Dear all,
I need help now. I run HPLC with one of the peptide, on RP with 0.1% TFA/ACN as mobi phases. There is an impurity peak came out. The weird thing about this peak is 1) did not show in my blank and system suitability standard. 2) peak area/height is not changing when sample is diluted. 3)did not show up in the dilution buffer inject (10x more), 4) this peak give the same area if I double the injection, though my peptide peak is doubled.
what could be the cause of this peak? Anybody have experience of this lipid problem in HPLC? I only have VWD detection.
thanks
Based on the information provided, it is possible that the impurity peak is caused by a secondary reaction that occurred during peptide synthesis or due to post-translational modifications of the peptide. The impurity peak may be structurally similar to the peptide and could be co-eluting with the peptide peak.
It is also possible that the impurity peak is caused by a minor component in the sample matrix that is not detected in the blank or dilution buffer.
To further investigate the cause of the impurity peak, you could try the following:
1. Change the mobile phase: Try changing the mobile phase or adjusting the gradient to see if the impurity peak disappears or shifts. This could help determine if the impurity peak is related to the mobile phase composition.
2. Try a different column: If the impurity peak persists, try a different column to see if it is specific to the column you are currently using.
3. Analyze the impurity: Collect the impurity peak and try to isolate and identify the impurity using techniques such as mass spectrometry or NMR spectroscopy.
4. Try a different detection method: If possible, try using a different detection method such as UV-Vis or fluorescence to see if the impurity peak is detected by other methods.
Regarding the possibility of the impurity peak being caused by lipids, it is possible, but it would be unlikely to cause a peak that is not affected by dilution or changes in injection volume. Lipid contamination typically causes peak tailing, and adding an organic modifier to the mobile phase, as previously mentioned, can help to address this issue.
Thank you for your help! Lamia Fatima . One thing I forgot mention, we tested this peptide 7 months ago, the strange peak was there before but not as strong as now. We only tested one sample at that moment because the purity pass the spec. Thanks
It could be a 'gradient' peak when Mobile Phase A switches over to Mobile Phase B. Typically, it occurs at the same time (see your timetable), is not related to your injection volume, and depends on the composition of your Mobile Phases.
Bruce Neagle HI Bruce, I have a smooth gradient that runs from 15% B (0.1% TFA in ACN) to 35% in 25 min. But the peak is not show up in the blank.
Thanks
Nancy
A peak is still possible. It is probably due to the difference in 'refractive indices (RI)' between the Mobile Phases (thus no UV peak). You can probably ignore it if it is away from any other peaks of interest OR you can reduce it by inserting a transitory ramp or slow down the current ramp.
Lamia Fatima
Problem solved. It turns out that it was caused by the guard column gasket. Somehow it is reacted with only this peptide because all the other peptides were good.
When I washed and changed the gasket, the peak disappeared. Thank you all for your help.
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