Chromatography Forum

I am new in HPLC troubleshooting, and I would like to know the possible reasons for the decreasing of the peak area when the injection comes from the same vial.

Any other changes in the chromatography? For example:
- does the retention time remain constant?
- does the peak tailing get worse?

Possible reason can be:

1. When punctering the vail septum a small hole is made and the sample simply evaporates between injections.

2. Sample decomposition. Your sample may not be stable at the temperatur which the sample is stored in the autosample and therefore you get lower and lower peak height on following runs.

3. Maybe you detector lamp is nearing it's end. So it gives a smaller and smaller area on all following runs. Somewhere in you software there is propably a way to check lamp intensity.. try this and see if the lamp is work according to specs.

These are just a few possible reasons. There are many more, more or less, exotic reasons that can explain what you are seeing.

Hope it helps!

I've checked the temperature and the pressure on my system and it's stable. On the other hand I tested the lamp as well and it's fine too.

Another problem related with this is that the retention time decrease gradually after 7-8 injections.

Need more information in order to make informed suggestions.

How much is the area changing (by what %)?
Does the area decrease by approximately the same amount with each injection?
How much is the retention time changing (%)?
Does the retention time decrease by approximately the same amount with each injection.

Depending on your detector and integrator settings, a decrease in retention time could cause the area to decrease (in principle, it should not; in practice it can).

The decrease of retention time can explain the decrease of peak area.
Your peak is getting broader so your integration is getting less accurate.

Have you checked your mobile phase?
Does the solvent evaporate so the mobile phase is getting more polair during analyses, for example by using helium flow.

Once I experienced that my mobile phase separated in two layers (without seeing). I solved the problem by stirring the mobile phase during the analyses.

Okkie

Okkie, you mean narrower? If anything, an increase of integration accuracy thus a change in area?

An increase in flowrate would also explain both of the observations.

1. When punctering the vail septum a small hole is made and the sample simply evaporates between injections.

3. Maybe you detector lamp is nearing it's end. So it gives a smaller and smaller area on all following runs. Somewhere in you software there is propably a way to check lamp intensity.. try this and see if the lamp is work according to specs.

Evaporation of solvent between replicate injections usually leads to larger areas, not smaller areas.

Detectors tend to show lower responses as lamps age, but not over the course of several consecutive injections. As lamps get Really old, you typically start to see excessive noise in the baseline punctuated bu occasional large spikes.

Changing retention times means that your system is nto yet stable, so you should not expect much in the way of reproducibility until you can at least have stable retention times. If you are using Triethylamine and a C-18 column (for example), it can take quite a while for things to fully equilibrate. I have been known to make a small batch of phase with 2-3x the prescribed TEA conc. and run it through a new column in a loop over night before using the column for a run w/ a TEA mobile phase. This sort of "breaks in" the column.

Maybe this is an injection problem if there is no fault on the hardware system.

I will see if any of the analytes stick to the needles, or syringe causing diminishing withdraw.

Also, i will check the stability of the article to see its stability.

You could be getting a partial vaccuum building up in the vial, if you are doing multiple injections from the same vial, especially if you have a larger (>50ul) volume.

Do you get the same problem if you perform a similar number of injections, but from separate vials?