increasing peak size with each run, no change in baseline

[magsterz]

Hi everyone:

I'm trying to get my system to give me consistent results with standards so I can start analyzing some samples. Right now I have a nice stable baseline with my mobile phase (basically 25% methanol, with SDS, citric acid, and EDTA). I've been injecting mobile phase onto the loop (100 ul) to see whether I can get rid of some unexplained peaks that I was seeing previously. I've flushed the injection valve, and I'm injecting mobile phase that has been withdrawn from the mobile phase bottle itself. I keep the syringe in the injection valve and have not removed it for the last four runs.

Here's the strange part. Every time I do an injection, I get a very similar signature (e.g., a small peak at 4 min, 6 min, and 10 min) but with each successive injection (of EXACTLY the same thing) the peaks are getting larger, especially the 10 min peak. Nothing is changing in the conditions -- at most there is an hour between injections.

Does anyone have any ideas about what might be happening??? I would be delighted to hear from you.

Maggie

[Kostas Petritis]

Is it isocratic or gradient elution?

[magsterz]

Isocratic.

[Kostas Petritis]

Maggie,

There was some similar discussion that replies partially your problem. The reply was from Chris Pohl and you can find it here http://www.sepsci.com/chromforum/viewto ... ight=#1327 but I also copy pasted below:

In addition of what Chris is explaining, the reason that you experience higher and higher peaks is because your system is not fully equilibrated. I would bet that if you wait several hours or leave your system to run overnight and inject again you will always have the peaks but this time their peak area will be constant.

The reason is that each time you inject you create a higher disturbance in your system as now you have more additives dynamically absorbed in your stationary phase.

That is my guess...

"If I understand you correctly, you are interested in understanding why you see a "ghost peak" for EDTA even though your sample didn't contain any EDTA. It is common to see a ghost peak when you add an eluent component which is visible to your detector. Whether it is negative or positive depends on the relative affinity of your analyte and your eluent additive. In the most common case, the presence of an observable peak in the chromatogram when working with an eluent system containing a detectable eluent component is caused by your sample disturbing the equilibrium between the stationary phase and the mobile phase with regard to this component. If the analyte has a higher affinity for the stationary phase than the eluent additive, this will displace at least some of the eluent additive into the mobile phase. This disturbance in equilibrium between the mobile phase and the stationary phase will be translated through the column as if you had injected the eluent component. In this case, since risendronate has a longer retention time than EDTA, it can be expected to operate in the manner described above. If you are interested in changing the location of the ghost peak, switch to a different chelating agent as your eluent additive. For example, NTA will undoubtedly elute significantly earlier than EDTA whereas DTPA can be expected to elute significantly after risendronate."

[magsterz]

Kostas:

Thanks very much for the reply. I think I get all of that. However, do you think it still holds if I am not injecting anything "new" into the system? The "sample" that's hitting the column has basically exactly the same composition as the mobile phase. I would imagine that injecting mobile phase ought to give me a basically flat line after the initial noise of the solvent front. The fact that I'm getting peaks at 4, 6 and 10 min has made me nervous.

In terms of the peaks changing, if this is a question of needing time to re-equilibrate, this also makes me nervous because my system is supposed to be able to analyze one sample after another if I'm using the autosampler. If it has to re-equilibrate after every injection, how do people do high-volume analysis?

[Uwe Neue]

How about the possibility that the solvents in your mobile phase in the sample vial are evaporating slowly and leaving the less volatile components behind, which you then see as positive peaks in the chromatogram that increase slowly in size?

[Kostas Petritis]

Maggie,

You have a point, and what Uwe suggest is a possibility, easy to verify if you just take a new sample from your mobile phase to inject.

Actually I was talking about the initial equilibration of your system and not the re-equilibration after each injection.

But indeed you shouldn't see these peaks if you are injecting your mobile phase and assuming that you are doing full loop injections etc...

[HW Mueller]

Maggie, what do you see when you actuate your injection valve just like during an injection without inserting a syringe, with a syringe (no piston movement) inserted, then injecting air and maybe also water?

[Vangelis]

Maggie,

assuming that you're doing full loop injections as Kostas said, it is indeed not explainable why you're getting these peaks. What HW Mueller suggested you try could give some explanation.

Regarding the peaks getting larger after every injection: could this be due to memory effects? Was the instrument used before by another person? Were there perhaps any high concentration samples passed through the system? If yes, was the system sufficiently flushed/washed after those injections, so that we exclude the possibility that leftovers have remained somewhere in the loop, the injector or somewhere else in the line and they're now driven into the column giving memory-effect- peaks?