calibration of hplc

[jiten37]

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?

[kubowicz.tomasz]

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

[gstaepels]

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.