Chromatography Forum

Hi All

my question is about the relation of pH and Ionization in the HPLC Reverse Phase separation.

Q. The pKa of the compound X is 2, Which pH (pH = 2 or 3) of the Mobile Phase will be suitable for this compound separation, because I want to suppress the ionization of the compound. Please explain the reason in each case.

Thanks you so muh.

The general rule is to be one pH unit below the pKa to have a fully protonated molecule. But not many columns can handle a mobile phase of pH 1, so that is probably out of the question in this case.

The peak shape is usually worst at the pKa, so maybe you should go for a fully deprotonated molecule (>pH 3) instead, under the condition that you still can get some retention.

The Idea is to keep molecule either totally ionized or totally unionized; at pH where both ionized and unionized species co-exist-- will usually give severe tailing.

for acids : %ionization=10^(pKa-pH)*100/(1+10^[pKa-pH])

for base : %ionization=10^(pH-pKa)*100/(1+10^[pH-pKa])

1. at pH=pKa---- ionization is 50%
2. at pH=pKa+1 (for acids) or pH=pKa-1 (for base)---- ionization is 91%
3. at pH=pKa+2 (for acids) or pH=pKa-2 (for base)---- ionization is 99%
4. at pH=pKa+1 (for acids) or pH=pKa-1 (for base)---- ionization is 99.9%

Change % ionization to % unionization by reversing the positive and negative signs in above list.

For a substance with pKa=2, and if it is acidic (most probably, though it can be a base as well) one needs pH of zero for keeping it 99% unionized. Which surly is too much acidic pH.
----Ion pairing may be a resort in case if its ionized species is too hydrophilic.

But pKa of the substance is not only thing one need to take care of. LogP value is important as well. An ionized species may be sufficiently lipophilic to retain on a reversed phase column.

Differential retention characteristics on C-18/C-8/CN columns can also be
exploited for separation.

Kodos to Jitender for excellent explanation.
Its obvious that todays available columns can't handle PH 1 or less. But in RP your aim is to supress ionic interaction of your sample with residual silanols. You may try working at PH somewhere 3-3.5 but try on columns with least residual silanols activity.
Highly endcapped columns ; base deactivated columns with maximum coverage of -Si-OH groups on silica; or polymeric bonded columns can still give desired separation. If you can't control the ionization of sample then control the silanols activity.

Still you have to keep your fingers crossed!!!

I just wonder if the formulas that Jitender wrote above are correct. Maybe I am wrong but I think they measure % of unionization or first formula should be for bases and second for acides.

"1) for acids : %ionization=10^(pKa-pH)*100/(1+10^[pKa-pH])"

"2) for base : %ionization=10^(pH-pKa)*100/(1+10^[pH-pKa])"

2. pH=pKa+1 (for acids) or pH=pKa-1 (for base)---- ionization is 91%
3. at pH=pKa+2 (for acids) or pH=pKa-2 (for base)---- ionization is 99%
4. at pH=pKa+1 (for acids) or pH=pKa-1 (for base)---- ionization is 99.9%

if you look at the first and last formula there is no difference then how is it possible that one giving 91% and the other giving 99.9% Ionization Can you explain please.

Thanks

Yes I've noticed. I think that in the last formula instead of 1 should be 3:

4. at pH=pKa+3 (for acids) or pH=pKa-3 (for base)---- ionization is 99.9%

Then it's correct.

So we can state that for maximum retention in RPLC one uses a buffer with pH 2 or more units away (in the appropriate direction) from the analyte pKa to confer neutrality. These pKa values are, however, not readily available under the conditions found inside the HPLC column, i.e. in water-organic mixtures, and thus one may never know if ionisation is completely suppressed at a given mobile phase pH.

With the information given so far, the mobile phase pH suitable for your compound separation is answerable only by experimentation. Ionisation does not necessarily have to be suppressed to achieve a good result but I would agree that the more retention you've got, the more possibility of success there is.

Given sound working practice, there is no need to shy away from working at or close to the pKa of the compound. Good peak shape can be achieved along with a degree of retention which represents a balance of the ionised and unionised retention times.

A couple of threads for reference,
http://www.sepsci.com/chromforum/viewtopic.php?t=4751
http://www.sepsci.com/chromforum/viewtopic.php?t=5549

Operating at the pKa also puts your peak near the maximum slope of the pH vs. k' curve. Your reproducibility of retention times will depend strongly on how carefully you prepare the buffer, and how carefully you proportion the organic solvent; that is you may have a ruggedness issue. In a complex separation, this may be unavoidable, but shouldn't be for a simple separation.

Mark, I found the pH vs. k' addition very informative.. thanks. Are you advocating to not work at/around the pKa if otherwise avoidable, e.g. if the compound or mixture doesn't possess a complex mix of functionality, or were you suggesting the problems with RT reproducibility might be unavoidable?

I advocate avoiding pH ~ pKa in a simple separation. If all the components are of the same class, or the number of components is small, you usually can find a pH away from pKa.

Another place to be careful is pH ~ pI for amphoteric substances by RP separation.

Sometimes when the separation is not simple, you find that one component has pKa near the pH that gives the best overall separation. For instance, water-soluble vitamins are a mix of acids, bases, and amphoterics. I once developed a method for water-soluble vitamins where the optimum pH for overall separation was 3.4; unfortunately that is close to pKa for niacinamide, and I had all kinds of nuisance with peak shape and retention instability. Later when I revisited that method, I used a two-buffer gradient and had less trouble with niacinamide. On the third pass, I tried ion-pairing.

I agree with Mark that there can be method stability problems, if the method pH chosen is close to the pKa of one or more of your analytes. However, even this can be handled wthe a suitable preparation method for the buffer. For example, if you weigh all buffer components, and prepare the buffer solution by again weighing the volumes, you can get a superb reproducibility even under these circumstances. It's only a little bit more work than using a pH meter.

I usually advocate starting method development at a pH away from the pKa (either high or low, as appropriate). If I can obtain adequate selectivity and retention by changing %organic, temperature, or solvent type, then I have avoided the potential robustness vs. pH problems.

If I can't obtain adequate results, then I'll use a pH closer to the pKa. There is nothing fatal about that situation, but it's not my first choice.

If you find yourself needing to operate near pKa, and you want someone else to be able to replicate your method, you need then to specify the tolerances. As Uwe says, weight is the preferred method of preparing buffers, but here too you need to set tolerances.

Also, the importance of the pH effect diminishes with lowering of the relative importance of the dissociating group in the molecule´s properties.