Could someone share with me information on how organic solvents in the mobile phase affect the pKas of analytes and the pH of buffers?

Thank you very much in advance.

G'day from WA
We routinely analyse for organic acids by RP-HPLc with a PO4 mobile at pH 2.50. We also use methanol gradient, and use the pump to mix bith eluents, thus the pH of the buffer is adjusted but not the methanol. Somewhere i have read that the addition of methanol to the aqueous buffer will slightly increase the pKa's of the acids. I have also collected eluate from the outlet of the detectro and re-read pH, which was still 2.50. So methanol seemed to have no effect on mobile pH, but it does affect the pKa's of acids being analysed.

What are you analysing, and how (ie column, mobile etc)

Hope that helps
Regards
Greg

There was an extensive discussion on pH and buffers some time back, with implications on this. Generally, pH is defined for aqueous solutions, adjusted in H20, and then tested for effectiveness in chromatography. The actual pH of the mobile phase is academic.

I raised this question because the following fact:

An amine (apparently I have worked on) has a pKa of 8.2 in aqeous medium. From experiments on SPE mixed-mode MCX cartridges, it turned out that the amine is half ionized when a pH 9.2 buffer is used (ammonium acetate). This indicates that the pKa of the amine and the buffer shifted under the experiment conditions. My question is: How can I choose a pH at which the amine would be completely ionized or protonated? In other words, how do we choose a buffer pH when analytes are in aq./organic mixture and therefore their pKas shift? As this IS NOT an academic matter, I really appreciate any guide from you.

Thank you, Greg and Dr. Mueller!

One can start by calculating, roughly, where the analyte is ionized , as wished, in H2O, then vary the pH and organic until optimum conditions. You donīt need to know what the pH etc, is. Are you going to throw the towel if you have no idea what the pK of your substance is?
Incidentally, how do you know that your amine was 1/2 ionized . . . .?

Greg and Dr. Mueller,

Aqueous vs Non-aqueous
In the old wet chem days aqueous meant even with a little water, the system was still aqueous that is the ionization constant of water controlled the chemistry until it contributed almost nothing to the concentration of the solution. Until that concentration, treat the solution as aqueous. A little methanol (75% is a little)won't change the pH much.

Finding the pKa of a compound.
Titrate it in the solution of interest. Prepare titratants with t-butyl hydroxide, and with perchloric acid.

Finding the endpoint.
Use a pH electrode with a bridge,

For a pKa of 8.2 at 9.2 it is still 10% ionized. At 10.2 1% at 11.2 0.1%.

Present your data for your titration and many chemists can explain your situation. That you know 50% ionization and still have your questions puzzles me.
Jim Gorum

On MCX cartridges, compounds ionized are not washed away by methanol but neutral ones are. That's how I estimated the % ionized.

We are industrial chemists and therefore need EDUCATED guess. Time is everything in the industry. Trial and error are the least favourable method.

Actually a colleague of mine conducted research on calculating pKa in aq/org mixture using dielectric constant and so on. However, he is not in the position to provide further details (projecyts many years ago). I would need some qualitative quidance.

Noname,
I appreciate the time factor, however, chemist trial and error, technician get told. If your method of elution is a good method for estimation of %ionized. then every pH unit up will reduce the washed off 10 times. So raise your pH 2 and 0.5% will wash off.
Jim

Here are a few simple rules, and some references to chase. The pKa of acids increases with the addition of organic solvents, the pKa of bases decreases. For more details, look for publications by Marti Roses and Elisabeth Bosch.
If I would have the problem that you are describing, I would get rid of the acetate and simply use ammonia to elute. You do not need to think of ammonia as a competing ion, you can simply use it to adjust the pH to a point where your analyte is not ionized any more. Of course, you need to have enough organic in your extraction solvent to elute your compound. BTW, this could be a part of your elution problem, not only the ion-exchange of the mixed-mode device...

Originally coming from the corner of physical organic chemistry I have immense problems with this "% ionized". Apparently, you donīt really mean, as I understood you (initial anonymous), that the solute is 50% ionized as soon as it contacts mobile phase, but rather, that 50% of your substance gets hung up on the column. The stationary phase will interfere with the equilibrium in solution (greetings from Le Chatelier) so that the extend of ionization in solution would be difficult to ascertain. If you increase the stationary phase your "% ionized" would have to increase (unchanged pH).
Now, if you figured out how to predict all this, let me know, but my suggestion remains: to keep your job: do educated trial and error until then. (Timesīs a wastin)

The original experiment design was: Many amines have pKa of 8-11. The analyte has a pKa of 8.2, that is, at the lower end of the pKa range. By selecting a right buffer (pH 9.2), we could separate it from many other amines in the samples. That's why I selected ammonium acetate, pH 9.2 (at its maximum capacity). The buffer was highly concentrated and dissolved in methanol (80% methanol).

The last response from Mueller was really interesting. Thank all for your invaluable input.

I have been off for a while.......weather's bad, ewes are lambing and the shepherd hasn't had much sleep.

If I understand the problem, one wants to make a particular amine neutral while leaving other more basic amines protonated in order to effect a separation. And the question is, can one calculate a "pH" to accomplish this separation. You may look up in prior post why I put the pH in quotes, it is a long story.

Once again, I have to agree with HWM. This would be a very difficult problem to calculate in general. For methanol-water mixtures the acid base theory has been developed, but for many solvents it has not. There are but a small number of systems where it might be possible to make the calculation you desire and there is probably no chance the data on your amines of interest have been studied in these systems. If your dielectric is sufficiently small you will have ion pairs and then things get more complex. It is likely the pkb's of your amines will no longer be in the same proportion to one another as they were in water, or even reversals are possible.

I believe an empirical approach to the separation will get you to a an answer the fastest. But, here is a somewhat less empirical approach. In the solvent for the separation, titrate the protonated amines and follow the titration with a pH meter. I would recommend putting the pH meter on a mv scale so you are not tempted to interpret the pH reading according to what you may know about water and pH. Estimate the mv reading at the end point, that is, where the amine now exists as the free base. Do the same for the other amines. If there are sufficient differences in the mv reading for the exactly neutralized amines, it means the amimes in the solvent have significantly different pkb's and this approach may work. Now adjust the eluent with base to the mv reading where the least basic amine is neutral and see if the separation works. My guess is that this approach will get you an approximation, but optimization will still be necessary. Aha, you say, but will not the mv reading change rapidly when near the end point of the titration, making it difficult to determine what mv reading corresponds to exactly neutralized amine. Well, yes and that is why this approach may work if the pKb are sufficiently different, and it will definitly not if they are similar.

We have some examples where we accomplished the type of trick that you are referring to using ammonia and different amines for elution. If you send me your email, I can send you some more background.

Do you know a database in a web page where I can found pKa or pKb of pharmaceutical substances?