Background peaks and trash in extraction

[ScientiaPotentiaEst]

Hey all,

I'm working to optimize and remove contaminants from an automatic extraction process for 525.2, that is based on using C-18 disks, EtOAc, DCM, MeOH, and DI water, and I've been having some issues nailing down the source(s?) of the contamination. I'd like your advice and input, if possible.

When I analyze a blank (using reagent water, or that uses no water at all) that I run through the extraction process, I get peaks that are tentatively identified as diethoxyacetic acid ethyl ester (a sometimes VERY large front end peak), a bunch of fatty acids ranging from Caprylic (C8) to Stearic (C18), some Acrylic Acid (ethylene diacrylate), DEH-Phthalate, Benzyl Butyl Phthalate, and some other minor stuff that varies from extraction to extraction.

Additionally, when a colleague uses this water for Haloacetic acid testing she's getting a consistent background peak that is coeluting with an analyte, causing her calibration curves to go sideways and QC to look terrible. Suspicion is that the contamination is coming from the water, but it's hard to know precisely what that contamination is or why it's happening.

The extractors that I use are several years old. For a period of time (about 6-9 months) before I joined the lab there was no one doing 525 testing and they were unused, but they had reagents sitting in the lines. Whenever we mix up 1:1 DCM:EtOAc, I've noted that it slowly turns brown (over the course of a month or so) and that if left for a LONG time (a year or more) that precipitate will drop out of solution and adhere to the glass jar. To this day, I don't know what precisely that precipitate IS.

When I run Tap water, DI water, and polished DI water blanks from several sources around the lab side-by-side, the tap has a bunch of other trash in it and the general pattern of all of them suggests that there is contamination in the water. It LOOKS like organic carbon in the water that isn't being filtered out by the DI system, reacting with acids either in the water already (our DI's normal pH is ~5.5) or acids in the ionizing beds to produce the fatty acids. Also, the presence of acrylic acid suggests to me that the ionizing bed is shedding monomers that might be causing problems.

When I run a blank using only the reagents from my extractors (and using commercial HPLC water for the DI water), I get many of those same contaminants. I don't get acrylic acid, and the levels of everything else are about 20-50% of what I get from a "real" blank, but the peaks are still there. I performed a test where I ran about 20 blank purges through the extractors, and then poured all of the volume generated into a single concentrator vessel then blew it down to 1 mL, and those contaminants were all there (with the exception of the acrylic acid) at fairly significant levels.

The lab's DI system has never had installed a significant Carbon bed or UV treatment to remove organic carbon from the water, so that's one big reason I think that's a problem. The only parameter we routinely test for is resistivity, which isn't really helpful when you're concerned about organic carbon contamination. I've suggested that we take samples of the water and send them off for TOC testing to confirm this, but that hasn't happened yet.

So, where could that sort of stuff be coming from? I have a few theories, but I'm not sure how big of a tinfoil hat I might be wearing with them. Do you folks have ideas?

[DR]

It is possible that some styrene monomer is getting through your water system. It happens. Depending on how much water you need, you may be able to clean it up via Empore Extraction Disks. I don;t think they'll grab everything you've listed, but they have helped get mystery peaks out of many gradient LC mobile phase preps. Search the forum - there are at least a few "how to" entries and discussions on their use.

Sounds like, long term, you need a decent water system as well as thorough cleaning and passivation of your rig's solvent lines.

[James_Ball]

If water was left in any of the lines or bottle for extended periods of time and algae was allowed to grow, then that could also be a source of the fatty acids.

Have you tried pulling some 10% nitric acid or 30% hydrogen peroxide through the water lines on the extractor to see if that helps? They will remove biological growth in the lines if that is present.

Also on our previous DI system with polisher that took it to 18megaohm water we had to run a chlorine tablet in the special fitting for it once a month as the required maintenance to keep the water pure. Biofilms are the bane of any water system for organic use.

[ScientiaPotentiaEst]

"If water was left in any of the lines or bottle for extended periods of time and algae was allowed to grow, then that could also be a source of the fatty acids.

Have you tried pulling some 10% nitric acid or 30% hydrogen peroxide through the water lines on the extractor to see if that helps? They will remove biological growth in the lines if that is present."

That's also possible. There's not a whole lot of point in doing the acid wash to try and get rid of it since we're replacing the whole system here fairly soon, and I don't have anything time-sensitive on the menu between now and then, but it's a good thing to keep in mind. What I'm doing right now is making sure to perform several blank purges every day, in the hopes that getting things moving will bring down the background to an acceptable level.

"Also on our previous DI system with polisher that took it to 18megaohm water we had to run a chlorine tablet in the special fitting for it once a month as the required maintenance to keep the water pure. Biofilms are the bane of any water system for organic use."

That's true. Mostly the system is designed to provide water for mol. bio. type stuff - PCRs, differential plating, etc - so there was a lot of consideration given to sterility and being bacteria-free, but we're only now coming around to realizing that it wasn't designed to serve the needs of the other labs in the building.