13 replies to this topic

[newbie99]

Hi, I've got a question:

How do I prepare 1M EDTA, pH 7.4? Only Tris Base is allowed for adjusting the pH, no HCl and NaOH can be added.

Is it even possible to do this? Because I can't seem to find a way.

Thanks for any help.

[doxorubicin]

So if you just put EDTA (Ethylenediaminetetraacetic acid) into solution at 1M, it will be acidic (pH < 7).  If you put Tris base into solution it will be basic (pH > 7; probably we can assume pH > 7.4).  Then there should be a way to mix these to arrive at pH 7.4.  I haven't heard of anyone having solutions of non-pH-ed Tris base lying around a lab, so I suspect you were allowed to adjust the pH of your Tris Base solution before you started.

[newbie99]

Tris base is in granule form.

But isn't it true EDTA fully dissolves only at pH 8.0?

I can only use EDTA (pure form, granules), Tris (pure form, granules), and water. No HCl, NaOH, HAc can be added. Is it possible? I mean in practice.

I know it's impossible to dissolve 1M EDTA without adding any NaOH.

Edited by newbie99, 09 February 2012 - 12:34 PM.

[bob1]

you should look at the solubility of EDTA!  And the pH thing is correct - EDTA will only dissolve at pH 8 or higher.

[newbie99]

I know EDTA has poor solubility in water, about 0.05 g/100 ml, and I know 1M EDTA is very hard, if not impossible, to make.

In theory, Tris + EDTA can be possible. But with the general rule that EDTA only dissolves at pH 8.0, I don't see how I can then bring the pH value down to 7.4 with only Tris base.

Any way around this?

Edited by newbie99, 09 February 2012 - 12:55 PM.

[bob1]

The simple answer is you can't.  The disodium salt (commonly found in the lab) is soluble at about 200g/l but for a 1 mol/l solution you need 372.25 g/l - impossible problem.

Tris base when dissolved forms a solution of about pH 11, though this varies depending on how much you are dissolving, as it has some buffering capacity that means that at low concentrations the pH will be dependent on the pH of the water (MilliQ is about pH 6 due to the deionisation process), so it is theoretically possible to make a pH 7.4 solution of tris with no addition of NaOH or HCl.   I can't remember how to do calculate this, but there will be websites around that can show you - look for buffering calculations.

[newbie99]

If this is possible, would that mean "dissolving at pH 8.0" also has exceptions?

I find it very hard to dissolve 1M EDTA with Tris base, and I only got the granules to fully go into solution with heating. The solution I have now is more like a very thick paste.

Looks like there's no easy way out.

Edited by newbie99, 09 February 2012 - 01:27 PM.

[mdfenko]

edta does go into solution at a pH below 8, but above 7 (i routinely prepare 0.2M edta to pH 7.4). it will be slower to dissolve than at pH 8.

1M, on the other hand might be a little difficult to dissolve completely.

you can try adding water to the edta powder to near the final volume and add tris base powder slowly until the edta finishes solubilizing (monitor the pH so that it doesn't rise above 7.4). then adjust to final volume if it all goes into solution (it may not).

0.5M may be more realistic.

[newbie99]

Thanks for all the help. I'm going to try your suggestions.

[newbie99]

One more question:

If I used HCl to adjust the final pH, would that affect the experiment?

The protocol was pulled from Nature Protocols: Make 1 L of 1M EDTA, pH 7.4, adjust pH using Tris powder.

[phage434]

You can't make 1 M EDTA.  In my experience, this is the typical sort of error I find in Nature Protocols.  It's pretty clear they don't actually test them in the way they are actually documented, but rather reconstruct them as they think they could possibly be implemented, if things worked out.

[newbie99]

Thanks. I just realized that the authors of the Nature article probably didn't prepare the EDTA solution at 1M, rather at a more realistic molarity, because all EDTA mentioned in the paper are in diluted form.
It makes me wonder why they would include the recipe for a 1L 1M EDTA in the "Materials" section.

[ickypimp]

bob1, on 09 February 2012 - 01:11 PM, said:

...... the pH will be dependent on the pH of the water (MilliQ is about pH 6 due to the deionisation process)...

Ro water has no buffering capacity so this is irrelevent

[mdfenko]

ickypimp, on 16 February 2012 - 03:40 PM, said:

bob1, on 09 February 2012 - 01:11 PM, said:

...... the pH will be dependent on the pH of the water (MilliQ is about pH 6 due to the deionisation process)...

Ro water has no buffering capacity so this is irrelevent

true, but not irrelevant. it is acidified and will be adding acid to the buffering agent.

Edited by mdfenko, 17 February 2012 - 09:29 AM.