Both buffers appear to have sufficient buffering capacity for the pH values you mentioned. However, the pH rise you’re observing is unusual. If I understand correctly, the pH of the buffer solution increases when remeasured after 24 hours, even in the absence of protein?

If this occurs after protein dissolution, it’s worth noting that proteins contain acid/base groups that can significantly influence the pH of the buffered solution. In this case, you may need to adjust the pH after dissolving the protein or consider increasing the buffer concentration to stabilize the pH.

Temperature differences might also contribute to this issue. If you measure the pH of the buffer when it’s cold and then remeasure it at room temperature, the pH can shift because pH is temperature-dependent. However, the discrepancies you describe seem larger than what would typically result from temperature changes alone.

The addition of NaCl could also play a role. While NaCl itself is neither acidic nor basic, it affects the ionic strength of the solution, which can influence the pH. If you’re adding NaCl after the initial pH measurement, this might explain the changes (at least in part).

Another possibility is that your buffer concentration is too low. With a low buffer concentration, it becomes harder to maintain a stable pH, as even small fluctuations in [H+] can cause significant changes in pH due to insufficient buffering capacity.

Lastly, if you suspect your pH electrode is causing the issue (which to me it seems it may be the case), you can verify its accuracy by measuring a solution of known pH, such as a pH 7 calibration buffer, daily. Significant variations between days might indicate a problem with the electrode. Regular recalibration is essential, though errors in calibration typically result in consistently incorrect pH values rather than fluctuations.

Thank you for your answer. There is no protein in the solutions. The changes are happening in 1M Tris-HCL; 20mM Tris-HCL + 1mM EDTA + 600mM NaCl; 20mM NaPO4+30mM Imidazole +600mM NaCl. Buffers are made at RT and when pH i stable for 30 minutes we consider it to be complete.

We are using a new pH electrode and have also ordered another one. I am checking the pH meter itself today.

Are you calibrating the pH electrode with standard buffers before each measurement?

Yes we are.

Based on your reply I would be quite confident that the problem must be with the electrode then. I myself experienced some pH electrode problems, especially if the electrode was stored for a longer time without use or if it partially dried. In these cases soaking electrode in KCl or other appropriate solution usually resolved problems but the recovery can last up to several days. Also when handling protein solutions it is generally advisable to wash electrode occasionally with some protein-degrading detergent but for a new electrode this is of course not a problem.

Tomislav, thank you. We have come to the same conclusion. The electrode was unused - backup that had been in the cabinet for a few years. WE have decided to order new electrode. We do not put the electrode in protein solutions only buffers. Hopefully this will resolve the problem with a new electrode. I will update you when I have news.

You are welcome, I am very glad if my comments were at least somewhat helpful. Wishing you the very best in your future scientific endeavors.