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Phosphate Buffer at pH 8, when evaporated and re-dissolved in water always has pH <6.5, why?

It is 50mM phosphate buffer in titer-plates and the evaporation occurs over 2-3 days in a 37C incubator. I am suspicious of carbon dioxide being a contributor but this buffer is at a pretty high concentration. Has anybody experienced this? I would have never thought that CO2 would have this dramatic effect and it doesn't on non-evaporated plates. What is happening with the evaporated ones? Just using fresh buffer is not useful, I am working on reconstituting dry plates that have to be shipped but they need to have the same properties. I am looking into a faster evaporation method.

Public Comments

1. Try evaporation in a vacuum. You did not give the details of what ingredients the phosphate buffer contained. There are various phosphate buffers. Are you certain that you started at ph = 8 ? You might just need to increase the buffer concentration to improve the buffer capacity, but at ph 8 it will always want to soak up CO2. Degassed water, to remove the CO2 might be considered, but first I would try the evaporation under a vacuum.
2. I tend to think you're getting some CO2 incursion. Even with buffer at a high concentration, you're looking at a long incubation period (2-3 days). Don't forget that the CO2 that is consumed by the buffer is constantly replaced by CO2 that diffuses in from the surrounding atmosphere. So this acts like a CO2 sink. Do your best to exclude CO2!