Chromatography Forum

I have six replicate samples. My first three trial has largest impurity at different retention time compared to my last three samples. Let say on my first three trial, I'm getting it at 4 minutes, whereas on my last three samples, I'm getting it at 10 minutes though it has 4 minutes impurity also but lesser compared than my 10 minutes. Am I right of saying, that what I'm just going to consider on my calculation are those largest impurity coming regardless of what retention time they have? thanks.

make sure that what ever you see is not impurity coming from a previous run.

Hello

If you inject the same sample 6 times retention time for impurity should be the same (even if peak area is not reproducible). It looks like you have problem with pump (flow or eluents mixing). I'd suspect: ball valves, cartidges in inlet valve or gradient valve.

Regards

Tomasz Kubowicz

Hello

If you inject the same sample 6 times retention time for impurity should be the same (even if peak area is not reproducible). It looks like you have problem with pump (flow or eluents mixing). I'd suspect: ball valves, cartidges in inlet valve or gradient valve.

Regards

Tomasz Kubowicz

what i mean here is six sample preparations? the first three sample prep has largest impurity on 4 minutes let say but the remaining three is at 10 minutes

what i mean here is six sample preparations? the first three sample prep has largest impurity on 4 minutes let say but the remaining three is at 10 minutes

Is it six preparations of the same sample or six preparations of six different samples??

Hello

You have 2 possible situations:

1.You have six different samples. For 3 injections you have ret. time for IMP about 4 min and for other 3 injections ret. time is about 10min. It means that you have 2 different impurities. Normally under the same conditions retention time is consistence for the same analyte (if pump is working fine!)

2.You have six injection of the same sample (prepared separately). Retention time for impurity should be the same even if concentration (peak area) is slightly different because of errors in preparation step (extraction, cleanup etc.). If retention time drifts you have problem with pump (flow, mixing, gradient)

Regards

Tomasz Kubowicz

Hello

You have 2 possible situations:

1.You have six different samples. For 3 injections you have ret. time for IMP about 4 min and for other 3 injections ret. time is about 10min. It means that you have 2 different impurities. Normally under the same conditions retention time is consistence for the same analyte (if pump is working fine!)

2.You have six injection of the same sample (prepared separately). Retention time for impurity should be the same even if concentration (peak area) is slightly different because of errors in preparation step (extraction, cleanup etc.). If retention time drifts you have problem with pump (flow, mixing, gradient)

Regards

Tomasz Kubowicz

so let say i fall over situation number 1 for my first three sample prep, i also have 10 minutes but 4minute impurity has larger area. so which one should i report as single largest? thanks

I'm not really sure if I got it
You have six independent samples, right? It's NOT one sample with six independent preparations?
Your API(s) peak(s) show consistent retention times over the whole sample set?

So you have six different samples of which some show different impurity profiles...and what's the problem, now? If your SOP says to report the largest impurity, then you should report the largest impurity. If the largest impurity is a different one in different samples, then, well, you have to report different impurities for the different samples.