Chromatography Forum

I am attempting to purify a peptide. I am using a Phenomenex Jupiter Proteo column. I have been using this same analytical system (Waters System/ 0.1% TFA in HPLC HOH and 0.1% TFA in HPLC ACN) for several months without anything like this happening. I have two different systems to check my samples. One analytical column is only a couple of weeks old, the other is a few months old. Recently I discovered my Reference Standard that I use to be overloaded. The peak are is twice what it normally is. Keep in mind that I run a Ref STD every week on my systems to check the content of my pools. Also, after the peak eludes from the column the baseline has shifted up, and there is a new impurity that I haven't detected previously.

To Keep you inform I have:
- Changed my buffer and by this I mean I have made fresh buffer,
- I have used a separate analytical system independent of the ones that I normally use
- Switched analytical columns using a different Phenomenex Jupiter Proteo column that I have never used

Still experiencing the same shift in baseline, overloaded peak area, and new impurity profile

Can anyone give me any ideas?

Is your reference solution fresh, is it possible that it has concentrated, or wasn't initially fully-soluble, or the sample solvent is not adequately buffered, or different to the initial mobile phase?. Is the needle rinse effective - a blank after the reference sample flatlines?.

Based on information to date, the most likely cause ( assuming that your baseline drift increases with CH3CN ) is that the mobile phase CH3CN or Water quality has changed.

Please keep having fun,

Bruce Hamilton

I figured it out. I ran a blank sample on my column and discovered that my aqueous phase buffer was contaminated. There was an additional peak in the blank that matched the exact retention time of my peptide, which would account for the area being double what it normally is. After making fresh buffer and running a blank there was no evidence of contamination. Thanks for your suggestions.

Thanks for reporting back after solving the problem. I have done similar things, it is hard to believe that one bodged a solution prep. . . one has the tendency to blame everything and everybody but oneself.

I think that you are using the same containers to make your analytical buffer and your prep collections. It seems that this may be the reason you are seeing peptide in your blanks. Sorry, dont mean to come down on the new guy! Just my 2 cents...