Chromatography Forum

Hello friends-
I am developing a method to resolve Zoledronic Acid from its impurities using a pH 3.5 aqueous mobile phase (H2O with triethylamine pH'ed to 3.5 with H3PO4) and using a Waters Atlantis dC18 column.
I am having the following issues, and wonder if anyone has any insight?
I seem to lose response over time, sometimes including completely absent peaks for PQL injections.
Can TEA or TEAP destroy the dC18 stationary phase over time, and can these columns be regenerated?
Also, I sometimes see negative peaks and strange system peaks in blanks with clean mobile phase. Does the grade of TEA make such a huge difference? Are there specific impurities in TEA (or TEA itself) that can interract with zoledronic acid and / or the column so strongly?
Any other thoughts about zoledronic acid in general?
Thank you!

zoledronic acid, like all other bis-phosphonates are very challenging chromatographically due to their incredibly strong chelating character. TEAP at pH 3.5 has very poor buffer capacity since the pKa of phosphate is 2.1. Ideally the buffer pH should be a maximum of 1 pH unit away from the pKa of the buffering species so pH 2.5 would likely give you much better reproducibility. Also, do not titrate your buffer with the pH probe immersed in the buffer (contaminates the buffer). Instead, titrate an aliquot to determine the recipe then make your buffer gravimetrically and only use the pH meter to confirm the pH of an aliquot (do not immerse the probe in the bulk buffer to avoid contamination)

Also, the buffer concentration (primarily the TEA in your case, use H3PO4 to adjust pH) should be adjusted to give reproducible behavior on different batches of sorbent. The TEA will ion pair with the phosphate groups and moderate the chelating behavior. 20mM is a good starting point and you can adjust from there depending on the selectivity and retention needed.

Hope this helps