Oxidation-Reduction Potential and pH Changes in Manometric Vessels Method for Continuous Recording KENT WIGHT and DEAN BURK National Cancer Institute, National Instituter of Health, Setherda, Md.
exchange, oxidation-reduction potential, and pH with specific reference to Chlorella photosynthesis. Thus a few attempts have been made to messurc the oxidationreduction potential, pH, and gas exchange simultitneously in dynamic biochemical systems. I n all such cases, however, only two of the variables wcre ever measured simultltneoudy. I n this research a method is described in which all three variables are measured simultaneously. Little or no attempt is made to interpret the ob
This investigation was carried out with the purpose of developing a technique for measuring and recording oxidation-reduotion potential and pH changes in vessels undergoing manometrio pressure changes. A large number of biochemical and analytioal reaotions may be studied in the manner indioated; glycolysis, respiration, or melanin formation in tumors, tissues, or homogenate preparations; and anaerobic or aerobic production or consumption of such gases as carbon dioxide, hydrogen, and oxygen by haeteria or algae, either thermally or photochemically.
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f o a reports exist in the literature concerning the simultaneous measurement of oxidation-reduction potential (0-R potential), pH, and manometric change. Cannan, Cohen, and Clark (3, 6) described a method of recording potential changes with a Leeds and Sorthrop recording potentiometer in bacterial cultures (Proteus vulgaris nnd Bacterium coli). The pH was measured separately by withdruniq samples a t intervals. Elema, Kluyver, and van Dalfsen (4,5), using Micrococcus denilrifieans, followed potential and pH changes manually during growth in an alcohol-sodium nitrate medium. Where nitrogen was being produced, the minometric changes were also measured. Kluyver and Hoogerheide (7) describd a modified Warburg vessel which contained a gold electrode and a capillary tube for measurement of oxidation-reduction potential, They folloned the potential changes of a number of bacterial suspensions. Wassink (10) manually recorded oxidation-reduction potential and manometric change in cultures of Chromatium by means of stationary electrodes contiuuallq immersed in the medium, dthough the p H determination involved periodic removal of aliquots of the cell suspension. Arnold et al. (1)have described a recording manometric apparatus. Since this paper was prepared, an extensive artiole by Spruit (9)has come to hand reporting simultaneous measurement of gas B
Ordinary single-arm vvaruurg Y C J J ~ ~(aayaciuy J 1 " LU f i w., ~ were modified as illustrated in Figure 1. Glass and platinum electrodes (Figure 1, A and B ) were fitted t o two arms of each vessel. A capillary tube (end diameter 0.75 mm. or less) fitted to a third side arm (Figure 1, C) contained saturated potassium chloride-agar. This capillary was connected by means of a potassium chloride-agsr filled plastic tube t o a bath of saturated potassium chloride in which two calomel electrodes (Figure 2) were placed
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AUTOMATIC SELECTOR SWITCH
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Figure 2. Diagram of Circuit for Measurement of pH and Oxidation-Reduction Potential of Bioehernioal Systems
The two calomel electrodes, glass electrode, and platinum electrode are in turn connected t o an assembly of instruments in the following manner: The glass and one calomel electrode are in .~. .~ ~~~. ~. ~~~
Figure 1.
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trode and other calomz electrode are wired t o aiother selector switch which is in contact with a separate oxidation-reduction amplifier and multiple recorder (circuit diagram in Figure 2). The inStNment8 for measurement of pH are: the Beckman automatic multiple electrode switch No. 1700, Beckman Model R glass electrode pH meter, and the Brown Electronik strip chart recorder (Figure 3 B'; Model No. Y 153 X 64 ,X 6-X41CY range 0 t o 35 mv.). For the determinatmn of oxidationreduction potential, the Beckman automatic multiple electrode switch No. 1700, Beckman Model RM millivolt indicator, and the Brown Electronik strip ohart recorder (Figure 3, B; Model No, Y 153 X 64 X 6-X-41CV, range -2.5 t o +42.5 mv.) were utilized.
Side View of Modified Warburg Vessel
C. Side arm for Glass aleourode B. Platinum electrode inulrporation of capillary tube for pofrsaiurn chloride-sg-sr bridge
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ANALYTICAL CHEMISTRY
By means of the selector switches and multiple recorders, the pH and Eh of the materials in as many as six modified Warburg
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one cell and sKort:circuithg the terminals. Therecording mad; is constant; if it deviates it can be adjusted. This is not true when the cell is in the circuit because of the possible variation of the electrodes. The entire circuit is diagrammatically represented in Figure 2, and the entire assembly of instruments is illustrated in Figure 3.
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the recordina is made can be understood. Furthermore. in such ~
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Printing O C C U ~ Severy 15 seconds. When the recording is automatic for six vessels, the pH and oxidation-reduction potential are recorded every 1.5 minutes; this can be varied. The limitations of accuracy of recording of pH measurements are =tO.O5 unit; for oxidation-reduction potentials, +4 mv. The oxidation-reduction potential can be measured from -700 to +700 mv.; the pH range covered is from 3.0 to 10.0. The pH and oxidation-reduction potentials of most biological systems fall within theselimits. The electrodes are assembled in the vessels, each vessel with its own electrodes (Figure 1); they are held in place with rubber
Figure 3. Assembly of I n s t r u m e n t s for Recording pH a n d Oxidation-Reduction Potential of Systems Undergoing Manometric Chanae
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Figure 5. Manometric and pH Changes of Anaerobic GIyeolysis (95% Nitrogen-5% Carbon Dioxide) h y Ascites Tumors in Warburg-Okamoto Ringer's Solution
Figure 6 . M a n o m e t r i c a n d pH Changes of Anaerobic Glycolysis (95% Nitrogen-5% Carb o n Dioxide) b v Ascites T u m o r s in WarbureOl&m>to Ringer's Solution I
bands. The glass electrode is previously standardized with buffer; for the platinum electrode a solution of potassium ferricyanide-sodium ferrocyanide is used. RESULTS
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