Chromatography Forum

Hi everyone. I'm having trouble analysing related substances of aspartame by Ph.Eur. First of all, I'm wondering if anyone else have had trouble with this, and how you ended up solving it?

The problem I'm facing is that the references seem to elute at the same time as the injectionpeak/voidpeak(or what do you call it?) or at the same time as another reference (B), L-phenylalanine (from Sigma-Aldrich). However, we are not certain of this, it might also be so that one of the references (D) have been decomposed. The reference is L-aspartyl-L-phenylalanine from Aldrich, but is it probable that it could have decomposed? It was first used in august 2008, so it is quite old, but still.

I was thinking it would be easy to check this by analysing them separately, but I don't seem to get any peaks from these in the chromatogram, even though I have been getting a peak for reference B earlier. This makes me believe that the reference (D) has been decomposed.
It's very strange, since the retentions times also seem to change quite a bit between difference sequence runs.

I have tried two different LC-systems (Both Shimadzu), three different columns (MZ analytical perfect sil C18 250x4,6mmx5um; Phenomenex Gemini C18 250x4,6mmx5um; Supelco Ascentis C18 250x4,6mmx5um) with one of them having been used succesfully before (the Phenomenex column) and I've mixed several new mobile phases. None of this have worked and from what we know I'm doing exactly the same as the analyst who did this analysis before and succeded with it. The only thing different is that I'm not using the same LC, however they are almost the same in components. Also, I dont get the same retention times for all references as the previous analyst received.

If anyone has any ideas, it would be very welcome!

Hi

First a link: http://www.edqm.eu/en/Databases-10.html

the knowledge database and reference database is open and sometimes provides good info.

Have no knowledge of your analysis but the above databses indicates that references (both aspatrtame and impurities) may be sensitive overtime (store at +°5C, shipping may be ambient), so unless you have control of the references I strongly recommend to get new ones and store them accordingly.

Databases only gave one column brand/packing name back: kromasil C18

Good luck

Chris

Thank you Chris. The aspartam impurity A is stored in the fridge, but the autosampler has been run at 20C. This has been changed now.

Yesterday i ran a sequence of only reference B (phenylalanine) and reference D (L-aspartyl-L-phenylalanine) separately. I then received peaks at 14 and 28 min respectively, which was the expected tR. I then started the whole sequence, with samples and all references. However, this morning I noticed that the peaks had different tR and in some cases, there were no peaks at all. This seems to be due to a pressure drop, which lasted ~30 sec before stable and normal pressure was established again.
Also in the run where the pressure dropped, the pressure dropped at ~35 min and peak was expected at 14 min. This peak did not show at all. Why is it a peak before this pressure drop does not show?

I've been getting more and more suspicious to the mobile phase, rather than anything else for each run being done. Could it be the mobile phase which messes up the chromatograms? We're running 90 % KH2PO4 which is set at pH=3,7 with H3PO4, and 10 % Acetonitrile. Both systems I've used for this haven't shown this behaviour of pressure drops before, so I doubt it's any mechanical issue but any suggestions would be appreciated.