Chromatography Forum

I mistakenly injected high amount of high concentration tryptic digest of protein mixture. Now in every blank I see a huge chromatographic peak. This is a peptide of MW 3403 Da, confirmed from mass spectrometry data. I have washed the column by 95% Acetonitrile for overnight. Even then I see the same peak. The most surprising part is that the peak intensity is increasing! Any solution?

Hello. It seems that mobile phase is better solvent than 95 % AcN. Could the column be washed 1) with the best solvent 2) reversed?

Thank you very much. By mobile phase dd you mean water: ACN = 98:2? And what is the best solvent?

It depends on the analyte's nature. I suppose that 95 % AcN is a bad solvent for most of charged polar analytes such as aminoacids or peptides. By mobile phase I mean the mobile phase of the method is used when "the peak intensity is increasing".

Hi, Dap. I am running a simple gradient of Water and ACN. It starts with 2% ACN and in 60 minutes it reaches 95% ACN. The peak I'm seeing is around 60% ACN.

Are you suggesting me to wash the column with that particular % of ACN where the peak is eluting?

Yep, it looks a good idea.

Thanks a lot Dap. Your suggestion helped me get out of the trouble. What I did:

Disconnected from MS. Increased the flow to 1000 uL/min and started with 5% ACN in water. Passed 10 column volumes and then brought down the flow to 200 uL/min. Connected to MS and checked the spectra. Repeated this step with subsequent increase in ACN, 10, 20, 30, etc. up to 95%. I saw the peptide is coming out at 20% ACN and as I increase the ACN percentage, the intensity of the peptide becomes less. So, I washed the column with 20% ACN in water at 1000 uL/min for 2 hours. When I ran the next blank the peak is now very less. That means this method works. I'm going to wash a bit more and see the blank again.

Thanks again.

Welcome!