Hi Everyone,

Background: I don't have much experience with HPLC (less than 2 years), but my employer has given me the complete rights to the HPLC system in the lab. This comes with cleaning the instrument, making sure it has little to no downtime, cleaning and storing columns, preparing mobile phase, etc. (within their means). I received this position from the prior BIOLOGIST (they are an almost exclusively biological lab, and I'm the first chemist they've hired) who explained some of the preliminary concepts behind chromatography (which I later discovered were mostly incorrect) and described to me his methods for mobile phase prep, as well as some other things like the flow-path, the different columns, some of the methods, etc.
Recently, I took one of the columns out of "retirement" (I have no data on standards, past injections, old samples ran, mobile phase storage conditions, etc.) and have noticed a peculiar systemic issue with this column. Whenever I wash the column (morning, end of day, end of runs), there is always a strongly absorbing impurity that elutes off the column in various amounts. I need help identifying what is causing this issue and potential solutions.

Instrument specifications:
I use a Shimadzu system with a CBM-20A (communications), SPD-M20A (diode array, UV abs), 20AR binary pumps, and the column is a Synergi Polar RP column with the dimensions: 150mm x 10 mm. I'm not sure about a lot of the qualities of the column prior to my use, it does not have high pressure, but I do not know the age or exactly what was run on it prior to my experiments.
The mobile phases I use are MPA: 99.85% DI H2O with 0.15% Formic Acid
And MPB: 99.85% Acetonitrile with 0.15% Formic Acid.

Method Specifications:
The method file I use for my wash is pretty simply described, so I will not write our specifically each step. This method has a flow-rate of 3ml/min, and a general average psi of 1100. The wash begins and ends in a 100% aqueous portion (100% MPA) as I analyze ultra polar compounds (Note: even on standard washes without the 100% AQ starting and ending conditions, this problem still persists, as well as if I'm injecting different samples (This is a wash from yesterday on my preparative column after a few samples :Image). This step is followed by five pillars of wash where the method file goes up to 70% MPB five times in the span of 10 minutes, while shooting back down to 10% MPB in the "troughs". This was pretty standard for all of the washes that we used, and my mobile phase is relatively standard across all of my columns and I have never seen this particular issue.

The issue itself:
I've got a few images included as I did a "contamination" test described by various authors on this website where I let my column equilibrate for 20 minutes following the end of my method, followed by another wash and then equilibration for 40 minutes followed by another wash.
The following image is of the wash I took this morning (one zoomed in, one not)
Followed by the wash I did on the same column from yesterday morning:
Now the two test contamination washes are as followed: the first is of the 20 minutes extra eq, and the second is of the 40 minute eq:


If you have trouble opening/seeing the images, right click on the icon and select "open in new tab". I attempted to make them visible, but there is certainly something I've screwed up!

Conclusion: Anyway, from what other authors have described regarding the look of contamination on a column, my chromatograms certainly give me the impression that I am dealing with that. This is strictly a column issue as I have never seen any issues with the same mobile phase (prepped yesterday, used on both this column and another (that had no contamination peaks)), or when I put a different column on the instrument (same frits, etc. too). The only real difference that I can think of is this specific column. Do you guys think that this column can be cleaned? Is this not specifically contamination but perhaps the column itself degrading in these conditions? The Synergi Polar RP column is stable to a pH much less than mine, but perhaps (certainly) the last user mistreated it and introduced some instability? Where should I go from here/what cleaning techniques do you guys recommended? Should I cease using this column altogether?

Thanks for the advice-- sorry for the long post! Please don't be afraid to ask me to run more tests, or provide more information. I'm trying to be as succinct as possible.

Tyler S.