EPA Method 530 Extraction problems

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

6 posts Page 1 of 1
I am wondering if anyone else is doing the EPA 530 analysis and SPE extraction. I have been having a problem with the o-Toluidine and the deuterated Surrogate falling out very quickly after extraction. I normally have to extract, concentrate and run the sample the same day or else the surrogate begins to fall out. After 48 hours I can lose almost half of the surrogate recovery I had after immediately analyzing the extract.

I noticed that the extract is mostly Acetone, since you elute with 2ml acetone, then 5 ml Methylene Chloride then another 5ml Methylene Chloride then concentrate at 40C with nitrogen flow. This leaves an extract that is mostly acetone due to the higher boiling point. I have noticed the most stable extracts are the ones I have blown down to about 0.5ml or a little less while I have trouble with ones that I stop at about 0.9 ml. Could o-Toluidine react with acetone? I can leave the standards which are prepared in methylene chloride on the instrument for days at room temp and not see any loss of analyte, but even when kept in the cooler the sample extracts will lose surrogate concentration.

Just wondering if anyone else has had a similar problem.
The past is there to guide us into the future, not to dwell in.
I am not familiar with this method, but I noticed when I did solvent recycling that wet acetone seemed to make the loss of HCl from DCM a lot worse.
o-Toluidine plus HCl will make a salt.
It's things like this that make me question the robustness of mixed solvent blow down steps, especially when they can't be easily dried.
I did an extraction on Friday and ran two test samples without the acetone step. They didn't recover any better but they also didn't recover any worse. Only three of the extracts had low surrogate recovery, well low enough to fail at 50% recovery limit. The fact the limit is so low compared to 70-130% for most other surrogates in the new methods for UCMR4 shows that there is a problem inherent in the method.

I am doing another set of samples today and will reinject the samples from Friday without acetone and see if they lost surrogate or not. If not, then I will know that part of the problem is the acetone. Just figuring out how to get around it will be the next thing to figure out if that is the case.
The past is there to guide us into the future, not to dwell in.
Just received this reply from EPA regarding method 530 extractions.

,”The acetone in the 530 method is used to help the methylene chloride elute through the sorbent, due to the fact water was put through it previously. If the acetone is causing issue, I would recommend a different brand, or higher quality. You can take the acetone step out, but some sample cartridges would exhibit difficulty with the methylene chloride moving through them.”

I noticed it did take a little more to get the elution flow started with only methylene chloride but it wasn't terrible (using Waters HLB cartridges) so my next extraction set I believe I am going to skip the acetone and see what happens.
The past is there to guide us into the future, not to dwell in.

Full extraction batch without Acetone and all the recovery passed. First full batch to have passing surrogate recoveries so far. Reshot the extracts the next day and surrogate recoveries were within 1% of the original if not exactly the same. Seems without Acetone there is no breakdown over time of the o-Toluidine or the deuterated surrogate.
The past is there to guide us into the future, not to dwell in.
Update to the Update.

We still have random failures of the o-Toluidiene-d9 surrogate. More passing than failing but still have failures and if the samples sit on the instrument we are seeing loss over time even without the acetone present.

We had one bottle extracted that was preserved for 525.3 instead of for 530 and that sample had great recovery and almost no garbage in the chromatogram, while the bottles preserved for 530 have background peaks that are 10x to 100x greater than the internal standards spiked at 1ppm.

Beginning to wonder if it is possible that Diazolidinyl Urea or the Trisma buffer could be reacting with o-Toluidiene.

https://en.wikipedia.org/wiki/Diazolidinyl_urea This molecule has so many active sites on it it would not surprise me if it is reacting in some way. The preservatives are buffering the sample to pH7 so there shouldn't be acid or base catalyzed reactions going on.

The samples with the lowest recoveries have a peak just after the surrogate peak which I believe is the reaction product from the loss if that analyte, but I need to analyze in full scan instead of SIM to try and identify the peak. It seems other labs are having the same problem and we are trying to figure this out, but nothing so far.
The past is there to guide us into the future, not to dwell in.
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