Chromatography Forum

Dear all,
I am trying to replicate a method for the analysis of amoxicillin on a HPLC-MS. The compound is diluted in water and the mobile phase is A:0.1% of formic acid in water and B: ACN. The gradient starts with 5% of ACN and it increases and the flow is 0,250 ml/min and it increases up to 100% of ACN in 12 min.
The column temperature is set up at 40ºC and I can’t control the temperature of the autosampler.
The peak shape are horrible and I am not able to get reproducibility. I am only able to get correct the first inyection.
Any idea or suggestion??
Thanks in advance

We need more information to help you.
What column do you use ?
When you say you're no able to get reproducibility, what do you mean ? Different retention time at each analysis ?
Do you let enough time after the end of the analysis for the column to get back to its initial state ?

The column is in the title it is a sunfire from waters.
I mean that after three inyection the compounds disapear..Is that possible?

What type of SunFire column do you use (C8, C18 ?...)
What are its dimensions ?
What is the volume of injection ?
Do you have an example of chromatogram ?
When you say the compound disappear after 3 injection, does that mean the 3 injections are identical and then you can't detect anything ?

Do you optimise the amoxicillin, i.e. infused directly to MS and get the optimum parameters?
Do you know that amoxicillin is ionised in negative mode?