A GLASS ELECTRODE APPARATUS FOR MEASURING THE pH VALUES OF VERY SMALL VOLUMES OF SOLUTION

A glass electrode apparatus is described with which pH measurements can be made with as small volumes as 2 drops (about 0.14 cc.) of solution. Using this apparatus the change of pH of the vacuolar sap of Nitella, due to the penetration of brilliant cresyl blue, has been readily followed. The sap and the dye have been found to poison the usual type of hydrogen electrode.     

A glass-membrane pH microelectrode.

By miniaturizing the original MacInnes and Dole glass-membrane pH electrode a new pH microelectrode has been developed. The technique developed utilizes the tip of a high electrical resistance glass pipet that can be sealed with a thin membrane of H⁺-sensitive glass. Single-barreled electrodes have been made with tip diameters ranging from 1.5 to 100 μm and double-barreled electrodes with tip diameters from 2 to 28 μm. The glass-membrane pH microelectrode provided a means for sensing the pH of biological solutions with an electrode having theoretical slope and tip configurational control. The most unique characteristics of the electrode were: the pH sensing surface was quite small, the tip diameter could be controlled, and the problem of electrode insulation was eliminated.     

Microelectrode of the Thomas type using a liquid membrane electrode

By replacing the glass-based pH electrode (L. R. Pucacco, S. K. Corona, H. R. Jacobson, and N. W. Carter (1986) Anal. Biochem. 153, 251-261) with a liquid membrane-based pH electrode, a relatively easy-to-manufacture modified Thomas electrode has been developed. The liquid membrane-based modified Thomas electrode can be manufactured without the special equipment (forge) and materials (glass) required to make the glass membrane pH microelectrode (L. R. Pucacco and N. W. Carter (1976) Anal. Biochem. 73, 501-512). The sensitivity (57.4 +/- 0.22 mV/pH unit), response time (20.0 +/- 2.67 s), and electrical resistance (3.48 +/- 0.67 X 10(11) ohm) of this electrode are similar to those of the glass-based version.     

Studying the drift of in line pH measurements in cell culture

The culture of Chinese hamster ovary (CHO) cells to produce monoclonal antibodies (MAb) requires accurate measurement and control of pH. Unwanted pH drifts in cell culture can adversely affect process performance, product quality, and product yield. To measure and control pH throughout the length of a culture, most cell culture processes use traditional glass pH probes. Several variables can affect the design and performance of glass pH electrodes and lead to drift in the measurement. Understanding these variables and their effects on pH performance can lead to design improvements and potentially reduce the drift. In this study, a set of Rosemount Analytical glass pH probes was investigated in cell culture operations. Electrochemical properties of the probes were monitored throughout the experiments. Experimental results show that the glass membrane potential experiences the biggest change during cell culture operations. Changes in the reference electrode potential are small compared with the changes in glass membrane potential. The glass membranes are affected by the steam sterilization process and this is the main cause for drift in the probe sensing signal during cell culture operations. Steam sterilization can cause the potential of glass membranes to change by up to 15 mV (～ 0.25 pH units). This change in membrane potential can be observed as an undesirable pH drift in bioreactors.     

Rapid pH change due to bacteriorhodopsin measured with a tin-oxide electrode.

The photocurrent transient generated by bacteriorhodopsin (bR) on a tin-oxide electrode is due to pH change and not to charge displacement as previously assumed. Films of either randomly oriented or highly oriented purple membranes were deposited on transparent electrodes made of tin-oxide-coated glass. The membranes contained either wild-type or D96N-mutant bR. When excited with yellow light through the glass, the bR pumps protons across the membrane. The result is a rapid local pH change as well as a charge displacement. Experiments with these films show that it is the pH change rather than the displacement that produces the current transient. The calibration for the transient pH measurement is given. The sensitivity of a tin-oxide electrode to a transient pH change is very much larger than its sensitivity to a steady-state pH change.     

Fabrication of pH-sensitive implantable electrode by thick film hybrid technology.

We report preliminary results of our experiments directed at fabricating pH-sensitive electrodes suitable for in vivo use by means of thick film screening techniques. Our results show that glass membranes of suitable thickness and possessing nearly theoretical sensitivity to pH can be fabricated by this process. A hybrid electrode structure permits the incorporation of a source follower FET amplifier directly adjacent to the pH membrane, significantly reducing response time and noise pick-up. Extension of the basic electrode structure to accommodate membranes sensitive to other ions is discussed.     

Iridium oxide pH microelectrode

The manufacture, calibration, and signal conditioning during construction of an iridium/iridium oxide pH microsensor is described. The microsensor was designed to be used extracellularly, primarily in biofilm research. The sensing tip diameters were typically in the range of 3-15 mum. The iridium oxide was formed by potential cycling in dilute sulfuric acid. A pH profule across a denitrifying biofilm was measured as an example of an application. The higher Nernstian slope (70-80 mV/pH for fresh electrodes), increased rigidity, and restriction of the sensing tip to the outermost end of the electrode are features which make the iridium/iridium oxide pH microelectrode superior to a glass microelectrode. (c) 1992 John Wiley & Sons, Inc.     

Electrode and cuvette for rapid continuous CO2 tension and pH measurement in blood

An electrode and cuvette system has been developed for the continuous and rapid measurement of either blood CO2 tension or pH. The CO2 electrode consists of a 1.5-mm-diameter flat-tip glass pH electrode covered by a film of carbonic anhydrase solution, over which a 25-micron-thick dimethyl silicone membrane is attached. Porous ceramic filled with 20% polyacrylamide, equilibrated with a salt solution, serves as a salt bridge between a Ag-AgCl reference electrode and the pH electrode surface. The electrode is housed in a four-port cuvette assembly. Blood from a vessel of interest is delivered to the cuvette by means of an occlusive roller pump. The cuvette maintains the electrode and blood at a constant temperature and directs a continuous jet of blood against the electrode surface. The cuvette also allows for easy and frequent calibration of the electrode with either gas or liquid standards. The 90% response time of the CO2 electrode is 3.0 s for liquids and 1.3 s for gases. Removal of the dimethyl silicone membrane and carbonic anhydrase film yields a pH electrode that can continuously measure blood pH with a 90% response time of 1.6 s.     

ONTOGENY OF CHICKEN HEMOGLOBIN II. CHROMATOGRAPHIC STUDY OF THE HETEROGENEITY OF HEMOGLOBIN IN DEVELOPMENT

Some properties of the carbonmonoxyhemoglobin (HbCO) from chicken embryos of ages 5, 10 and 15 days of incubation, from 1-day posthatching and from adult chickens have been investigated by chromatography on carboxymethylcellulose (CM-cellulose) column and by starch gel electrophoresis.
Chromatogram of the hemoglobin (Hb) from 5-day chicken embryos has shown that it consists of at least 6 components. Starch gel electrophoresis of each isolated component from the column in phosphate (pH 6.8), in borate (pH 8.6) and in formate buffer (pH 1.9) has shown later that there are 3–4 embryonic type Hb components in 5-day embryos.
Chromatogram of the hemoglobin from adult chickens has shown that it consists of at least 4 components, but the examination of each isolated component from the column by electrophoresis in phosphate (pH 6.8), in borate (pH 8.6) and in formate buffer (pH 1.9) has shown that there are 4–6 adult type Hb components in adults.
In ontogenic process, embryonic Hb type is detectable in embryos up to 15 days of incubation. Fetal Hb type, which is not detectable in adult chickens, can be first found in 10-day embryos.     

Agarose Gel Electrophoretic Separation of Blood Serum Proteins in Cattle

The agarose gel electrophoresis described by Johansson (1972) was modified so that a buffer of pH 7.9 was used in the gel, whereas the buffer in the electrode vessels had a pH of 8.6. The cattle blood serum protein picture is described in detail. The β1-globulin zone shows a very distinct picture of the genetically polymorphic bovine transferrins. The region between the α- and β-globulins shows a number of faint and often very distinct bands. A faint background staining over the whole electrophoretogram may partly be caused by a rather strong lipoprotein in the α1-region, lipids thus having migrated all over the electrophoretogram. The modified method described is well suited as a “screen electrophoresis” for cattle serum and is also useful e.g. in studying bovine transferrin polymorphism.     

Fundamental aspects of antimony thin film electrodes for pH measurement.

This report concerns the investigation of the sensitivity, temperature dependence, accuracy, and the standard electrode potential EO of an antimony thin film pH electrode which was prepared with electron beam evaporation techniques. The air-formed oxide film on antimony thin film electrodes has been proved by both the cathodic reduction method and electron spectroscopy for chemical analysis (ESCA). The antimony thin film electrode responded rapidly to pH changes and its sensitivity was slightly changed depending on the buffer composition. The accuracy of this electrode was compared with that of the glass electrode. Temperature had some influence on the function of this electrode. The standard electrode potential of this electrode was discussed together with that of other forms of antimony electrodes. The structure and thickness of the surface oxide on antimony thin film electrodes was confirmed by cathodic reduction and ESCA. It was clear that the surface oxide governs the electrode reactions. Possible applications of the antimony thin film electrode are discussed stating some limitations in the use.     

Use of ribozyme cleavage kinetics to measure salt-induced changes in solution pH

Several ribozymes are active in molar concentrations of monovalent salts, and pH rate curves under such conditions are consistent with mechanisms involving general acid–base catalysis. Interpreting the apparent pK_a values requires an accurate estimation of solution pH, which can be difficult to obtain using a typical glass pH electrode in the presence of high salt concentrations. In the current work we have used the VS ribozyme as a “biological pH meter” to evaluate the effects of molar concentrations of monovalent salts on changes in solution pH. We find that almost all of the measured change in pH observed in high concentrations of LiCl is due to a real change in solution pH. In contrast, high concentrations of NaCl cause errors in pH measurement in addition to changes in actual pH. Different buffer systems affect both the direction and the magnitude of pH changes: we observed changes in measured pH of up to 1 pH unit, which require proper interpretation to avoid adverse effects on the conclusions drawn from pH rate and other experiments that utilize very high salt concentrations.     

A micro pCO2 electrode

By utilizing a previously developed micro pH glass electrode it has been possible to make a micro pCO₂ electrode with a tip diameter of about 10 μm. This was accomplished by placing the micro pH electrode in a conical tube containing a weak NaHCO₃ solution. The tip of the conical tube was closed with Teflon^® oil wax mixture. This closure prevented the flow of solution, but allowed CO₂ to pass into the NaHCO₃ solution thus altering the pH of this solution. Changes in pH were seen and measured by the micro pH electrode and could be related to the pCO₂ of gas or solution in which the total electrode system was placed. This electrode, principally because of its small size, has many possible applications in biological research.     

EFFECTS OF ACETATE ON THE GROWTH AND CHLOROPHYLL CONTENT OF GOLENKINIA1

Growth and chlorophyll synthesis by the green alga Golenkinia cease after approximately 60-hr incubation in 0.01 M sodium acetate. These effects are immediately preceded by a rapid rise of the pH of the medium to 8.6–8.8 due to acetate uptake. The pH kinetics are due to a gradual loss of the phosphate buffer, not to the induction or activation of acetate assimilatory enzymes. However, neither high pH nor sodium acetate alone is sufficient to inhibit cell division and bleach the algae; both must be present. Additional experiments support the hypothesis that acetate alters the cells so that they become sensitive to high concentrations of OH^? ions.     

THE R?LE OF THE ACTIVITY COEFFICIENT OF THE HYDROGEN ION IN THE HYDROLYSIS OF GELATIN

1. The hydrolysis of gelatin at a constant hydrogen ion concentration follows the course of a monomolecular reaction for about one-third of the reaction. 2. If the hydrogen ion concentration is not kept constant the amount of hydrolysis in certain ranges of acidity is proportional to the square root of the time (Schütz''s rule). 3. The velocity of hydrolysis in strongly acid solution (pH less than 2.0) is directly proportional to the hydrogen ion concentration as determined by the hydrogen electrode i.e., the "activity;" it is not proportional to the hydrogen ion concentration as determined by the conductivity ratio. 4. The addition of neutral salts increases the velocity of hydrolysis and the hydrogen ion concentration (as determined by the hydrogen electrode) to approximately the same extent. 5. The velocity in strongly alkaline solutions (pH greater than 10) is directly proportional to the hydroxyl ion concentration. 6. Between pH 2.0 and pH 10.0 the rate of hydrolysis is approximately constant and very much greater than would be calculated from the hydrogen and hydroxyl ion concentration. This may be roughly accounted for by the assumption that the uncombined gelatin hydrolyzes much more rapidly than the gelatin salt.     

Direct measurement of proton binding to the active ternary complex of pig heart lactate dehydrogenase (Short Communication)

With the use of a glass electrode it is shown that one proton is taken up from the solvent when o-nitrophenylpyruvate forms a ternary complex with lactate dehydrogenase and NADH at pH8 and before the complex has rearranged to give products.     

Diary

Abstract

The acid-base properties of hydrous ferric oxide were studied by glass electrode potentiometry. From the Potentiometrie data, surface protonation constants were derived according to the Diffuse Double Layer convention. Chemical equilibrium modelling of the experimental system suggests that titration points below pH 4 should not be used for the determination of protonation constants because of potential HFO dissolution. Surface protonation constant, PZC and binding site estimates agree excellently with currently available best estimates.     

EFFECT OF THE HYDROGEN ION CONCENTRATION ON THE PHAGOCYTOSIS AND ADHESIVENESS OF LEUCOCYTES

1. Immediately after coming into contact with glass, leucocytes are most adhesive at pH 8.0 or > 8.0. 2. Agglutination of leucocytes increases with increasing H ion concentration from pH 8.0 to 6.0. 3. In phagocytosis experiments where leucocytes creep about on the slide picking up articles the optimum pH is 7.0. Here ameboid movement is probably the limiting factor. 4. The optimum for phagocytosis of quartz from suspension is on the acid side of neutrality at or near pH 6.7. 5. Phagocytosis of quartz increases with the acidity, while adhesiveness of leucocytes to glass increases with the alkalinity.     

An antimony-silver/silver chloride electrode system for measuring pH in insect gut contents

An electrode system consisting of a cast antimony rod with a silver chloride-coated wire as a reference electrode has been developed to measure pH in insect gut contents. The electrode works well with buffer volumes down to 0.1 μl. Response to pH is linear between pH 4 and 11 and equates to a change of 52.3 mV per pH unit at 18–20°C. Electrical resistance is low (0.25 MΩ), so the electrode can be used with low-impedance meters and does not require shielding from induced currents. Its useful range lies between redox potentials −330 mV (pH 7) and +297 mV to 350 mV, corresponding to a pe + pH range of 1.4 to about 10.8. This covers reported gut pH values for most insects so far examined. Consequently the electrode is suitable for measuring pH of gut contents from many insects that are too small to be analysed with current commercial electrodes.     

Effect of changing venoarterial pH difference on in vivo arterial pH

Plasma pH has been postulated to change slowly in blood leaving the pulmonary capillaries because of the uncatalyzed dehydration of CO2. If so, there could be a difference between in vivo and in vitro arterial pH, the magnitude of which would be dependent on the venoarterial pH difference (v-aDpH). We tested this hypothesis in anesthetized dogs by changing v-aDpH by airway CO2 loading and by comparing arterial pH measured in vivo by a rapidly responding intravascular pH electrode with that measured in vitro by a conventional glass electrode. Using a multiple regression analysis, we found a small but significant contribution of venous pH to in vivo arterial pH, with a regression coefficient of 0.0718 (P less than 0.0001), suggesting a postcapillary increase of in vivo arterial pH. When carbonic anhydrase was inhibited by the administration of acetazolamide, the effect of venous pH on arterial pH was abolished, and a unique relationship between in vivo and in vitro arterial pH was established (regression coefficient 1.02; P greater than 0.05, comparison with unity). These results could be accounted for in a computer simulation of gas exchange among alveolus, plasma, and erythrocyte. We conclude that there exists a small but measurable postcapillary increase in arterial pH.