Chromatography Forum

Hi all, can I ask you one question? I have a problem with retention time of salicylic acid . It shift with 1 minutes dispite the operating circumstances are the same .

Mobile phase: A 5% acetic acid , B : AcN
column C18 , flow rate 0.800 mil min

In the begining at 1mg ml concentration the retention time was 31,2 min , but afterwards in lower concentration it is 32.1. What is the reason ?


Thank you!

I assume that you are using a wavelength near 304nm. Your choice of solvents is fine. Bump the % ACN up to about 60% to start, get that sucker off the column sooner !!!

This is a straightforward assay.

Welvelenght set at 280 nm. I use gradient starting from 86% DA and 14% B , as percentage of B increase . but I notice ,that the retention time of standart shift with 1 min. If previously Rt of standart is 31 min in next run it is 32 min.

Why shift RT? Condition are the same . I can't build a calibration curve!

Quite possibly you are not allowing enough time for column equilibration between runs. That said, 32 minutes is a very long retention time for a salicylic acid analysis (as suggested by Consumer Products Guy). Your peak may be eluting after the gradient has finished.

You have told us the flow rate, but what are your gradient time, column dimensions, and dwell volume?

are you getting the lower concentration because you are injecting less on the column?

i agree with the others here, 31-32 min for your compound is a lot.
i have seen applications at 10-15 minutes generally

We don't know the injection volume or column dimensions, but it is possible that the shorter retention time at the higher conc is due to overloading.