calcium chloride in a phosphate buffer - how to avoid calcium phosphate precipitates (Sep/26/2008 )
so this is my first post in my first forum - I guess my question is kind of important to me ;-)
I want to do a Calcium Imaging experiment - first following a group's described method step by step. They use a sodium phosphate buffer. For a pH of 7.25 they want me to mix 8 mM of the monobase and 2 mM of the dibase sodium phosphate. This adds up to a 10 mM phosphate solution of the desired pH. According to a source from our beloved internet it is possible to dissolve up to 0,3 mM Calcium chloride in that solution. Unfortunately the paper says: 1,5 mM!!! Theory and practice go hand in hand as I can't avoid having the precipitates. I tried warming, coming from different pH and I changed the order of putting the solutions together.
What am I doing wrong?
- The group published two papers (Neuroscience and Cell Calcium) and in both the recipe of the buffer is identical. So I assume they didn't do a mistake in writing it down.
- The first author is not to be found anywhere on the internet and I don't want to annoy the professor with a buffer question.
- Other groups use HEPES buffer instead. So did I - and couldn't reproduce the groups' results. So I want to try it with phosphate buffer.
- One option would be to go down with the phosphate concentration to a total 2 mM. Then 1,5 mM Calcium chloride should be possible to solve. But what about the buffer capacity? I assume it would be too weak then, wouldn't it?
I'm excited to listen to your comments,
thanks a lot.
calcium ions and phosphate tend to precipitate; you have to calculate the amount of free calcium ions in a Pi-buffer with an appropriate program f.i. Eqcal; temp, pH and other ions are also relevant
You could try dissolving the calcium in half the volume (at 2x concentration), the phosphate in the other half (also at 2x), and mixing. Local concentrations of calcium will be much higher if you add a concentrated solution to an existing phosphate buffer. This still may not (likely will not) solve your problem. I like the HEPES approach. Perhaps the paper is wrong, or they ignored the precipitate.
I agree with phage 434 suggestion. the immediate local concentration of ca ion upon addition of the stock solution will cause precipitation.
Aside from Phage434's suggestion, might the paper have used a chelator to bind to the calcium? Something like EDTA will keep the Ca ions soluble by chelating ion, preventing it form actually binding to the phosphate ion and precipitating.