Metabolic study on different HPLC gradients

[Nuclearama]

Hi,

we performed a in vivo study with a radiopeptide, but the problem occured was, that if we use a gradient starting with 10% ACN-55% ACN 30min we get more peaks and one very high peak at about 4 min, whereas starting with 1% ACN - 55% ACN 30 min we get a lower amount of peaks and the very high peak at the beginning is missing.
Could anybody explain why we get so different results in the different gradient systems? An overlay of peaks was not observable...

greets Anton

[HPLC chemist]

Sounds like your detector is not selective enough (detects everything!). Since you are using a radiopeptide you should be using a scintillation detector or radioactive detector or MS (selective ion)

[James_Ball]

The high peak at 4 minutes when starting with 10% organic could be multiple analytes eluting together due to them not being as retained as they are when starting with 1% organic.

You need to inject a very high concentration of the labeled analyte in a clean matrix to establish the retention time for it in each gradient, then inject the sample and look for that peak at its proper retention time.