Chromatography Forum

Dear
Pls tell me how to start method development for new formulation?
How to select MP- pH based on the pKa? How analyte ionization works in the mobile phase pH?

If your pKa is 5 try pH3 and pH7 and 70% ACN 30% buffer and than see what`s coming out. That`s the short version. For more details try to find some literature in the WWW or go and check websites of HPLC column manufacturers, try to get Uwe Neue`s book, participate in a training course or contact your prefered supplier of HPLC columns. In India for example you can contact Genetix Biotech in Delhi, or their local office in Mumbai. Good luck.

As a matter of personal preference, I always try to keep the mobile phase pH within one unit of the analyte pka if possible. Gerhard's answer is absolutely correct; like I said ... personal perference.

However, the real reason for the reply is, pH considerations aside, for HPLC what you will want to do is run a gradient from 90% water / 10% organic to 100% organic over 30 min and see what happens. What you do next largely depends upon the results you get from that run. Running this gradient will also help ensure that you retain any of your less retentive compounds. Acetonitrile is the first choice for the organic because of it's lower column pressure; however methanol works just as well if your column/flow rate combination can withstand the higher pressure.

Why would you try and analyse near your analyte's pKa? For the most robust performance you want your analyte either in a fully charged or fully uncharged state, operating near the pKa of the analyte will give you an equilibrium mixture of charged and uncharged molecules which will be difficult to separate robustly

Robustness is also imparted by using a good buffer, that way you have the full (depending on the column) scale of pH available to optimize your method.

Why would you try and analyse near your analyte's pKa?

Because I was only half thinking when I replied and was truly thinking of something completely differennt.

To be honest, I was thinking about buffer catacity when I typed that reply.