pka values

[nitish]

hello, how we know pka values of any compound and what is correlation of pH of mobile phase diluent and pka values

[amaryl]

hello, how we know pka values of any compound and what is correlation of pH of mobile phase diluent and pka values

For pKa value search in books or articles pertaining to your analyte.

The pH of the mobile phase should be atleast 2 units away from the pKa of analyte (to keep it in one form basically its being kept in unionised form)... for acidic drugs move to acidic pH and for basic drugs at basic pH not to exceed the pH limit mentioned for your column usually for RP its 2 to 7. There are even columns that can be operated at higher pH range having different packing then ODS.

Hope it helps.

Regards,

Amaryl.

[Uwe Neue]

There is software around that can predict the pKa, if you give it the structure. There are several options. A commercial version of the software can be obtained from ADC.

But once you have experience, you can make some pretty good guesses as to the range of pK values, which is all you need for HPLC: alphatic carboxylic acids and anilines at 4.75, aliphatic amines around 9 to 10 etc.

I do not agree that it is important to stay by 2 pH units away from the pK of your analytes. If you control the pH well (see the discussions on the other posts), you will get reproducible results in the interim range as well. In addition, the pK of your analyte and the pK of your buffer will cahnge as you add organic, therefore this rule can rarely be followed properly.

[amaryl]

I do not agree that it is important to stay by 2 pH units away from the pK of your analytes. If you control the pH well (see the discussions on the other posts), you will get reproducible results in the interim range as well. In addition, the pK of your analyte and the pK of your buffer will cahnge as you add organic, therefore this rule can rarely be followed properly.

Could you please elaborate on this Sir.

Thanks and Regards,

Amaryl.

[Uwe Neue]

The change in pH and pK with the addition of the organic solvent is nothing to worry about for the usual reversed-phase applications. If you have set up a buffer close to the pK of the buffer in water, it will have essentially the same buffer capacity in the presence of the organic solvent (at a constant concentration). For practical method development, this is therefore not important.

However, studies mostly by the Roses-Bosch team have shown that the pH scale and the pK of compounds change with the addition of an organic solvent. Bill can elaborate in detail. For practical purposes, the pK of acids increases and the pK of bases decreases when an organic solvent is added.

[Bill Tindall]

The effects on pKa of adding an organic solvent to water were investigated more than 50 years before Roses and Bosch rediscoverd them. This work is summarized nicely in Bates' book, "Determination of pH" first published in 1954. The Roses and Bosch papers are easy to obtain, and they write in an easily understood style, so the suggestion to go there for more information is a good one. This material is way to involved to explain with short notes on this Forum. As they say around these parts, "I'm give out" from the last discussion.

[rc_12321]

If you prepare a buffer solution with its Ph =PK , then it will have maximum buffering capacity.I do know on what basis Amaryl suggested the concept keeping PH 2 units away from the Pk of analyte .
Hence , it is the matter of how preciselyand accurately you maintain your PH of solution that decides reproducibility of your method.Addition organic compounds to buffer will affect its PKa, but to what extent? However as long as your method is validated for robustness and ruggedness, the composition changes of your mobile phase do not affect reproducibility.

[amaryl]

The change in pH and pK with the addition of the organic solvent is nothing to worry about for the usual reversed-phase applications. If you have set up a buffer close to the pK of the buffer in water, it will have essentially the same buffer capacity in the presence of the organic solvent (at a constant concentration). For practical method development, this is therefore not important.

However, studies mostly by the Roses-Bosch team have shown that the pH scale and the pK of compounds change with the addition of an organic solvent. Bill can elaborate in detail. For practical purposes, the pK of acids increases and the pK of bases decreases when an organic solvent is added.

Pk of acids increases with addition of organic solvent so the statement of working at acidic conditions do holds supportively. We are moving away from the pk of analyte by working at acidic conditions even if the addition of organic solvent to acidic buffer raises pH.

Am I wrong in interpretation...

Thanks and Regards,

Amaryl.

[syx]

In normal or reverse phase chromatography, the analyte should be kept in unionized state to get sufficient retention. Ionized form tends more soluble in water because it is more polar. We should control the pH of mobile phase below pKa value of acid substances or above of basic substances. One point under/above pKa is sufficient but two points give better.
Due to column packing stability in limited pH-range, we could not get this ideal condition. If the pKa is too low/high and the retention is very short, we could use another option of separation: ion-chromatographic using ion-pair reagent. In this type of chromatography, the analyte is kept in ionized state to perform interaction to ionic site of the reagent.
Buffer selection is based on pH value that is needed by analyte as told above. The effective pH range of buffer is 1 point below to 1 point above of its pKa value.

For example:
The pKa of an acid substance is 4.5
Theoretical pH value needed for the mobile phase is 4.5 – 2.0 = 2.5
Buffer that is available for this pH is phosphate (pKa = 2.1; effective pH range 1.1 – 3.1)

[rc_12321]

If your solution contains compounds different Pka then how do apply this concept?

[Bill Tindall]

There is a simplier way......

Adjusting the mobile phase for separating acidic compounds is normally vastly easier than the above discussion implies. The best thing to do is make the water about 0.05%, or less, in phosphoric acid and do the separation. This approach works in our labs for a great variety of aliphatic acids as well as aromatic acids with from one to several carboxyllic acids groups. Tens of thousands of separations on dozens of different organic acids have been done with this mobile phase. There is no worry about buffer salts precipitating, the solution is trivial to prepare and stable forever.

This approach works because this soution is an adequate buffer so long as the sample is not basic. For basic samples add a squirt of phosphoric acid to them prior to analysis. The resulting pH is sufficient to protonate typical aromatic acids, while not so acidic as to destroy typical RP columns. It is only necessary to diddle with pH in these separations if one wants to take advantage of differences in pKa to effect a separation, something I have never had to resort to.

I suspect that a good many amines could be separated with the same eluant.

[Uwe Neue]

Bill,

You are right that under your conditions, things are reproducible, as long as your column and you analytes are stable. But you are missing out on a wonderful opportunity to change the selectivity of separations by playing with pH. You can get MUCH more selectivity changes from a pH manipulation than you can get from changing from methanol to acetonitrile, if your analyte has a pH dependent ionization.

I think that pH is a very nice tool to play with.

[syx]

Here is an example of the influence of pH to separation:
Dexchlorpheniramine is a basic compound and Betamethasone is a neutral (nonionized).

[Alex Buske]

Bill,

Phosphoric acid is a great buffer, easy to use and also suitable for diluted bases.
However, the pH is around 2 and column stability might be an issue. I once developed a method using phosphoric acid and everything seemed to be o.k., When I wrote up validation results I noticed that retention times were decreasing over time. Just a few seconds. Nothing to worry about as long as you do just a validation and from time to time some release or stability testing, For use on a daily base I would expect column lifetimes < 500 injections.
And yes, it was a Type-B silica ODS column with endcapping.

Alex

[tom jupille]

The "2 pH units away from the pKa" rule of thumb is just that: a rule of thumb. The idea is to stay away from a region where retention and/or selectivity are pH-dependent. If you can manipulate the other parameters (solvent strength, solvent type, temperature, etc.) to get the necessary selectivity, you will be ahead of the game, and you will have a lot of leeway in buffer selection. Syx's example provides a nice illustration.

If you can't get the necessary selectivity, then (as Uwe points out) you can often get a great deal of control of peak spacing by controlling pH. To a certain extent, the choice is influenced by what you know about retention behavior from past experience. If you are confident that retention won't change dramatically, then manipulating pH becomes much more attractive (to my mind at least).