hi,I would like to get advice on is it by using different HPLC system but same column would give us different chromatogram?I got this problem recently,I cant get the same chromatogram os a extract with different HPLC system.Thank you.

Please give us some more information so we can help you.

What column are you using, and what mobile phase? Is this an isocratic separation or a gradient? What analytes are you separating, and how are the results different on the two different systems - different retention times, different peak areas, different order of elution?

What are the two HPLC systems - Waters, Agilent, Shimadzu, Dionex, ...?

Getting different results on different HPLC systems is not unusual for some separations, but we need more information before we can decide if you have a similar problem.

What column are you using,

- is Ascentis c18 column with id 4.6mm , mobile phase is A: 0.1% TFA B: Acetonitrile
Is this an isocratic separation or a gradient?

-ya, I am doing gradient

What analytes are you separating,

- I am trying to separet a crude fruit pee; ethanolic extract

and how are the results different on the two different systems - different retention times, different peak areas, different order of elution?
-I get very different HPLC profile.different in retention time,peak area and even the peaks.

What are the two HPLC systems - Waters, Agilent, Shimadzu, Dionex, ...?

-using SHidmazu HPLC system & GIlson HPLC system

And another different thing is that For SHidmazu system I set temperature of the column compartment at 40degree while with Gilson System I only put it at room temperature.



Thank you.

Gradient separations on different HPLC systems are always a problem. Every system has a different way of mixing the solvents for the gradient. Some systems mix before the pump (low pressure mixing) and some mix after the pump (high pressure mixing). Each system has a different "delay volume". This is the volume of liquid between the mixer and the column, and this volume causes a time delay between what you program at the mixer and when the mixture travels to the column. If the delay volume is different on two different HPLC systems, then the time delay will be different, and the chromatograms will be different. If you have a very fast gradient (less than five minutes long), then you may see bigger differences. However, usually you only see differences in resolution. The same peaks are present, but some are not separated from each other on one system.

Another problem is the difference in temperature for the two systems. Temperature can cause a change in "selectivity" which means the relative retention times can change. You may see compounds eluting in a different order. This may explain some of your differences in chromatograms.

So what should you do?

1. Please send me more information on the gradient - percent A and B and time.
2. Repeat the gradients at the same temperature.
3. Try a slower gradient - make the change over a longer time.

If thiese suggestions do not help, send a picture of the chromatograms, and we will try to help you again.