For bicarbonate/CO2 buffered (HEPES free) DMEM media being stored in the fridge, the immediate CO2 environment is just atmospheric CO2 concentrations (i.e. low). Because of this, the bicarbonate should pick up H+, form carbonic acid, dissociate into H2O + CO2 and release CO2 into the air in the bottle.
This results in the media becoming more alkaline over time, but would it not also result in a reduction in the moles of bicarbonate present in the media? So over time, the concentration of bicarbonate in the media would decrease?
Would this then have the effect of reducing the overall buffering capacity of the media over time?
Based on this, if you then used aged media in cell culture at 5% CO2, would aged media acidify more under these elevated CO2 conditions than media that had been made up fresh, having lost some of its buffering capacity? (All else held constant.)
So could the media become more acidic over time?
I have done the same experiment several times and it seems like the pH of the media (even when in the incubator) was changing over time. It would make more sense with my results if it was becoming more acidic. Could the mechanism described above explain this phenomenon?