Reducing variability and struggling with pH
I introduced the previous blog about our InnovATEBIO webinar (To Reduce Variability/Enhance Reproducibility: Begin at the Beginning) with a story about pH measurement. In the experience described in that blog, I was trying to measure the pH of a difficult solution, one with a high pH and containing urea. As it turns out, problems with pH measurement have continued to bubble up periodically in my life, even when my knowledge of such measurements is considerably more sophisticated than it was long ago. The table below shows data from a team of my students who were exploring variability in pH measurements.
Student data for project exploring pH measurement. This table shows the results that a team of careful students obtained when they investigated variability and pH. Using bottles of two buffers, one presumably pH 9.0, the other 5.0, they looked at five different pH meters. They also took a single pH meter, shown in the top line, and checked the results with three different pH probes (combination electrodes).
In the student data, looking across the first line, where three different pH probes were checked, it appears that probe #3 was not functioning properly, based on the buffer presumed to be pH 9.0. However, probe #3 provided a more accurate value for the buffer presumed to be pH 5.0. At this point, the students realized that there were various problems. The three probes provided different values. Also, the variability in readings led the students to wonder if the pH 9.0 buffer really was pH 9.0, and if the pH 5.0 buffer really was pH 5.0. These presumed pH values are those obtained by the lab manager using a particular pH meter and probe. A couple of things to note here: First, the students properly calibrated each probe/meter with fresh, unexpired pH standards. In theory, calibration should compensate for differences in the performance of different probes. Second, all the pH probes in our laboratory are “Tris compatible.” (Not all pH probes are compatible with Tris buffers, and this can be a cause of error.) While it is difficult to say how much variability or error in any measurement is acceptable, an error of 0.4 pH units, as the students observed, could certainly have biological significance in many situations. We do not want that much uncertainty in our measurements.
Our students needed to move to other projects, and never were able to corral their pH data. So, Jeanette Mowery and I decided to take on the challenge and spent several summer days in the laboratory exploring the details of pH measurement. Jeanette and I eventually were able to obtain consistency in our readings of ± 0.1 pH units, both for Tris buffers and commercially prepared standards. Issues that we found were important included allowing ample time for equilibration of solutions after mixing, using a two point calibration (some of our meters had a three point calibration mode that we found to be less accurate), carefully compensating for solution temperature, using only fresh calibration buffers, and using only probes with high efficiencies [1]. What did we learn from our time in the laboratory? Perhaps nothing that we didn’t already know, but we did convince ourselves that getting good pH measurements is dependent on good technique – and patience.
[1] pH probe efficiency is defined as: % Efficiency= change in (mV/pH unit)/(-60 mV/pH unit) X 100. Assuming your meter will read millivolts, it is possible to determine probe efficiency by graphing millivolts (on the Y-axis) versus the pH of a series of standards (on the X-axis). The slope of the resulting line ideally is close to -60 mV/1 pH unit. As an electrode ages, the slope becomes less steep. We discard electrodes whose efficiency is less than 85%.