Peak Fronting Issue

[olaniyi]

Hi all,

I ran a sample ( containing sugar estate, secondary amine and steroid ) on the Waters Alliance system a couple of months ago. I obtained good chromatogram for the amine and steroid compounds. When same sample was run on same method recently, I obtained bad peak shape ( peak fronting ) for the secondary amine compound. The method was ran at temperature of 50 degree celsius. The peak shape issue was only resolved after reducing the temperature to 35 degree celsius.

My intention is to obtain a comparable chromatogram on Agilent and Waters alliance system using the same method parameters. How can I obtain good peak shape for the amine compound at temperature of 50 degree celsius which is what gave good chromatogram on the Agilent system and on the waters system 3 months ago. Why is the amine compound now fronting on waters system and not on the Agilent system at all despite using the same method parameters, same column e.t.c

I'll appreciate your comments and answer.

Thanks.
Ola

[HW Mueller]

Same problem as in "Same column different LC" of Sept. 12?

[Mark Tracy]

In this case, I suspect it has to do with temperature control. You are running mobile phase at 25C into a column at 50C, and therefore the temperature is nonuniform in the column. The nonuniformity is less severe when you operate the column at 35C. A mobile phase preheater might solve the problem.

[DR]

If a preheater is not available for the Waters system (1100s preheat the mobile phase by default), running as much of the precolumn tubing into the oven as possible will help. You may even want to add a small length of narrow bore tubing and form it into a couple of loops.

[Sallybeetle]

I agree with DR's solution. I have fashioned different lengths of coiled stainless steel HPLC tubing to fit in the column heater. The coil is placed inline immediately prior to the column. I minimized the length of straight tubing between the injector and coil, or left a minimum amount of straight tubing prior to the coil. I then used the length of coil that gave the best chromatography for the HPLC assay. This solution has worked wonders for fronting, tailing, and retention time drift.

[ym3142]

Yes, Waters and Agilent have different heating mechnism. The former heats on column and the later on mobile phase, as u may have known.

[Rob Burgess]

I work with Ola, and it appears that this issue has been resolved partially (or at least we think so).

What Ola did with the Waters Alliance system was to use access and run direct function setting called "condition the column". I wasn't too sure what this was but the manual says it performs a gradient through the column.

When he next did a run at 50°C the peak shape problems for the basic amine have dissappeared. I was wondering if there was some bound material stuck to the stationary phase that was causing the problem? When the column was "conditioned" i.e. strong solvent wash placed through it this may have washed away any strongly retained substances and improved the column performance. Is this possible I ask?

The final point I wanted to get across to Ola (and others here) is that to really find out what any potential HPLC related problem is to employ the much talked about: "change one thing at a time rule". In realitywe weren't comparing like with like (i.e. Agilent vs. Waters) because we were using different columns and mobile phases (thus there are 3 different factors here). If we had tested the same column and mobile phase batch on both systems we would proably have got to the root cause of the problem much sooner!

[olaniyi]

Thanks for all your wonderful comments. I appreciate it all. Like Rob mentioned earlier, problem was solved by "conditioning the column" which is part of the various tools available on the kit. This may be something really important to do before running at high temperature of 50 degree celsius.