Automatic Drop Timer for Use with Dropping Mercury Electrodes LOUIS 3IEITES A~YDJULIAX M. STURTEVANT Sterling Chemistry Laboratory, Yale University, New Haven, Conn. ADDITIOX to its importance in purely polarographic I Xmeasurements, the drop time a t a dropping mercury electrode
has recently been used to secure information concerning the potential of the electrocapillary maximum (4)and the polarographic critical concentrations of surface-active materials ( 1 , 6). Unfortunately, the manual determination of a large number of drop times, Rhen great precision is required, is a very tedious procedure. This communication describes an automatic apparatus for making these maasurements, and compares data on the average drop time thus secured with data on the lives of individual drops. VI 12AX7
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drop falls, the pull-in coil of S5 is actuated and its contacts complete the register and electric stop-clock circuits. S4 is then returned to the neutral position. The contacts of S5 remain closed until S4 is turned to the stop position. This actuates the release coil of S5 a t the fall of the next drop and thus opens the register and clock circuits. The operation of S5 is faster than that of the register, so t h a t the drop whose fall actuates the pull-in coil of S5 is counted, but that which actuates the pull-out coil is not. Accordingly, the total number of counts is correct. Sufficient gain is available to ensure reliable triggering with maximum cell currents as small as 0.2 to 0.3 pa. If considerably larger cell currents are employed, a series resistor smaller than 1000 ohms can be used. Thus the iR drop through the timer can easily be kept below 0.5 mv., and the appropriate correction can be applied t o the total applied potential if the cell current is knoLvn.
I
Figure 1. Circuit Schematic for Drop Timer All resistors 0.5 watt, 10% tolerance except a s noted; i n p u t resistor should be 1% tolerance. Capacitance values of 100 or above in micromicrofarads all others i n microfarads
Goldsmith ( 3 ) has described a drop counter, which does not include provision for timing the drops. His instrument uses a 22-henry inductor as the voltage-developing element. The effective impedance of the inductor changes during the drop life, which makes correction for the tR drop through the apparatus difficult. It has seemed preferable to insert a constant impedance in the cell circuit, and the authors have therefore used a 1000-ohm resistor as the voltage-developing element.
7000
7010
7015
D R O P TIME. S E C S
Figure 2.
DESCRlPTION OF C l R C U l T
Referring to Figure 1, the voltage drop across the 1000-ohm resistor in series v i t h the dropping electrode is amplified by three stages of conventional resistance-rapacitance coupled amplification, with differentiation by means of small coupling time constants. A germanium diode is inserted betn-een the second and third stages to remove the pulse produced by the rapid increase in the diffusion current during the early part of the drop life. The positive pulse output from V 2 A triggers a singly-biased multivibrator composed of V 2 B and V 3 A , and the positive output pulse from the multivibrator is fed through a cathode follower to a sensitive relay, 53, and a register (Mercury electromagnetic register, Production Instrument Co., Chicago, Ill.). At the start of a measurement, 5 4 is in the neutral position and the contacts of the latch-in relay (obtainable from Potter and Brumfield Manufacturing Co., Princeton, Ind.), 55, are open. The amplifier gain is increased slightly beyond the point a t n-hich 53 is heard to click at the fall of each drop. After one of these clicks is heard, S4 is put in the start position. When the next
7005
Drop Times in 1 F Potassium
Chloride A.
E.
By measurement of ten drop lives with drop timer By measurement of individual drop lives with millisecond timer
As little attention is ordinarily given to the possibility of picking up appreciable 60-cycle voltage in a polarographic circuit, it is advisable to check with an oscilloscope that the magnitude of any such voltage is considerably less than that of the voltage change which is intended to trigger the amplifier. A convenient point for this observation is betu-een the plate of VlA and the negative input terminal. The polarographic circuit should be so arranged that this terminal can be connected to a water pipe or other good ground. Figure 2, A , s h o w the data secured in 150 measurements of ten drop lives, using the apparatus described. The applied 1183
ANALYTICAL CHEMISTRY
1184
potential was -0.520 volt VS. S.C.E.; this is very near to the potential of the electrocapillary maximum of mercury in 1 F potassium chloride, so that dt/Ed e is neaily zero and the effect of small fluctuations in the applied potential is negligible. The maximum current during the drop life was about 0.3 Ha. These data show that the mean drop life is 7.0097 seconds, with a standard deviation of 1.2 milliseconds. Twenty determinations of ten drop lives by the conventional manual technique gave a mean drop time of 7.007 seconds, with a standard deviation of 4 milliseconds. The slightly lower mean value secured by the manual method reflects the vibrations set up by the manual manipulation of the clock. A more fundamental comparison was secured by timing 150 individual drops, under exactly the same conditions, Jvith the millisecond timer recently described by Sturtevant ( 7 ) and used in measurements of the instantaneous diffusion current by Grant, Mcites, and Sturtevant (6). With this instrument, the position of the sharp drop in current at the end of the drop life clan emily be determined u i t h a precision of better than a millisecond. However, because the action of the timer is not initiated until an indeterminate instant within the first niillisecond after the fall of the preceding drop, the measured time n i l 1 always be smaller than the true drop time. The average difference between the true and measured drop times in a large number of measurements would be expected to be about 0.5 millisecond. I n good agreement with this prediction, the mean drop time found from these data (B, Figure 2) was 7.0090 seconds with a standard deviation of 2.5 milliseconds. That this standard deviation is smaller than that which would be predicted from the data secured with the drop timer is probably due to fluctuations in the power line frequency. This would cause the indications of the electric stop clock to be in error, but would not affect the frequency of the tuning fork which serves as the standard of time in the millisecond timer. .4s a further test of the accuracy of the drop timer, the temperature coefficient of the drop time was determined. Five measurements of ten drop lives were made a t each of ten temperature.; between 10" and 40" in deaerated 0.1 F potassium chloride a t -0.520 volt vs. S.C.E. The data, shown in Table I, give the mean temperature coefficient of the drop time as - 2.16 ( f0.11) x 10-3 per degree. This mean deviation corresponds to an error of only a few tenths of a millisecond in the average drop time at each temperature.
Table I. Temperature Coefficient of Drop T i m e Temperature, C. t , See. ( - ~ t / t ax ~ )103 9.9
3.7526
12.9
3.7266
16.3
3.6976
19.5
3.6706
22.85
2 6-164
26. Oj
3.6202
29 3
3 5941
33,l
3,5646
2.30 2.32 2.24 2 00 2 25 2 20 2.17
The room in which these measurements were made has two solid brick walls about 15 inches thick and a very heavy steelreinforced concrete floor. It ie therefore extraordinarily free from vibration, so that these data probably provide a close approximation to the inherent precision of the drop time a t a dropping electrode of conventional form. It is evident that, under ideal conditions, the drop time is far more reproducible than has hitherto been believed ( 2 ) . ACKNOWLEDGMENT
The authors are indebted t o Stephen Boyan for his assistance in the design and construction of the circuit described in this paper. LITERATURE C l T E D
Colichman, E. L., J . Am. Chem. Soc., 73, 1795 (1951). English, F. L., Ax.4~.CHEM.,20, 889 (1948). Goldsmith, K., J. Sci. Instrumenls, 25, 385 (1948). Grahame, D. C., Larsen, R. P., and Poth, M.A., J . Am. Chenz. Soc., 71, 2978 (1949). Grant, D. IT.,hleites, L.. and Sturtevant, J. M.,Division of Analytical Chemistry, Symposium on Polarographic Methods, 120th Meeting, ~ E R I C . I N CHEMICALSOCIETY,Piew York, x. I-. hleites, L., J . Am. Chem. Soc., 73, 177 (1951). Sturtevant, J. M., Rev. Sei. Instrumenlu, 22, 359 (1951). RECEIVED for review December 6, l%l. Accepted March 1, 1952. Conmibution No. 1087 from t h e Department of Chemistry of Yale University.
Determination of Inorganic Phosphate Produced by Arterial Enzymatic Action FREDERICK K. BELL, C. JELLEFF CARR, AND JOHN C. KRANTZ, J R . School of Medicine, Unicersity of Maryland, Baltimore, ;Md.
THE authors have been interested in micromethods for thc 1 determination of inorganic phosphate in connection with studies of the adenosine triphosphatase activity of arterial tissue ( 3 ) . Experience with available methods was not satisfactory. The major disadvantages of these methods are: ( 1 ) the hydrolysis of adenosine triphosphate (ATP) by trichloroacetic acid; (2) the uncontrolled color development even in the absence of inorganic phosphate; and (3) the capricious color development in the final steps by the excess adenosine triphosphate substratc ( 2 ) . These difficulties thwarted the precise and absolute determination of the adenosine triphosphate activity of tissues of the vascular system, where the total amount of enzyme is small and the quantity of tissue is limited. A new method published by Griswold, Humoller, and McIntyre ( 2 ) for the estimation of inorganic phosphate in biological material involves several radical departures from earlier methods and appeared t o solve some of the difficulties. The inorganic phos-
phate is precipitated and isolated as magnesium ammonium phosphate, which is then converted, under dcfinite conditions, into the heteropoly molybdenum blue compound. The solution of this substance is very stable, displays a sharp absorption maximum, and is therefore suitable for accurate spectrophotometric analysis. By this method the excess adenosine triphosphate substrate can be removed from the reaction mixture and its subPequent interference with color development eliminated. Lowry and Lopez ( 4 ) used a buffer mixture of sodium acetate and ammonium sulfate for the precipitation of tissue protein. This reduces the capricious hydrolysis of adenosine triphosphate resulting from acid precipitation. The authors considered the probability that this step in their procedure could be carried out in this medium. This communication describes a method incorporating the advantages of the reaction mixture of DuBois and Potter ( I ) , the protein Precipitation procedure of Lowry and Lopez, and the
