Method development

[sapna]

If the Pka of a drug is 4.3,then which pH of buffer can be used to have more intensity(response) and why?
If the Pka of a drug is 9.2,then which pH of buffer can be used to have more intensity(response) and why?

[DR]

Altering the pH of your mobile phase will do little or nothing to the level of response from your analyte, that's a function of the compound's structure, number & types of double bonds etc. (I'm assuming UV detection here).

Keeping the pH of your mobile phase a unit or two away from your analyte's pKa will prevent poor peak shape due to fractional ionization of the analyte. To the extent that the partion coefficients of your analyte's ionized and unionized forms differ, you can separate them. This separation is rarely complete, so the result is a broader or somewhat split peak.

[tom jupille]

(I'm assuming UV detection here).

Given that this is posted on the LC-MS board, MS would be more likely.

That said, sapna, are your compounds acids or bases, are you referring to ESI or APCI, and + or - ionization?

[Jim Shen]

If the Pka of a drug is 4.3,then which pH of buffer can be used to have more intensity(response) and why?
If the Pka of a drug is 9.2,then which pH of buffer can be used to have more intensity(response) and why?

For APCI, solution phase pH would be close to meaningless in predicating ionization behavior. Because APCI is a gas phase process the most dominated factor will be gas phase basicity of the compound.

For ESI, if you are 2 units above or below the pKa of the drug it will be either completely ionized or un-ionized. However because you are driving some couple thousand volts into them ionization will occur even if you are not ionized in the solvent.

[sapna]

But my question is that for LC-MS/MS,what should be the pH of buffer for Mobile phase?

[Kostas Petritis]

That will depend on the ionization mode and the type of the analytes. You should be more specific as Tom requested and provide more info on the nature of your analyte...

[JA]

...but you didn't answer my question with the limited information supplied so I have left this uninformative board

I am guilty of it too, but isn't it frustrating when people drop their own threads.

And for a complete waste of time, I will add that if it was UV the chromaphore could change with pH and so therefore could the lambda max and signal intensity.

[jag]

Hi Sapna,
Altering the pH will not increase or decrease the repsonce in ESI significantly as the high voltage (~5000V) is applied in the source for ionisation of the molecules.

However pH will effect the retention in the column. Let us take the first example compound pKa is 4.3 and assume the analyte is acid. At around pH 2.5 most of the molecules are in non-ionizable form which gives higher retention in the column. at pH 6.3 most of the molecules in ionizable form which gives lower retention.

There were some considerations saying that when the pH is above the pKa resulting molecules are in ionizable form may improve the response as the retention/resolution is not a major concern in MRM mode. But I haven't observed this response increase. It may work in APCI or for some specific molecules, but I am not sure.