Could someone please provide me with information, and if possible references, in relation to the merits and demerits associated with adjusting the pH of the mobile phase in a reversed phase HPLC method before and after adding the organic solvent.

The method in question uses a gradient program consisting of three mobile phases. Mobile phase A is 100% buffer, mobile phase B is composed of 20% buffer/80% methanol and mobile phase C is composed of 20% buffer/80% acetonitrile. The pH of all three mobile phases is adjusted to the same level. Furthermore, the pH of mobile phases B and C is adjusted after the addition of the organic solvents.

The method has been fully validated and it has been used for over a year without any problems. Nevertheless, following a recent meeting a suggestion was made to change the method so that the pH of mobile phases B and C is adjusted prior to the addition of the organic solvents.

Considering that the method has been used successfully for a significant period of time, can a change to the conditions of the mobile phase preparation as suggested be justified?

Jim Alexiou
Product Development

Jim,
The convention is to adjust pH of buffers before adding organic modifiers. pH is defined on an aqueous system only. The measurement has no meaning in a mixed system.
However, if your method is validated as it is, I would not change it. Reverting to standard practice would probably change your separation and would require revalidation. If it ain't broke don't fix it.

I agree: never trouble trouble until trouble troubles you.

While I agree that one should not go looking for trouble, I think that you should do a few experiments with the aim of bringing your procedure in line with standard practice. I would prepare three mobile phases, all with the pH's adjusted prior to addition of your organic modifier. With the first, I would adjust the pH to your specification, then I would bracket that pH with the other 2 MP's. Using these three, check the effect of pH upon your separation, and proceed accordingly.

SOP's are SOP's and should be followed, but they should also be revised when necessary, and this seems to me to be one that may bear looking into.

I wouldn't be surprised to see ruggedness issues crop up in the future - what'll happen when you change your pH probe?

Merely my two cents...

In addition to juddc's comments above, how about measuring the pH before and after organic addition? This would tell you the apparent effect of the organic addition on your pH meter.

If nothing else, it will add a little interest to the analysis' history.