Is this Contamination?

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

8 posts Page 1 of 1
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)
Image
Image
Followed by the wash I did on the same column from yesterday morning:
Image
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:

Image
Image

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.
Why is this a problem at all for you if the contamination peak only comes off during washes and not your method itself?
We would NEVER keep around a column that did NOT check out fine, we would dispose/recycle.

Your method has a flow-rate of 3ml/min, which is a lot for any assay within the last few decades, maybe that's from a USP or similar antiquated procedure. If you can, I'd move to a smaller bore column and lower flow rates.
The examples I gave of the wash are there just to display the absorbance of the impurity, which I did find out was stationary-phase eluting off the column. Unfortunately, in my lab, the last tech. did not record ANY information whatsoever on the conditions of many of our columns or their storage solutions so I have gone back in to assure their quality.
Additionally, my other columns do not retain my compounds too well, and it requires an 100% AQ stable column for the job-- this was the only one I could find within our lab that was stable in these running conditions so I had to see if the quality of the column and my sample were good.
As for the mobile-phase flow-rate, I am actually trying to purify enough of this new sample for an NMR/MS and was trying to save time on a higher flow-rate. But, I clearly have not gotten that far as injecting samples. I actually did move to a smaller column, but am running it at a slightly higher flow rate but have not noticed any plate# disruption since switching from it's normal flow-rate. Both being well below the limits of the column and pumps.
This was an antiquated column, and I did get rid of it after I confirmed with the manufacturer the cause of my issue. Unfortunately, I have not spent much time in the chromatography space, and my predecessor did not have any records of the runs or standards used on the column prior to my arrival so I had no reference to compare it to. I'm not even sure of the exact mobile phase running conditions or samples injected previously on the column. Anyway, thank you guys for the help, and I will be sure to use your advice in the future!
The examples I gave of the wash are there just to display the absorbance of the impurity, which I did find out was stationary-phase eluting off the column. Unfortunately, in my lab, the last tech. did not record ANY information whatsoever on the conditions of many of our columns or their storage solutions so I have gone back in to assure their quality.
Additionally, my other columns do not retain my compounds too well, and it requires an 100% AQ stable column for the job-- this was the only one I could find within our lab that was stable in these running conditions so I had to see if the quality of the column and my sample were good.
As for the mobile-phase flow-rate, I am actually trying to purify enough of this new sample for an NMR/MS and was trying to save time on a higher flow-rate. But, I clearly have not gotten that far as injecting samples. I actually did move to a smaller column, but am running it at a slightly higher flow rate but have not noticed any plate# disruption since switching from it's normal flow-rate. Both being well below the limits of the column and pumps.
This was an antiquated column, and I did get rid of it after I confirmed with the manufacturer the cause of my issue. Unfortunately, I have not spent much time in the chromatography space, and my predecessor did not have any records of the runs or standards used on the column prior to my arrival so I had no reference to compare it to. I'm not even sure of the exact mobile phase running conditions or samples injected previously on the column. Anyway, thank you guys for the help, and I will be sure to use your advice in the future!
If you've got a target compound that's so hard to retain that you're having to resort to 100% aqueous, can I suggest looking at alternatives? First, if your compound is peppered in amine groups, consider running it in mildly alkaline conditions on a column that will tolerate them (if you're using Phenomenex, the Kinetex EVO C18 would be worth considering; other manufacturers have equivalents, I know Waters do decent alkaline-stable reverse phase columns too). Second, if it's so hydrophilic, consider Hilic.
Things that need 100% aqueous are a nightmare because the ultimate method will be teetering on the brink of failure, by definition, and you can't go any further to make it robust. It will fail if the sample composition changes slightly (because any organic solvent in the sample matrix will mess up peak-shape because it's going to wash the peak a long way into the column before it's diluted out enough for binding), it will fail if the method is transferred to a loop-injection system and no one thinks about whatever solution fills the part of the loop not occupied by sample; it may also fail on transfer to another system if the column equilibration time is a bit short, and you're relying on the autosampler's delay time to augment the equilibration (and even if you're not planning on transferring the method to another instrument, if you publish your work and someone tries to repeat it and it fails, it sort of creates a bad impression). But I do sympathise; Hilic's a pain to set up, and it's annoying having to find alkaline buffers rather than just doing the standard formic-acid-plus-water mix.
Hi Imh,

Thank you for the response! I'll take what you're saying and apply it to all my chromatography in the future. I believe I am in a unique situation within my laboratory. Without revealing any proprietary knowledge, the last chemist (I am the only one, and so was he) identified "active" compounds from a plant extract, purified them well enough to get a structure, and my goal was to use his synthesis and purify enough for in-vitro and in-vivo testing. Unfortunately, the active molecules he described and identified were never assured prior to testing in-vitro or in-vivo and what I have discovered is that the molecules he previously described are not the ones that provide the activity. We had been having a hard time getting consistent results from our biological assays and we found this to be the root cause. Unfortunately for me, I have to basically back-track to where he started and identify the active from a large range of these break-down/active products. Surprisingly, from one molecule (actually two, as it's epimeric) we get around 10-15 different active molecules. Some of them are really hydrophillic (explaining the 100% AQ starting conditions), while others are very hyrophobic. As you stated, those first few peaks are a nightmare when it comes to consistency, but as the molecules get more time on the column and in the MP the peak shape, consistency, etc. gets significantly better. Thus far, it seems that the molecules eluting in the fully-aqueous portion are non-actives and can be ignored for now thankfully. Thank you for your help and everyone else here's help as well-- I am always open to more suggestions or criticism!
Hi Imh,

Thank you for the response! I'll take what you're saying and apply it to all my chromatography in the future. I believe I am in a unique situation within my laboratory. Without revealing any proprietary knowledge, the last chemist (I am the only one, and so was he) identified "active" compounds from a plant extract, purified them well enough to get a structure, and my goal was to use his synthesis and purify enough for in-vitro and in-vivo testing. Unfortunately, the active molecules he described and identified were never assured prior to testing in-vitro or in-vivo and what I have discovered is that the molecules he previously described are not the ones that provide the activity. We had been having a hard time getting consistent results from our biological assays and we found this to be the root cause. Unfortunately for me, I have to basically back-track to where he started and identify the active from a large range of these break-down/active products. Surprisingly, from one molecule (actually two, as it's epimeric) we get around 10-15 different active molecules. Some of them are really hydrophillic (explaining the 100% AQ starting conditions), while others are very hyrophobic. As you stated, those first few peaks are a nightmare when it comes to consistency, but as the molecules get more time on the column and in the MP the peak shape, consistency, etc. gets significantly better. Thus far, it seems that the molecules eluting in the fully-aqueous portion are non-actives and can be ignored for now thankfully. Thank you for your help and everyone else here's help as well-- I am always open to more suggestions or criticism!
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