Chromatography Forum

Hi Members,
What would you do if impurity peaks starting to merge with the main peak? (the initial resolution between impurity and main peak is about 1.5) column: Waters xterrra C8, mobile phase H2O and ACN, gradient 90% water to 10% water after 40 minutes. The main compound is testosterone palmitate and the impurity is a little tiny unknown peak. I noticed the same behavior in two "new" Waters C8 columns (about a month old). I had no choice but to buy another two columns to complete the validation work.
Thanks in advance,

It appears that something in your sample matrix is tying up the active sites in your column. Without more information, it is difficult to say exactly what is happening. You can try to clean the columns up by following the manufactirers cleaning procedure that came with the column.

Tough to tell. The sample matrix is a possibility. Also for such small changes the column temperature can be relevant. Also, consider going back to store the column in the same solvent in which it came (acetonitrile).