Self-Equilibrating Electrolytic Method for Determination of Acid Production Rates DAVID W. EINSEL, JR., HANS 1. TRURNIT', SEYMOUR D. SILVER, and EDWlN C. STEINER Chemical Corps M e d i c a l Laboratories, Army Chemical Center,
As a method for measuring cholinesterase activity quickly and accurately was desired, an automatic electrolytic titrator was developed. The titrator is designed to measure directly the rates of acid-producing reactions, but with appropriate modifications it may be used to measure the rates of other chemical reactions or dynamic processes such as diffusion or solution.
The electrolytic cell is a modified veision of the one used by Epstein, Sober, and Silver (4).I t consists of two compartments, A and B, separated by a semipermeable (parchment) membrane C, and each equipped nith a platinum electrode, D,E . The cathode compartment, A , is further equipped with a stirrer (magnetic or Vibro-Mix), F , pH electrodes G, gas inlet H , and thermometer if desired. Compartment A is sealed and a slight positive pressure of nitrogen is maintained over it to prevent diffusion of carbon dioxide into the cell. Its size may be varied according to the needs of the reaction being studied; 1 5 , S O - , and 75-ml. capncities have been used in this laboratory.
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N R E C E S T years numerous articles have described coulometric titration techniques for analyzing oxidizing or reducing compounds, ions such as hydrogen, copper, or chloride, and some organic compounds such as mercaptans. Cooke and Furman ( 2 ) have given an excellent review of these methods up to 1950. In most cases to date the techniques have been used to determine amounts or concentrations of these substances-Le., batch method of analysis. I n certain instances (1, 3, 4 , 8 ) they have been used for continuous titrations, with automatic devices to follow changing amounts of the titrants, but the technique has not been used to follow the kinetics of chemical reactions taking place in the electrolytic cell. For this purpose the instrument described has been developed. Specifically, it has been developed to determine the rate at a-hirh cholinesterase (ChE) catalyzes the hydrolysis of acetylcholine chloride (AcCh), but with slight modifications it could be used to measure other reactions. When studying enzyme systems it is often very difficult or impossible to determine the concentration of active enzyme present in a tissue preparation, but it is sometimes possible to measure a value that is proportional to the concentration. This value, called activity, is obtained by measuring the rate at which the enzyme acts upon its substrate under conditions which make the reaction peeudo-zero order. In the case of cholinesterase the substrate is acetylcholine and the reaction proceeds as folloll-s:
The type of platinum electrode used in the cathode compartment is critical. Beckman platinum electrodes (KO. 281) have been found satisfactory, but ordinary platinum wire may be unsatisfactory unless the exposed surface is kept very small. If a large surface of platinum is exposed, the electrolj sis may appear to be much less than 100% efficient, and there may be marked pH drift when the electrolysis is terminated. No explanation for these phenomena has been found.
II
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J m I-
+---1
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ELECTROLYTIC
The activity of tissue preparations containing cholinesterase may be determined in two general n-a?s. The rate of consumption of substrate may be measured colorimetrically ( 5 ) or the rate of production of acetic acid may be measured by allowing the reaction to proceed in the presence of a bicarbonate buffer in a Warburg apparatus. Carbon dioxide is given off as the acid appears and the rate can be measured manometrically by measuring the rate of change in pH in a specially buffered reaction mixture ( 7 ) ) or by measuring the rate a t which alkali must be added to an unbuffered reaction mixture to maintain constant pH (9). The last method may be done manually or automatically. The method described here is similar to the last method, except that constant pH is maintained by removing hydrogen ions electrolytically at the same rate as they are formed by the enzyme reaction. This is accomplished automatically by a feed-back system somewhat resembling that described by Lingane ( 6 ) .
Figure 1.
RECORDER
A i
I I
L
I
CELL
Schematic diagram of automatic electrolytic titrator
RI.
10,000 ohms, 0.5 watt Helipot 10-turn 1000 ohms (Beckman) Potentibmeter, i0,OOO ohms tapped a t 9000 ohms (General Radio) 20,000 ohms, 5 watts Rs. 530 ohms 0.5 watt XI. .\Idtiran& milliammeter, 0-1, 0-5, 0-10, 0-25, 0-50 ma. M g . .\lilliammeter, 60 ma. R2. Rz. Ra.
The pH electrodes are used in the usual manner with a Beckman pH meter (Model G), some-what modified. Its design requires that a definite current pass through the null meter, I, in order to bring it to the zero or balanced position. The voltage drop existing across the meter is utilized to tactivate a BrownHoneywell strip-chart recorder in such a way that the recorder follows the movements of the null meter needle. The two units are coupled through a voltage divider, J , which permits adjustment of the position of the recorder pen relative to the magnitude of the current passing through the null meter. Some calomel reference electrodes are sensitive to a pressure differential between the inside and outside of the electrodes. This trouble was eliminated by connecting the electrode opening to the nitrogen inlet. The feed-back system consists of a potentiometer, L, coupled
IN STRUM ENTATION
A schematic diagram of the instrument is shonm in Figure 1. 1
BROWN
pH METER
ChE (C H ~ ) & C H ~ C H Z O ~ C C H H20 ~ + ( C H ~ ) ~ N C H ~ C H Q OCH3C02H H
+
Md.
Present address, RIAS, Inc , P.0 B o x 1574, Baltimore 3, Md.
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V O L U M E 28, NO. 3, M A R C H 1 9 5 6 mechanically to the pen drive of the recorder in such a \yay that an increasing voltage is applied to the electrolytic cell as the pen, K , moves to the left. OPERATION
I n general, the instrument is operated by placing a suitable electrolyte containing an enzyme sample and its substrate in the cathode compartment] A , and the electrolyte only in the anode compartment, B. The pH meter and recorder are activated and the battery circuit is closed. The instrument then equilibrates in such a way as to keep the pH constant. When the pH is constant] the rate of hydrogen ion production due to the enzymatic reaction may be equated to the rate of removal of hydrogen ions by electrolvsis, which in turn may be calculated by applying Faraday's law to the equilibrium current. Thus, multiplying the equilibrium current in milliamperes by the factor 0.621 peq. per ma.-minute gives the reaction rate in microequivalents per minute.
c
0 ._ c
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v1
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a c l
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desired value and following the readjustment procedure outlined above. The time to reach each equilibrium ranges from 0.5 to 3 minutes with cell volume of 13 ml. The p H during a determination is maintained accurately to within 0.01 p H unit. CALIBRATION
The instrument was calibrated by adding standardized hydrochloric acid to the cathode compartment a t measured rates. The current necessary to keep the pH constant mas recorded and compared with the rate of acid addition. I n a series of five runs using electrodes \T-ith small surface areas, the error ranged from 0 2 to 2.8%, the electrolj-tic value being too high in all cases. Some trouble was encountered in this type of calibration due to fluctuations in the pH meter caused by inhomogeneous concentration in the cell, These troubles do not occur in studying reactions in which hydrogen ions are produced throughout the solution. That the cholinesterase activity measured by the instrument is proportional to the concentration of enzyme in the cell was shown by varying the amount of enzyme source (blood) added to the cell and plotting it against the observed equilibrium current (see Figure 3). The media used in the cell for these determinations included 0.131 to 2.031 solutions of sodium chloride, sodium bromide, potassium chloride, and potassium bromide, with and without added 0.0154 magnesium chloride. Sample volumes up to 2.0 ml. 11-ere used in a cell containing 68 ml. All measurements were made a t pH 7.3, n-ith a substrate concentration of 0.004M acetylcholine chloride (Merck). All the graphs of concentration 21s. activity obtained under these limited conditions iyere linear. The slopes of the graphs varied with the specific activity of the enzyme.
Time Figure 2.
Typical curve obtained during process of making measurement
The way in which automatic control of pH is achieved may be seen from the schematic diagram (Figure 1).
If no acid is being produced in compartment A and the pH in the compartment is the same as that set on the pH meter, the null meter will be steady a t the zero mark. If voltage divider J is adjusted so that the recorder pen and potentiometer L are a t the electrical zero of the potentiometer, the battery switch may be clossd n-ithout causing a change in the cell. If, now, acid is produced a t a constant rate in A , the decrease in pH will be reflected in the leftn-ard movement of the null meter needle, the recorder pen, and the potentiometer contact. This causzs a voltage to appear a t the platinum electrodes, resulting in electrolytic removal of hydrogen ions from the cathode compartment. The pH will continue to decrease and the voltage to increase until the removal of hydrogen ions is just as fast as the production of hydrogen ions. At this point the pH n-ill remain constant and a dynamic equilibrium vi11 have been reached. In practice it may be necessary to measure the rate of a process at a precisely knom-n pH The equilibrium described above occurs at a pH lower than that set at the p H meter. I n order to equilibrate a t the set pH the instrument must be manipulated as follows (see Figure 2): After the generation of acid has been started, 1, and the instrument has reached its preliminary equilibrium, 2, the glass electrode is temporarily disconnected by releasing the push button on the pH dial. This brings the pointer of null meter Z and consequently the position of the recorder pen back to zero, 3. Immediately, potentiometer J is adjusted, 4, to bring the pen to its preliminary equilibrium position. The push button is then depressed, 5, and the instrument allowed to equilibrate, 6.
If, after a determination is completed a t a given pH, another is desired at a different pH, the new determination may be obtained with the same sample by setting the drum of the pH meter to the
0.2
Volume
0.4
0.6
0.8
of S a m p l e (m13
Figure 3. Relationship betw-een enzyme sample volume and apparent activity of dog whole blood I n 0.5M S a C l , 0.01.11 IlgClz, 0.004.11 AcCh solution a t p H 7.3
The intercept of the graph a t zero sample volume corresponds to the rate a t which acetylcholine chloride is split in the absence of cholinesterase in a solution of pH 7 . 3 . I n routine determinations of enzyme activity this value is considered as a blank and is subtracted from the observed equilibrium current. It is reproducible and depends upon the concentration of acetylcholine chloride and upon the total amount of solution in the cell.
ANALYTICAL CHEMISTRY
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The precision of the instrument has been determined by measuring the cholinesterase activity of 10 aliquot portions of a preparation of goat red blood cells. The standard deviation was found to be ~ t 0 . 0 4peq. per minute. DISCUSSION
Thus far, the instrument has been used only in studying the enzyme system cholinesterase. The enzyme activity of the blood of humans, dogs, rats, goats, and rabbits, and of rabbit brain has been studied. The instrument's operation has been equally satisfactory with these different tissues and it should be satisfactory with homogenates of other tissues. A particularly useful application of the instrument in the field of enzymes is the study of enzymatic activity as a function of pH, temperature, or ionic strength. The procedure for varying pH was described above. Its advantages with respect to time and sample economy are obvious. Temperature could be varied systematically if the electrolytic cell rn ere thermostated, and the change of enzyme activity with temperature could easily be determined. Ionic strength could be varied by adding calculated amounts of salts to the cell after each equilibration. Although the instrument has been designed to measure pseudozero-order reaction velocities, it could be used to measure firstorder reaction velocities. Rate constants could be evaluated in the usual way, except that the milliammeter readings would be substituted for concentrations of reactive species as follo5-s:
where
CO z1and 2 2 u1 and
u2
= initial concentration of reactant = amounts reacted a t time tl and t 2 , respectively = reaction velocities a t time tl and t2, respectively
I I and I2 = current readings a t time t l and f2, respectively k
= specific rate constant
In this type of determination the pH must necessarily increase. However, the increase may be made so small that it will not appreciably affect the reaction nor the observed current. Dynamic processes other than chemical reactions, such m gaseous, ionic, or liquid diffusion or solution rates, could be studied in this instrument after suitable modifications of the electrolytic cell, the sensing element, and the electrodes. LITERATURE CITED
Austin, R. R., Turner, G. K., Persy, L. E., Instruments 22, 588 (1049). --, \ - -
Cooke, W. D., Furman, K. W., ANAL.CHEM.22, 8 9 6 9 (1950). Eckfeldt, E. L., U.S.Patent 2,621,671 (Dee. 16, 1952). Eustein, J., Sober, H. 9., Silver, S. D., ANAL.CHEY.19, 675-7 (1947).
Fleisher, J. H., Pope, E. J., Arch. Ind. Hug. and Occupational Med. 9, 323-34 (1954).
Lingane, J. J., ANAL. CHEM.21, 497-9 (1949). Michel, H. O., J . Lab. Clin. Med. 34, 1564-8 (1949). Shaffer, P. A., Jr., Briglio, A , , Jr., Brockman, J. A, Jr., ANAL. CHEM.20, 1008 (1948). Tammelin, L. E., Low, H. E., Acta Chem. Scand. 5, 322 (1951). RECEIVED for review May 18. 1955. Accepted December 3, 1955. Requests for reprints should be addressed to Hans J. Trurnit, e t RIAS, Inc., P.O. Box 1574. Baltimore 3, M d .
Ultraviolet Spectrophotometric Determination Phosgene with An iIine WARREN B. CRUMMET" M a i n Laboratory, The Dow Chemical Co., Midland, Mich.
An ultraviolet spectrophotometric method has been developed for the determination of phosgene based upon the absorption of the 1,3-diphenylurea formed when phosgene is allowed to react with aniline in aqueous solution. The method is sensitive, specific, and capable of good precision.
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HE aniline method for the determination of phosgene was first described by Kling and Schmutz (6) and shortly afterwards was successfully used by Biesalski ( 1 ) in studying the thermal decomposition of carbon tetrachloride. The method consisted in bubbling the gas to be tested through water saturated with aniline and weighing the 1,3-diphenylurea (carbanilide) formed. Olsen and coworkers ( 7 ) studied this method and found that 100 ml. of the aqueous aniline solution dissolves about 5.5 mg. of 1,3-diphenylurea. They therefore modified the procedure by first saturating the solution with diphenylurea to eliminate solubility errors. Comparing this technique with the sodium hydroxide (8), silver nitrate ( 7 ) , and sodium iodideacetone methods (4), these investigators recommended their procedure and that involving the use of sodium iodide and acetone. General acceptance of the aniline method by industry testifies to its practicality. However, it has some inherent weaknesses. Very small precipitates are difficult to handle. Some foreign materials may precipitate, giving high results. The solubility of diphenylurea may vary m-ith the conditions under which the sample is taken. On consideration of these variables it seemed
of interest to develop a new method which would be more sensitive and more specific. The variation in the absorption spectrum of aniline with pH has been studied by Tischler and Howard (9) from 305 to 255 mp. In acid solution the absorbance is much smaller than in basic solution and the spectrum is similar to that of benzene (3, 6). The ultraviolet absorption spectrum of l13-diphenylurea in ethanol has been reported by Schroeder and coworkers (8). The spectra of these compounds in methanol were studied in the present investigation (Figures 1 and 2). It was found that fairly small quantities of l13-diphenylurea can be determined in the presence of relatively large amounts of aniline in acidic methanol. Although both compounds exhibit absorption maxima a t 254.5 mp, the absorbance of 1,3-diphenylurea on a weight basis is 93.6 times as intense as that of aniline. Aniline also exhibits a sharp absorption peak a t 260.5 mp, which can conveniently be used to determine the quantity remaining after the phosgene has reacted. APPARATUS AND REAGENTS
A Cary recording spectrophotometer, hlodel 11M5, with matched 1-cm. silica cells, was used for absorbance measurements. The slit control was set to produce a slit width of 0.12 mm. at 254.5 mp. A manually operated spectrophotometer may be used, if enough points are plotted. The spectrum of 1,3-diphenylurea was determined on solutiona of a recrystallized product, melting point 235.5-7' C . Fresh1 distilled aniline was used to prepare solutions in water, in sucg concentrations that 50 ml. contained about 2 mg. per mg. of
