Chromatography Forum

I was calibtrating hplc.In calibration of pump, we use gradient flow calibration. In this we are getting less area and improper peak shape during first two injection. Then 3 injection are giving proper peak shape n area. What may be the reason for improper peak shape in gradient flow calibration, choking of mobile phase filters or system instabilisation?

Hello

First of all it is quite strange that you're using peak area to calibrate pump...
The best test to check pump (Quat/binary) for mixing is "gradient test" - all you need is marker (eg. acetone in water).
Optionally you can create gradient method and run some mixture (3-6 compunds) and check retention times drift (it will show you problems with mixing/forming gradient if retention time RSD is high)

Regards

Tomasz Kubowicz

In some cases "saturating" the column is a good way to get to a stable system. I often do a first injection with the sample and overload the column slightly. I a lot of cases this help to get a stable system fast.