Hi, just a general solutions question:

If you need to make up 100mLs of a 10mM Tris, 1mM EDTA solution and you had a 10mM Tris and 1mM EDTA on hand, could you combine 50mLs from each to give you 100mLs? Is there a dilution necessary?

Thanks!

No, combining 50 ml of 10 mM tris with 50 ml of 1 mM EDTA will give 100 ml of 5 mM tris 0.5 mM EDTA solution. You are effectively diluting each component by a factor of two.

Hi!
50ml of 10mM Tris +50ml of 1mM EDTA =100ml of 5mM Tris and 0.5mMEDTA buffer which is already diluted.

Hi,

Thank you both for your help! Just one last thing: Do either of you (or anyone for that matter) have any idea how much 12N HCl is needed to adjust the pH to 8.0 for a 500mL 1M Tris solution ( to give 1M Tris-HCl)?

Thanks so much!

Equimolar amounts of Tris base and Tris-HCl will have a pH equal to the pKa of the buffer. For Tris, pKa is 8.3, close enough to 8. So, you need a little less than 1/2 the molarity of HCl as you have Tris. With 1M Tris-base you will need 500 mM HCl. You can get that by adding about (1/2) * (1/12) = 1/24 th volume of 12 N HCl. Note that you will increase the volume of your solution, and you will no longer have a 1M solution. An easy way around this is to mix up 1M Tris-Base and 1M Tris-HCl (the fully acidified version) and mix them in the correct proportions (in this case, about 1:1). This leaves you with 1M Tris-HCl in all cases, allowing you to adjust the pH while keeping the molarity constant.
Note that Tris, in particular, has a large change in pH with temperature, so you should measure it at the temperature at which you want to control the pH.

See http://home.planet.nl/~skok/techniques/lab...ry/pka_pkb.html for some simple buffer discussion, or lookup "pKa Tris" with google.

Hi,

Wow, who would have thought making up buffer was so complicated! I understand the reasoning except for one thing: why is it (1/2) * (1/12th) and not (1/2) *(12N) ? And, what do you mean by the fully acidified version? How will you know you have achieved this version?

Thanks so much, again.

It's really not all that complicated. You could just ignore what I said and use your pH meter, but it is a very good idea to understand *why* you are doing things.

(1/2) * (1/12th) and not (1/2) *(12N) ?

This is what I meant. Sorry for the confusing notation.

You can buy Tris-Base powder, and you can also buy Tris-HCl powder (the salt). The salt has equimolar amounts of HCl and Tris-Base, since there is no free HCl (prepared by acidifying Tris-Base with an excess of HCl and drying). Dissolved equimolar amounts will have pH = pKa(tris) = 8.3 at 20C.

You should learn about how buffers work -- by resisting the change in pH through the slight shift in the relative amounts of the free base and salt in solution. You should also understand why this works well when the solution pH is near the pKa of the buffer and very poorly when you are 2 pH units away from the pKa.