I measured the pH of M9 buffer and found that it was acidic. I made a new one and measured the pH immediately after the addition of MgSO4 and it is still acidic. There was no precipitation in the solution.
What is the pH of M9 buffer and how can I ensure that the pH is neutral or close to 7?
Hi Reza, The pH of M9 is set by the ratio of KH2P04 (3g/L) to Na2HPO4 (6g/L). I think the simplest explanation (other than a faulty pH meter) is that your Na2HPO4 is in hydrated form, in which case you will have to add more than 6 g to achieve the proper stoichiometry.
Hi Eric,
Thanks for the feedback.
How can I know if the Na2HPO4 is or is not in hydrated form? I opened a new bottle but it was received about 2 years ago.
And how can I calculate how much more of it to add to achieve the right pH?
I calibrated the pH meter several times and used pH paper and another pH meter so the result was the same so the problem is not the pH meter which I wished it was!
Reza
Hi Reza,
Sodium phosphate is often sold in hydrated form because it is easier to dissolve than the anhydrous. This is indicated in the formula on the bottle and the name of the salt. For example, Na2HPO4•7H2O is the heptahydrate form. To compensate, divide the formula weight of the hydrated version by 141.6 (MW of anhydrous) and multiply this times 6 to get the number of grams to add to one liter. I guess you should also check your KH2O4 hydration status. Alternatively, make up a new batch of M9 the same way as before (but only add water up to 900 ml), then add concentrated NaOH (e.g. 10N) drop by drop while stirring and checking on the pH meter. Top up to one liter with water when you achieve your desired pH. Best, Eric
Hi Eric,
Thank you so much for the detailed answer! I appreciate it a lot.
Neither the Na2HPO4 nor KH2PO4 I used are hydrated and it is what confuses me. I could not observe any clump as a sign of absorbed humidity/hydration therefore hydration. If this is a problem why most labs do not use the hydrated form to avoid this problem, I wonder!
I also mentioned that they do not report anywhere about this problem or measure the pH of M9.
Kind regards,
Reza
Hi Reza,
Worms will tolerate a fairly wide pH range, so I guess most people are just happy when the M9 performs from an empirical perspective (I’m in that category). If you look up “Lidstrom:buffers” on google you will find an OpenWetWare link that has some handy tables and useful info about phosphate buffers. For example, NaCl will decrease pH because the Na+ ions liberate more H+.
Best,
Eric
Hi Eric,
Thank you again for the valuable information. Yes, I have realized that the pH is not a major issue for most but unfortunately, it showed to play a very important role in the assays I run. It was after strange results and variations in results I could trace and relate these differences to the pH of the buffer. I do not know how to calculate the expected pH in a solution consisting of more than a single chemical compound but I received feedback today from Sigma-Milipore that the pH of M9 should be 5.8. I send them the list (cat log and Lot nr) of the chemical I used delivered by them (Sigma-Aldrich). I do not know how they calculated the pH. They also meant that NaCl and MgSO4 are not important in contributing to pH variations in this case.
Best,
Reza