Adjusting pH for a specific proteins pKa

[esbena]

I´m trying to setup a method for measuring creatinine in mice serum and urin by UPLC.

For this I´m using a Zorbax SCX300 2.1mm x 50mm, 5u HPLC column with a 5 micron guard column in front.

My running solvent is : 5mM sodium acetate pH 4.1

My creatinine standard is from Sigma Aldrich: creatinine anhydrous, ≥98%, 82.03 g/mol, pKa 12.309.

And I´m make a two-fold dilution from 128 uM - 0.5uM

Acquity parameters:

- Flow: 0.3 ml/min
- Column temp 45C
- Autosampler temp 18C
- Inject vol: 3 ul
- Run time: 10 min
- UV: 225 nm

Back pressure was around 740 psi during entire run.

I get an average retention time of 7.5 +/- 0.05 min, with a broad peak....

I would like to get a retention time of 3.6 +/- 0.05 min and a narrow peak...

Any suggestions/help??

[tom jupille]

If you want to decrease ion exchange retention, you move the pH *toward* the pKa. The low pKa for creatinine is 4.8, so that means increasing the pH a bit. How much is "a bit"? The only way to find out is to do the experiment(s). As you get close to the pKa, retention can change dramatically, so my suggestion would be to make a small change (0.2 units, maybe?) and see what happens. You can "fine tune" from there.

[esbena]

I found a pKa of 12.3 for creatinine...?

But thanks..

How about the other parameters? Are there any I should try first?

[tom jupille]

I found a pKa of 12.3 for creatinine...?

That's the second ionization. The first is variously given as 5.01 (http://www.clinchem.org/content/29/2/256.full.pdf) or 4.8 (http://www.zirchrom.com/organic.htm) [that zirchrom reference gives the second pKa as 9.2]

How about the other parameters? Are there any I should try first?

Probably the best thing to do would be to sit down and learn a bit more about ion exchange in general. That will give you a feel for what is important and what is not. I can recommend "Practical HPLC and CE of Biomolecules" by Cunico, Gooding, and Wehr. You can get used copies on Amazon for under $50:
http://www.amazon.com/exec/obidos/tg/de ... 1076360802

The short answer is that increasing the ionic strength will decrease retention, as will increasing the temperature.

And, by the way, creatinine is not a protein.