[keetner]

[Edit: Sorry, realized this probably would've been better suited under a general chem sub-forum. I was thinking about it in the context of proteins and the like.]

Hi guys,

I was hoping to get a bit of help with this question regarding ionic-strengths and buffers. I have little idea as far as how I should approach this, so any help would be appreciated. We were asked to determine the ionic strength of 50mM phosphate buffer at pH 7.5, using NaCl as the salt. Purpose of this is for protein purification.

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So just to briefly go over what I did so far (and I don't know how much background info might be needed)

Since pH is 7.5; pkA 7.2:
H₂PO₄^2- HPO₄^2- + H+

After using Henderson-Hasselbach:
[HPO₄^2-] = 0.0333M
[H₂PO₄^2- = 0.0167M

Ionic strength (phosphate and sodium ions):

(1/2)x([0.0333](-2)² + [0.0167](-1)² + [0.0333x2](1)² + [0.0167](1)²) = 0.116M

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Now for my question:
Even though we have 0.116M, I need to make it up to 0.15M (physiological conditions for the protein), but how can this be done? I mean, I'm guessing it's not as simple as 0.15-0.116M, since we have a mixed species here. I am thinking you could adjust by adding in either more of the phosphate or salt (I'm leaning towards salt, though).

Regardless of which is added, how would this affect the charge of your buffer, though? Because if you have to dump in a lot of extra phosphate or NaCl...would that not skew it to either being more positive or negative in nature? As the charges might not be neutralized properly any more.

That's about it, though. I don't know if I'm going about this the right way (my chemistry is pretty terrible). Again, any help would be appreciated -- thanks!

[Borek]

You are on the right track. All you need to do is to add some NaCl.

No idea what you mean by "charge of the buffer", but it is definitely some kind of misconception you have. Buffer is not "charged", and every solution is always electrically neutral - it contains exactly the same amount of charge in positive and negative ions. NaCl that you will add is also electrically neutral.

[keetner]

Hm, I think I see what I did wrong; I was focussing more on the context of Na+ versus NaCl.

One more question -- with the addition of more NaCl, how would this affect your phosphate ions? Because if you solely add in more NaCl, would you not technically have the same amount of Na+ interacting with the phosphate...? If this is the case, I'm not entirely clear how this would affect the ionic strength of the buffer, unless I can assume that NaCl completely dissociates? Sorry, I'm not really sure what to make of it.

Thanks for the response, though -- it's appreciated.

[Borek]

NaCl is fully dissociated and - for most practical purposes - you can assume there are no interactions between Na⁺ and phosphate ions.