Chromatography Forum

Hello,
We would like to transfer UHPLC method developed on one system to second identical system in another lab. However, system 1 runs Empower and system 2 is under EZchrom. Receiving lab set the same method parameters, columns and solvents from the same batch, both systems have comparable dwell volume, however the receiving lab gets worse resolution (you can see it without integration) for the peak of interest than the same sample prep analysed in system 1. So I wonder maybe there is a difference how both software handle data and plot chromatograms, or even how both execute method parameters during run ??

Before suspecting the software I'd rather have a thourough look at the hardware if there are any differences.
- Are those two UHPLC machines exactly the same or just "comparable"? UHPLC methods tend do be even more hardware-specific than usual HPLC-methods. A few microliters more or less of system or dwell volume might create huge differences in chromatography.
- Is it possible to swap the columns between both machines? So that you're absolutely sure it's not column-related.
- Concerning the software, I'd double- and triple check the method parameters. Especially have a look at detector parameters. Data aquisition rate and response time can have a huge influence on fast chromatography.
- From the chromatogram, can you see what's the root cause of that worse resolution? Are those peaks closer together (aka a change in selectivity) or are they broader (change in efficiency)?

Before suspecting the software I'd rather have a thorough look at the hardware if there are any differences.

- Is it possible to swap the columns between both machines? So that you're absolutely sure it's not column-related.

That's exactly what I would do. Sometimes it's the little things that are big (Yogi Berra).

Thanks guys, I'll have a look at these potential issues.

Hi, among many other possible issues as mantioned above, make sure that Empower and EZchrome systems have the same gradient curve and if the curve is set by a identical number, check that the number is really for the same gradient shape. Chromeleon and Empower for instance differ in this respect.

Let me back up a bit here. Given similar overall retention, resolution depends on selectivity (relative retention) and efficiency (peak width). So, when resolution was "worse", which of those changed?

If the change was in relative retention, but the peak widths were comparable, then the data system is *not* the problem; it must be in the chromatography: either the controller is delivering a different gradient profile (I'm assuming gradient, since you mentioned consistent dwell volume), or the proportioning system is malfunctioning, or the temperature is different.

If the change was in peak width but the spacing of the peaks was comparable, then it *might* be in the data system (check particularly things like sampling rate, bunching rate, time constant, etc.) or it could be hardware related (differences in extra-column volume -- especially if you are using "UHPLC" columns), or even method-related (especially if the diluent is stronger than the initial mobile phase).

Hi, among many other possible issues as mantioned above, make sure that Empower and EZchrome systems have the same gradient curve and if the curve is set by a identical number, check that the number is really for the same gradient shape. Chromeleon and Empower for instance differ in this respect.

We operate Agilent UHPLC under Empower and I can't find any field in the Agilent Instrument control framework (Instrument Method) that would allow to select a gradient curve, besides "Flow ramp up" and "Flow ramp down" rate. I assume it must be a linear transition for all gradient steps.