O2k-pH ISE-Module
| Description | O2k-pH ISE-Module: two pH electrodes and reference electrodes and accessories, two black PEEK stoppers, supported by the O2k-Core. |
|---|---|
| Product ID | 12400-01 |
| Type | O2k, O2k-Module, MultiSensor, Stopper, Catalogue |
| Link | pH and Oxygen @OROBOROS |
| Image |
O2k-Catalogue: O2k-pH ISE-Module
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O2k-Guide
- A pH-Manual is delivered with the O2k-pH ISE-Module.
pH electrode
For general information about the Oroboros pH System, protocols, and templates see pH and Oxygen. Using the pH electrode in the O2k to measure proton production requires some modifications of standard protocols and in fact a certain degree of method development. Some of the encountered challenges are
- very small buffering capacity required
- calculation of proton flux from the observed parameters
Specifications
The specifications Oroboros Instruments uses for quality control of pH electrodes is drift (after 45 min stabilization, 37°C, buffer with 2 mM buffering capacity, integrated over 5 minutes) <= 20 µpH/s.
pH - Calibration and practical topics
For information how to calibrate your pH electrode with DatLab see manual MiPNet12.08 and protocol MiPNet08.16.
While working with the pX channel please always observe the guidelines for avoiding damage to the electronics by ESD.
pH electrodes have to be calibrated at the temperature intended for use and the correct pH values of the pH calibration buffer has to be used for the calibration. These values are sometimes difficult to come by. Sigma-Aldrich supplied us with temperature dependent pH values for the following products:
| T |
B5020 (pH4) |
B4770 (pH 7) |
B4985 (pH 10) |
|---|---|---|---|
| °C |
pH |
pH |
pH |
| 0 |
4.01 |
7.12 |
10.31 |
| 5 |
4.01 |
7.09 |
10.23 |
| 10 |
4.00 |
7.06 |
10.17 |
| 20 |
4.00 |
7.02 |
10.05 |
| 25 |
4.01 |
7.00 |
10.00 |
| 30 |
4.01 |
6.99 |
9.95 |
| 35 |
4.02 |
6.98 |
9.91 |
| 40 |
4.03 |
6.98 |
9.87 |
| 45 |
4.04 |
6.97 |
|
| 50 |
4.06 |
6.97 |
9.81 |
| 55 |
4.08 |
||
Please note however that a.) there seems to be a contradiction between the value stated for the pH4 buffer at 25 °C in this table and on the product itself ("pH 4.00 +-1") b.) this information is as supplied to Oroboros Instruments several years ago and with no responsibility by Sigma Aldrich for the correctness of this information.
pH-Stat
One approach we have developed is to use the TIP to run in a "pH stat" mode, i.e. keeping the pH constant by a feedback controlled automatic injection of base. Besides keeping the pH in the desired range this can actually be used to determine proton flow from the amount of base injected, circumventing the determination of buffering capacity, see below. The "pH-Stat" allows using very weakly buffered media (2 mM buffering substances) and might even make buffering obsolete. The feedback modus of the TIP can be used in two ways to achieve a pH-Stat modus:
- pH-Stat_strict: This program keeps the pH value strictly between user defined upper and lower limits. The difference between the upper and lower limit will determine the time between injections depending on the current proton flow. Therefore, the time between injections may vary drastically with changing proton fluxes.
- pH-Stat_interval: This program adjusts the pH value in certain time intervals back to the upper limit. The difference between the upper and lower limit is set extremely small but a defined minimum pause between injections of e.g. 180 s is defined. Therefore, usually a base injection will be done every 3 minutes and the pH value will oscillate between the upper limit and some (proton flux dependent) lower limit. The lower limit set in the program has no significance because the minimum pause time will not have elapsed when the lower limit is met.
While the pH-Stat_strict is necessary to keep the pH value in a precisely defined range the pH-Stat_interval program ensures defined periods undisturbed by any injection of base. Such periods are necessary for the calculation of proton flow from the observed pH change and theoretically also for measuring respiration (however, if a 100 mM KOH the disturbance of the oxygen signal by the small amounts of KOH added was usually very small).
Both mentioned TIP setups can be found in the DatLab template file DLTemplates_pH.dlt available for download here.
Calculating Proton Flow
Potential Applications
On the simultaneous measurement of O2 and pH, we may refer to the classical literature on bioenergetics and the discovery of the chemiosmotic coupling mechanism, the quantification of H+/O2 stoichiometric ratios for proton pumping (Peter Mitchell). Other groups (e.g. Eskil Elmer - http://www.oroboros.at/index.php?id=mipnet-sweden#c1588) have used the pH electrode in the O2k in conjunction with a study of mitochondrial permeability transition.
The majority of novel applications will address the problem of aerobic glycolysis in intact cells, using the measurement of proton production as an indirect but continuous record of lactate production and corresponding acidification of the medium, while simultaneously monitoring oxygen concentration and oxygen consumption. In a well buffered culture medium, the pH change is extremely small relative to the amount of protons (lactic acid) produced, hence a low-buffering capacity medium needs to be applied. A titration of acid (lactic acid or HCl) into the low-buffering capacity medium yields the pH-dependent buffering capacity (Delta H+ added/Delta H+ measured by the pH electrode). Under various metabolic conditions, lactic acid production is the dominant mechanism causing acidification, hence the pH measurement is a good indirect indicator of aerobic glycolysis.
See also
