sample preparation in HPLC method

[sudhakar]

I am developing HPLC method for tetrabenazine drug (pKa=6.51), using di-ammonium phosphate buffer and ACN in gradient elution. in all type of column drug peak is showing fronting shape. sample preparation done in 100% acetonitrile. how to improve the peak shape

Thanks for related information, but peak shape is not improving and it is observed that, using THF in mobile phase-improved the peak shape but interference observed at polar region and it is affecting the related compounds separation of tetrabenazine. pls guide me

[tom jupille]

Any time the diluent (what the sample is dissolved in) is stronger than the initial mobile phase, you have the potential for peak shape problems. The best approach would be to dilute the sample with buffer so that it more closely approximates the initial mobile phase composition. A second possibility is to limit the injection volume.

In addition, peak fronting can be a consequence of insufficient buffer capacity. This can easily happen if you are outside the effective pH range of your buffer (you didn't specify the pH in your original post).

[WillyWonka]

Hi there,

Also if you are working at a pH to close to the pKa of your molecule you may experience this. You need to work at a pH that is at least 1 units from the PKa. Not giving good result all the time but eliminate some problems!

Hope this helps!

Willy the ''GC''

[WillyWonka]

I forgot to say that the difference in pH of 1 unit is from mobile phase to the PKa of the molecule. I would go lower than 5.5 as buffer of mobile phase to keep your molecule in one state.

Willy the ''GC''