I synthesized a short hydrophobic peptide and confirmed its synthesis using MALDI_TOF analysis for the crude sample. Before purifying using a semi-prep column the sample was injected into the analytical column(Discovery® HS F5 HPLC Column) and showed a nice peak. However, when I collect the sharp peak as a fraction manually to confirm that it's the main product using MALDI_TOF no desired mass is seen!! Could you please help me with this?
Hi,
That is strange; are any other masses observed in the MALDI spectrum of the sample from the peak you collected?
Hi
Yes, there are some other signals. But higher or lower than desired mass.
Eef Dirksen
Hi
Yes. It was adjusted for 220 nm and 280 nm wavelength in the case of the peptide bond and aromatic amino acids absorption respectively.
Andrzej Wasik
The UV signal/peak may come from substrates or byproducts of your synthesis. Are you sure your compound gets eluted from the analytical column? Since we don't know the nature of your peptide nor the analysis conditions it's difficult to give any advices.
Is there only one peak present in the chromatogram? If so, it would be quite suspicious to get such perfect chromatogram for a a crude sample.
What is the molecular mass of your peptide? If its mol. mass is close to that of a matrix you use for MALDI-MS, the signal of your peptide can be masked by signals from the matrix. In this case ESI-MS is to be used.
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