Chromatography Forum

Hi,

I've collected fractions from a Shimadzu HPLC off an Organic Acids column in 25mM Kh2PO4, pH = 2.5. I want to run these fractions on a C18 column for further separation. What is the best way to precipitate out any phosphate so I don't harm my C18? The mobile phase for the C18 is 85% 0.25%-glacial acetic acid, pH = 3.5 and 15% Methanol. I've tried 1:1 Organic Acids mobile phase (KH2PO4):C18 mobile phase, then filtered. Theoretically the methanol should precipitate the phosphate, but I can't tell if it worked. Any suggestions?

Thank you!

Sounds like a cool experiment! I think people routinely use phosphate buffer with methanol. Especially at such low concentrations.
You might bump the pH up to your mobile phase buffer though. Silanes aren't terribly stable at pH 2.5

Theoretically the methanol should precipitate the phosphate, but I can't tell if it worked.

If I read your post correctly you only have 12.5 mM phosphate and 7.5% methanol in that 1:1 blend. Based on Carr's 2004 data (http://chromatographyonline.findanalyti ... rticle.pdf) I really wouldn't expect precipitation until you get up to around 80% methanol. And C18 columns are routinely used with 25 mM phosphate buffer, so I would not expect any damage from that aspect.