generally time consuming and may require a fairly large sample of acid or base. Belcher ( I ) describes buret titration of microgrRm amounts of acids and bases. In addition, it is sometimes necessary to prepare more than one concentration of standard solution. A microtitration procedure has been developed that overcomes all of the disadvantages described above. The description and application of this procedure, as well as the theory involved, are the objects of this report. The basic approach and necessary equipment for the acidbase microtitration has been adapted from a microdilutor system that has found extensive use for serial dilutions in microbiological assays (2-4). Figure 1 shows the entire microtitration system, which is composed of a transfer loop capable of holding and delivering a precise, constant volume of fluid, a titration plate consisting of a series of pits into which can be placed the same or differing volumes of fluid (this can be an ordinary spot plate), and a microburet that is capable of accurate delivery of small volumes of fluid. 2
Present address, Abbott Laboratories, North Chicago, Ill. Present address, Allergan Pharmaceuticals, Santa Ana, Calif.
(1) R. Belcher, “Submicro Methods of Organic Analysis,” Elsevier, Amsterdam, 1966. (2) W. F. Lewis and E. M. Knights, Jr., Microchem. J., 8, 349 (1964). (3) E. A. Edwards, J . Bacteriol., 87, 1254 (1964). (4) M. J. Rosenbaum, I. A. Phillips, E. J. Sullivan, E. A. Edwards, and L. F. Miller, Proc. SOC.Exp. Bio. Med., 113, 224 (1963).
TITRATION
PLATE
Figure 1. Equipment to carry out acid-base microtitrations by the serial-dilution approach
The titration is carried out by preparing a series of known acid concentrations via serial dilution and titrating an unknown concentration of base against these acid concentrations until an end point is reached. Specifically,a given volume of water plus indicator is placed in the pits using the microburet; known acid is then placed in the first pit with the aid of the transfer loop, and the resulting solution is serially diluted into the remaining pits. This serial dilution is carried out by transferring a given volume from the first pit to the second pit, mixing well, transferring from the second to the third pit and so forth, all of the moves being carried out with the transfer loop. After obtaining a variety of acid concentrations in the pits, a sample solution of base (unknown concentration) is placed on the transfer loop and serially titrated against the acid until an end point is achieved. ANALYTICAL CHEMISTRY, VOL. 42, NO. 4, APRIL 1970
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