V O L U M E 2 8 , NO. 7, J U L Y 1 9 5 6
1177
The results obtained for the spectrographic analysis of fire assay beads in the leaching experiments are shown in Tables 111, IV, and V. DISCUSSION
The variables that xere altered in an attempt to find the conditions which would result in maximum recovery of platinum metals can be seen in Table I. Attempts were made to correlate these variables with the results of analysis shown in Table 11. S o definite advantage was gained for the 0 or P samples by the use of large buttons, by reassaying, or by niter assays. In addition to these variables, large alterations were made in the amount of litharge used in the niter assays without obtaining improved recovery. Slterations in the amount of silica present in the flux, as well as oversize buttons and reassaying, failed to produce any outstanding advantage in the case of H samples. Results obtained by niter assay for H samples were very similar to those obtained by assaying the roasted samples. All the evidence above can be interpreted to mean that a wide variety of conditions give equally good recoveries of platinum, palladium, and rhodium from the samples tested. One result for an 0 sample mas deleted from Table 111. It was considered to be due to the presence of a grain of platinum metals mineral. The presence of small, isolated grains of sperrylite could also account for the lack of precision with the ore samples. Scattered results are characteristic of analyses performed on samples in which the constituent sought is not intimately dispersed throughout the sample. Ideally, a fen- micrograms of platinum metals mineral must be distributed evenly in 30 grams of ore. This is not possible with any knoxn methods of sampling and mixing. I t is thus necessary to attach significance only to
very large assays or to the averages of several small assays. Leaching and normal assay results agree closely when the ore is concentrated. Averages of platinum, palladium, and rhodium recovered from H samples by leaching (Table V) agree well with the normal assaying results. In the case of the 0 and P samples the data in Tables I11 and IV may be interpreted only to indicate that leaching processes do not, in general, provide values higher than those obtained by normal fire assay. The results are not intended to define the precision which may be obtained by leaching processes. Undoubtedly more precise values could be obtained with 0 and P samples through the use of larger samples. However, the difficulties incident to the wet treatment of vary large amounts of ore encouraged the authors to limit their objective here to the question of the superiority of the leaching process. The peculiar variations between the proportions of leachable and unleachable platinum metals are difficult to explain satisfactorily. Undoubtedly difference in grain size is an important factor. ACKNOWLEDGMENT
Appreciation is also expressed to the Canadian Department of Agriculture, Science Service, for financial support and leave of absence given to I. Hoffman. LITERATURE CITED
Hawley, J. E., Rimsaite, Y., Am. JIl.line?.aZogist38, 163 (1963). (2) Hoffman, I., Beamish, F. E., AXAL.CHEM.28, 1188 (1956). (1)
RECEIVED for review December 9, 1955. Accepted March 5, 1956. Work supported by a grant from the National Research Council (Canada).
Determination of Polyphenol Oxidase Activity by Rotating Platinum Electrode LLOYD L. INGRAHAM W e s t e r n Utilization Research &a&
U. S. Department o f Agriculture, Albany 70, Calif.
Use of a polarized rotating platinum electrode enables polyphenol oxidase activity to be measured at various ascorbic acid concentrations, which is not possible with the commonly used chronometric method of Miller and Dawson. With this new method a continuous potentiometer record of oxygen consumption can be made.
T
HE catalytic activity of polyphenol oxidase, which is responsible for enzymatic darkening of fruits, is commonly described by a chronometric method that measures the time required for oxidation of a given amount of ascorbic acid (3, 9) with catechol as substrate. During studies in this laboratory it became necessary to determine the activity of polyphenol oxidase a t various concentrations of ascorbic acid, which is impossible with the chronometric method. Because the rate of the reaction catalyzed by polyphenol oxidase is not constant, but falls off rapidly with time from reaction-inactivation (S), it is desirable to be able to measure the rate of reaction during the first few minutes. A polarized electrode for measuring the oxygen consumed in the reaction seemed to satisfy this requirement. The first attempt with a polarized electrode was made with an alternating polarizing and depolarizing potential (IO). Al-
though this method was stable and accurate, 5 or 6 minutes were required for the cell to reach equilibrium after addition of the enzyme or substrate to initiate the reaction. This method was developed for photosynthesis studies (b) and would probably serve well in any determination where the first 5 minutes of the reaction are not so critical as with polyphenol oxidase. However, a rotating polarized platinum electrode was found to reach equilibrium within 5 seconds. The use of a rotating platinum electrode to determine oxygen in solution is well known (6-7, 11); its use in determining polyphenol oxidase activity by measuring the oxygen consumed in the reaction is described here. EQUIPMENT
A schematic diagram of the equipment is shown in Figure 1. The reaction cell containing the rotating platinum electrode ia polarized from the potentiometer with 0.800 volt. The current is measured by measuring the iR drop across R with a recording potentiometer. The variable resistance, R, is a standard decade resistance box which may be varied from 0 to 2000 ohms. The recording potentiometer has a range from 0 to 10 mv. and chart speeds of 11/2 and 6 inches per minute. Switch S is added to prevent erratic motions of the recorder pen when the cell is filled or emptied. The cell, which contains 5 ml. of solution with the electrode inserted, is shown in Figure 2. The electrode is 2 mm. long and
ANALYTICAL CHEMISTRY
1178
0.26 mm. in diameter. Electrical contact is made with the electrode by means of the iron wire, B , dipping in the mercury pool. The 6-mm. glass shaft of the rotating platinum electrode, which is turned by a '/eo-hp. induction motor a t 1750 r.p.m., fits into the cell through a Teflon sleeve, C. The cell is emptied with an aspirator through the capillary tube, D, and is filled with a longneedled hypodermic syringe through capillary E. The excess solution and air bubbles exit through a hole, F , in the Teflon bearing. The cell is attached to a calomel cell through a salt bridge. The lower end of the salt bridge a t G is stoppered with a sintered-glass plate and an agar plug saturated with potassium chloride. The whole cell is immersed in a 25.0' C. constant temperature water bath to the level shown at H .
Potentiometer
m I C
I
accumulate in the cell because it,s polymerization products coat the electrode and cause the diffusion current t o decrease. coated electrode may be returned to approximately its original characteristics by a washing with dilute alkali or alcohol. Four or five volumes of reaction mixture are washed through the cell to eliminate any air bubbles. Resistance R is adjusted so that the recorder reads 9.5 mv. The enzyme, usually about 0.2 nil., is injected into the cell by means of a 0.25-ml. syringe. If the enzyme is not injected with sufficient force, the curves soriietiniea show a 1- or 2-second erratic period indicating a small mising time. If the supply of enzyme is ample, this procedure can lie reversed-Le., the enzyme is added to the original reaction mixture and the reaction is initiated by the addition of substrate. In Figure 3 is shown a tracing of recorder paper from an actual determination of enzyme activit.y. During the time A B the consumption of oxygen by the system without substrate is measured (blank). At point B the substrate catechol y a s added. Peak C is an initial time marker produced by momentarily closing switch S (Figure 1). The rest of the curve after C s h o w the uptake of oxygen by the system when catalyzed by polyphenol osidase. INTERPRETATION OF RESULTS
Recording Potentimeter Figure 1. Schematic diagram of apparatus used to determine activity of polyphenol oxidase
H -
The plots in Figure 3 show that the oxygen-uptake curves are not linear nith time but have a decided curvature. However, in the usual operation of the equipment, the curvature is not too great to determine the initial slope directly from the experimental curve by means of a prism or mirror. The most convenient range t o measure is from 1 to 5 niv. per minute when the recorder speed is 11/% inches per minute. From the solubility of osygen in water saturated with air [5.% nil. per liter (e)], the definition of a catecholase unit [lo cu. mm. of oxygen per minute ( S ) ] , the calibration curve, already described and the fact that air i3 670 less soluble in solutions 0.02.11 in citric acid and 0.04.lf in disodium phosphate than in water, one may calculate that 1 m v . per minute is equal to 0.40 catecholase unit. The chrononietric method requires a t least 50 catecholase units for an activity determination. This method requires considerably less enzyme, although 50 catecholase units itself is very little. Figure 4 shows six individual deterniinat'ions of activity by this method on solutions containing various volumes of a preparation of polyphenol oxidase from prunes. Experimental. The preparation of prune enzpnie parallels thsr
Figure 2. Cell used to determine polyphenol oxidase activity A . Electrode B . Electrical contact (iron wire) C. Teflon sleeve
D. Capillary for emptying cell E. F. G.
Capillary for filling cell Air a n d excess solution outlet Lower end of salt bridge H. Level of water bath C
Q U
The current, measured by the recording potentiometer, is linear with the amount of oxygen present in the solution when the electrode is polarized with 0.800 volt. The intercept a t 0% oxygen is the diffusion current for hydrogen ion in a solution which 1s 0.04M in disodium phosphate and 0.02M in citric acid. The solutions saturated with gases containing various percentages of oxygen were prepared by carefully adding a known volume of a solution saturated with air to a known volume of a solution saturated with nitrogen.
x >
0
METHOD
The 5-ml. cell is filled with a solution 0.02M in citric acid, 0.04M in disodium phosphate, and 2 to 3+f in ascqrbic acid. The solution is saturated with air and contains varylng amounts of substrate depending upon which substrate is used. The pH of this soluhon is 5.5. The ascorbic acid is added t o the reaction mixture t o prevent o-quinone formation, because the polarized electrode will measure the o-quinone concentration in addition to t h e oxygen concentration. o-Quinone should not be allowed to
TI me
Figure 3. Direct tracing of actual recorder track showing oxygen consumption vs. time
V O L U M E 28, NO. 7, J U L Y 1 9 5 6
1179
0.04X in disodium phosphate, relative to that in pure water, was determined x-ith the rotating electrode by comparing a s o h tion 0.02M in citric acid and 0.04X in disodium phosphate, saturated with air, yith a solution of 50% of 0.04M citric acid and 0.08M disodium phosphate, saturated with nitrogen, and 507, of pure water saturated with air. After subtracting the diffusion current for a solution saturated with nitrogen, the diffusion current in the first solution \vas 69& less than tn-ice that 111 the latter solution.
4 t
LITERATURE CITED
Enzyme
Figure 4. Rate of oxygen absorption measured by rotating platinum electrode at various amounts of polyphenol oxidase from prunes Enzyme amounts are milliliters of enzyme per 100 ml. of reaction mixture; catechulase units would be 20 times t h a t shown for undiluted enzyme
used to prepare potato enzyme by Baruah and Swain (I), except that 0.5% Pectin01 (Rohm gS Haas Co.) which contained no polyphenol oxidase activity was added to eliminate the pectin. The solubility of oxygen in the solution 0.02111 in citric acid and
(1) Baruah, I?., Swain, T., Biochem. J . 55, 392 (1953). (2) Brackett, F. S.,Olson, R. A., Crickard, R. G., J . Gen. P h ~ s i o l . 36, 529 (1953). (3) Ingraham, L. L., Makower, B., ANAL.CHEm 27, 916 (1955). (4) International Critical Tables, vol. 111, p. 258, RIcGraw-Hill, Kew York, 1928. ( 5 ) Laitinen, H. A, Kolthoff, I. hI., J . Phys. Chem. 45, 1061 (1941); Science 92, 152 (1940). ( 6 ) Longmuir, I. S., Biochem. J . 57, 81 (1954). (7) Marsh, G. A,, ANAL.CHEW23, 1427 (1951). (8) Miller, W. H., Damon, C. R., J . Am. Chem. soc. 63, 3375 (1941). (9) Miller, W. H., AIallette, I f . F., Roth, L. J., Dawson, C. R., Ibid.. 66. 514 (1944). (10) Olson, R. A.,Brackett, F. S., Crickard, R. G., J . Gen. Phvswl. 32, 681 (1949). (11) Warshowsky, B., Schantz, E. J., ANAL. CHEx 26, 1811 (1954).
RECEIVED for review October 8, 1955. Accepted March 29, 1Q56.
Preparation of Buffer Systems of Constant Ionic Strength PHILIP J. ELVING, JOSEPH M. MARKOWITZ, and ISADORE ROSENTHAL University o f Michigan, A n n Arbor, M i c h .
Directions are given for the preparation of McIlvaine buffer systems of constant ionic strength, including a table of data.
THE
importance of using adequately buffered solutions for the qtudy of many types of chemical phenomena has long been recognized. The preparation of a large variety of buffer systems rovering the usual range of pH is described in many reference and textbooks (1,4-8). For certain purposes it is necessary to maintain the ionic strength of the solution relatively constant while varying the pH by varying the composition of one buffer system, as well as by using different buffer systems. For example, the polarographic half-wave potentials of certain types of organic compounds have been shown to be markedly dependent on the ionic strength of the test solution ( 2 , 3). The diffusion currents are also affected, although to a much lesser degree, and the slope of the polarographic wave is in some cases sensitive to ionic strength. In the case of simple buffer systems such as those involving acetic acid-sodium acetate and ammonia-ammonium chloride, it is relatively simple to keep the ionic strength constant over the normal buffering range of the system corresponding to pK, f 1. Bates ( 1 ) has described the preparation of a number of monobasic weak acid and monoacid weak base buffer systems of a definite ionic strength. However, in the case of more complicated buffer systems, such as those involving citrate and phosphate, it is much more difficult t o keep the ionic strength constant, The usual directions for preparing these types of bufiers result in large variation of ionic strength over the normal buffering range. Because the ionic strength depends upon the square of the charges on the ions uresent. the effect miaht be serious in
Table I.
Preparation of Constant Ionic Strength RIcIlvaine Buffered Solutions
Composition, G /Liter PH Solution Desired NazHP04. HsCsH10i a t 2 j 0 C. 12H20 Hz0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5,6 5.8 6.0
6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0
1.43 4.44 7.80 11.35 14.7 17.7 20.4 21.5 25.4 27.6 29.7 31.6 33.4 35.3 36.9 38.4 40.0 41.5 43.3 45.2 47.5 49.6 52.1 55.4 58.9 62.3 65.0 67.2 68.6 69.6
20 6 19.7 18.7 17 7 16.7 15.8 15.0 14.2 13.6 12.9 12.3 11.7 11.2 10.7 10.2 9.75 9.29 8.72 8.32 7.74 7 12 6.47 5 72 4.79 3.70 2.74 1.91 1.35 0.893 0.589
$ :;:
~~~i~ Strength,
M 0 0108 0.0245 0.0410 0.0592 0.0771 0.0934 0.112 0.128 0.142 0 157 0.173 0.190 0.210 0.232 0.256 0,278 0,302 0.321 0.336 0.344 0.358 0,371 0.385 0.392 0.427 0.457 0.488 0.516 0.540 0.559
G. KC1 Added per Liter of Solution t o Produce IonicStrength of 1.OM 0.5.M 74.5 72.7 71.5 70.2 68.7 67.6 6G.2 64.9 64.0 62.8 61.7 60.4 58.9 57.2 55.5 53.8 52.1 50.6 49.5 48.9 47.9 46.9 45.8 44.5 42.7 40.4 38.2 36.0 34.3 32.9
37.2 35.4 34.2 32.9 31.4 30 3 28.9 27.6 26.7 25.5 24.4 23.1 21.6 19.9 18.2 16.5 14.8 13.3 12.2 11.6 10.6 9.62 8.50 7.23 5.44 3.10 0.488
