Peak Fronting (Co elution) Troubleshooting

[HPLCaddict]

Sorry for being cynical, but I'd say your main problem is that you are running a black box. You don't know what the mobile phases are??
The first thing I would do is to really push the supplier of that "method" and the materials. You are PAYING for a working method, right? It used to work in the past? It doesn't work now? You didn't change anything? How can you be sure that THEY didn't change anything with the supplies? If they're not willing (or able) to help with troubleshooting, I'd start looking for a new supplier...just a few thoughts.
Some more specific things: are you really injecting 250 ul? That's quite a bit and if that tiny disturbance at ~0.6 mins is your injection peak, it's definitely too much for your column. BTW what's all that junk eluting after your peaks of interest?
As already asked, what's about reequillibration?

[M Farooq]

Farooq, it is fronting! Remember that right after the 1. Control another one is injected (control 2) which is just a replicate. That control (2) doesn't exhibit the same fronting. Ergo a mobile phase composition inconsistancy is most probably the issue. More thorogh review of the method parameters including the gradiet table is the way forward.

Best regasrds

Dear Dancho, it still seems to me that there is a co-eluting component in the "tetra" peak, which is giving an impression of fronting. If the "tetra" peak were truling "fronting" due to the mobile phase/ stationary phase interactions, it would always front in each and every run. The peak should never change with time. This is not the case here.

Since the original poster is saying is that the "fronting" increases with time, it is very likely that a time dependent component is degrading, leading to a very definite shoulder on the tetra-peak.

The only solution is to find out that particular component by mass spec. A simple trick might help the original poster. Increase the injection volume till you see a large peak for this shoulder. Manually collect this fraction from the detector outlet into a very clean vial and send it for mass spectrometry to another facility. This is a poor man's way of LC-MS which works very nicely for simple problems like these.

Hope it helps.

[danko]

Control 2 inst exactly a replicate. Control 2 has a higher percentage of disialotransferrin, so in a sense control 1 is like a negative control where as control 2 is a positive control.

! Had it been a partially co-eluting peak, it would’ve been even more pronounced when the concentration is higher as it obvious is in the second control.

Dear Dancho, it still seems to me that there is a co-eluting component in the "tetra" peak, which is giving an impression of fronting. If the "tetra" peak were truling "fronting" due to the mobile phase/ stationary phase interactions, it would always front in each and every run. The peak should never change with time. This is not the case here.

! True, but I’m not convinced that the mobile phase composition during the elution is the same all the time. I’m having this feeling that the re equilibration is inconsistent – or The sample solutions are of varying pH and/or organic solvent content.

Since the original poster is saying is that the "fronting" increases with time, it is very likely that a time dependent component is degrading, leading to a very definite shoulder on the tetra-peak.

! Yeah but then it would’ve been the same for all samples and not only the first control.

I think an investigation of the elution conditions and sample solvent/s is essential her in order to solve the mystery.

Best Regards

[NO89]

Good news!

So yesterday I received a new LOT of controls from the company who provide us with the original ones. I ran the new control one and it work perfectly, no fronting / co elution.

I also ran an injection of the previous control from which we were having issues with and there was fronting in the tetra peak.

So it seems that there was definitely a stability issue with the control.

The provider of the control said that they had changed the sample matrix in the set set of controls to human serum. This seems to have done the trick.

It seems that my post had got everyone thinking here and seemed to have got a few of you debating here which I hope you all have something to take from.

So many thanks for all your input, I know I have learnt a few things here regarding trouble shooting some issues.

[danko]

Hmmmm, very stragne story.

You rote that you’ve been running this method for a year or so and the problem arrows all of a sudden.
Also you wrote that you tried a competitors control and experienced the same issue with it.
Finally, why doesn’t the second control exhibit the same behaviour as the first?

Best Regards

[NO89]

Danko, I agree it is very bizarre indeed.

I have ran the new control again today to test its stability after reconstitution and kept a 4 degree celcius over night and its still works fine.

And yes that is right, this method has now been run for 10 months, and the issue has raised over the past month. The original set of controls come in sealed packages, and made fresh every time, so its not like they have all of a sudden become contaminated at some point either.

Yes, i tried a control from a competitor (Chromsystems) and the exact same issue happened. I don't know if i am being too picky with my chromatography or not, but the fronting exhibited just isn't acceptable in my eyes. The only thing i can think of is that both products have been created using the same sample matrix.

I have done some research into this and i found the following paper, http://www.clinchem.org/content/49/11/1881.full.pdf

In their study they used samples from citrated plasma and they saw the exact same issue i did, seen in figured 2 D page 6 of the paper attached. Fronting in the terta peak.

With the new LOT of controls i have received I have been told that the sample matrix has been changed, and this has worked.

I think what we are seeing here is also a stability issue as control 2 is now beginning to behave in the same way. So the second control is actually exhibiting the same behavior as control 1.

Best Regards

[Gerhard Kratz]

It looks like you are running a gradient????? with a quaternary pump system??? Please check if all of your 4 magnetic check valves are working properly and flow rate is correct.

[danko]

Sorry for all the misspells in my previous post. I forgot to read it before sending it.

OK. Could be fun to measure pH in the solution with the “old” control formulation and compare it to the “new”. Also the “control 2” solution’s pH is of interest in my mind.

You see I still think there is something about the injection its self rather than degradation (i.e. additional peak)


Best Regards