Chromatography Forum

Hi

I'm doing now the determination of tylosin on hplc. Becose of short column life time (max 2 months) and bad chromatograms. I'm trying to change method to recive better chromatograms and longer life time for my columns. I use low concentration of sodium perchlorate in 40% acetonitrile in water MP with pH betwen 8 - 8.5. The column(RP 5 um 4.6*25 silica) pH range is 2 - 7.5.

My questions are:
*Can I replace sodium perchlorate with TFA?
*If yes what concentration? (0.5mM?)
*What pH? ( Need to be in column pH range) (2.5?)
*Should I change eluent strength, rt~21min, broad peaks, bad symetry (exp. 50%)?
*Or maybe I need to buy a new column (exp. geminin analytical column 5um 4.6*25 with pH range 1-12) and use my old MP ?

Thank you for any help!

There's plenty of columns nowadays that can cope with pH 8-8.5, (XBridge and Extend spring to mind, but a Google search will give you loads) so if that is what's best for your analyte then run at that. You need to know why that pH was chosen in the first place. If it was arbitrary then by all means change but if someone else investigated and found it was the best place to run you might be flogging a dead horse trying to develop a method at a different pH.

Seb@:

The USP method uses the following MP: 60% of 2mol/L sodium perchlorate, adjusted with HCl to pH=2.5 and 40% acetonitrile.

Did this MP give you the bad chromatography and short column life times or was it your modified MP?

coorect me if I am wrong,
this tylosin has many alkyl OH and one NR3. pka may at 9.5. any pH from 2 to 7.5 should be good . try 20 mM kH2PO4 pH to 2.5 by H3PO4 on Luna C18 150x4.6 column. 80-100A, organic MEOH first then ACN later if necessary
I never try this. Theoretically it should OK. Other wise try 300A column using the above condition
good luck