Chromatography Forum

Hi all

I ran a standard which has 21 peaks, the first and last 12 peaks are nice and sharp, but in the middle peaks have split, I changed the cartridge, column, eluent and buffer, it still showed spilt. When I injected different concentration, some of them are good, some have the issues, and this issues showed irregularity, which the same sample sometimes spilt sometimes not. Is there anyone knows why this happened?

It's a common problem in HPLC and it can have many causes meaning we can only guess without more information.

- Aren't you overloading your column?
- Are you using the right loop for the volume you are injecting?
- Are you using the right solvent for your standards with respect to the mobile phase composition at the start?
- .........

Check for instance http://www.chromatographyonline.com/spl ... &sk=&date=

Inject half the microliter amount, and see if the spitting disappears or improves.

Hi

Thank you all for help, I realised it might be related with my samples, Hypoxanthine can only solubility is low and easier have participated with long term stay, so it might be an effect of my injection.