525.2 Extraction Guidance

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

5 posts Page 1 of 1
Hey folks,

I've been working for the last several months (year, really) developing an EPA 525.2 method for my laboratory, and I feel like I could benefit from some guidance. My problems at this point are two-fold:

1) My IS/SS recoveries are generally somewhere between 50-80%, which on the high end is sufficient but not something I consider functional. My worst recoveries tend to be for Acenapthene-d10, and 1,4-DM2NB.

2) On any extracted sample I get substantial background. It consists mostly of poorly-resolved "abstract art" rather than sharp peaks, and is scattered across the whole chromatogram (but more on the back end than the front).

Preparing my samples consists of taking ~1 L of H2O and making sure pH is <2 and no Chlorine is present, dechlorinating and/or adjusting pH as necessary if they aren't. I then add 100 uL of 50 ppm IS/SS mix to 5 mL MeOH, and then adding that to the sample. I add the IS/SS mix to the MeOH because the IS/SS mix is suspended in EtOAc, and I observed that if I added it EtOAc directly to the water sample then it could bead and not mix into the water, whereas MeOH has little trouble doing so.

My extraction method is modeled on the EPA method, though my volumes are in general larger because the automated extractors I use cannot reliably measure volumes smaller than 8 mL. It is:

* Condition with 8 mL 1:1 DCM/EtOAc, Soak 60 sec, drain completely
* Condition with 16 mL MeOH, Soak 60 sec, start drain but immediately move on to next step
* Condition with 8 mL Reagent H2O (right now HPLC water), Soak 0 sec, drain 2 sec but then move on so that liquid remains above disk
* Load sample
* Wash sample container with 8 mL Reagent H2O, Soak 0 sec, move on immediately
* Wash sample container with 8 mL Reagent H2O, Soak 10 sec, drain completely
* Dry disk for 150 sec
* Wash sample container with 12 mL EtOAc, Soak 60 sec, drain at higher vacuum to pull solvent completely through disk even in the presence of residual H2O, for 10 sec to remove most of the EtOAc
* Wash sample container with 12 mL DCM, Soak 60 sec, drain 8 sec to remove most of the DCM
* Wash sample container with 12 mL 1:1 DCM/EtOAc, Soak 0 sec, drain completely and keep vacuum running to remove any residual solvent

After the extraction process I take the 36 mL of eluate and pour it through Sodium Sulfate (in a funnel stopped up with glass wool) and into an evaporation tube to dry it, then blow it down under heat until it's at a final volume of 0.75 mL. I transfer that volume to a GC vial, and rinse the evaporation tube with ~0.4 mL of EtOAc before transferring 0.25 mL of that to the GC vial (though generally 0.25 is the whole volume I get at the end because some sticks to the glass and some evaporates while I'm working with it).

Right at the end of the process I add 10 uL of 500 ppm p-Terphenyl-d14 to my sample.

Then, I run it on my GCMS. The chromatography/spectroscopy side of things works beautifully for calibration standards and other unextracted samples (RSDs at 5-15%, R^2's generally at 0.999+), so I'm not too worried that my instrument side might be giving me problems. What I'm worried about is the extraction side of things.

What I think I lack is simply experience with this method. Because of the way I was brought on, I had no one in the lab with experience with this method to ask for hints and pointers, to check if I'm doing things right, and so on. If there are people out there who could help, that would be very much appreciated. The method is straightforward enough, but that doesn't mean that I'm 100% confident I'm not missing some small-but-critical detail.

So, what do y'all think?
The two analytes you are not recovering well are the ones with the lowest boiling points so it could be lost in the blow down, or in the air drying of the disk.

If the spectra of the junk in the background looks like hydrocarbons, then it may be the sodium sulfate or glass wool. We place our glass wool in a Soxhlet extractor and extract with MeCl2 over night then dry it in an over and wrap in aluminum foil to make sure all residual oils and phthalates have been removed. We also wash our sodium sulfate with MeCl2 then filter and place it in a muffle furnace at 450C to burn off any residual oils and phthalates that might be left over from production.

We are moving away from 525.2 and going to 525.3 but most of the ideas are the same. We also use cartridge SPE instead of disks, which some see better recoveries with while others swear by the disks as the best recovery, disk do allow for faster extractions though since the water will flow through them more quickly.

When evaporating, I like to evaporate to near 1ml, then wash the tube down with a few ml of EtOAc and continue to evaporate to the 0.7ml volume, this way anything that may have adhered to the tube gets washed into the final volume and you get rid of more of the residual MeCl2 to better match your solvent based standards. There is also the option of extracting blank waters with no surrogate or internal standard added and processing to completion to make a blank "matrix" to use to make solvent standards with, which will better mimic the sample matrix and account for any enhancement or suppression of response due to matrix in the MS.

It sounds like you are close to having it worked out, just keep tweaking a little at a time. One other thing to try is reduce temperature and nitrogen flow during evaporation to help with recoveries. It takes longer to reach final volume but can help with recoveries. I also use 1ml volumetric tubes to bring to final volume to make sure I have exactly 1ml in the final extract to eliminate any variances in that might come from inconsistent final volumes.
The past is there to guide us into the future, not to dwell in.
James_Ball wrote:
The two analytes you are not recovering well are the ones with the lowest boiling points so it could be lost in the blow down, or in the air drying of the disk.


That's a fair point. I'll try adjusting my drying time downward - the factory method is 60 seconds, but the EPA method is 600 seconds - and see what effect that has on the end result.

James_Ball wrote:
If the spectra of the junk in the background looks like hydrocarbons, then it may be the sodium sulfate or glass wool. We place our glass wool in a Soxhlet extractor and extract with MeCl2 over night then dry it in an over and wrap in aluminum foil to make sure all residual oils and phthalates have been removed. We also wash our sodium sulfate with MeCl2 then filter and place it in a muffle furnace at 450C to burn off any residual oils and phthalates that might be left over from production.


Another idea to try. I'll take some Sodium Sulfate and glass wool and clean it with a procedure like that, and see if that has an effect on the end result. A library search on the background shows that it is various C8 to C18 hydrocarbons and fatty acids.

James_Ball wrote:
We are moving away from 525.2 and going to 525.3 but most of the ideas are the same. We also use cartridge SPE instead of disks, which some see better recoveries with while others swear by the disks as the best recovery, disk do allow for faster extractions though since the water will flow through them more quickly.


I tried to convince folks to move to .3 but I got overruled, sadly. It's a better procedure, but sometimes institutional inertia wins out.

James_Ball wrote:
When evaporating, I like to evaporate to near 1ml, then wash the tube down with a few ml of EtOAc and continue to evaporate to the 0.7ml volume, this way anything that may have adhered to the tube gets washed into the final volume and you get rid of more of the residual MeCl2 to better match your solvent based standards.


The rinse procedure you describe is nearly the same as what I do. Right now I rinse twice with EtOAc while concentrating, once at about 2-5 mL total volume and once when the volume first reaches 0.75 mL.

James_Ball wrote:
It sounds like you are close to having it worked out, just keep tweaking a little at a time. One other thing to try is reduce temperature and nitrogen flow during evaporation to help with recoveries. It takes longer to reach final volume but can help with recoveries. I also use 1ml volumetric tubes to bring to final volume to make sure I have exactly 1ml in the final extract to eliminate any variances in that might come from inconsistent final volumes.


I tried reducing the N2 flow last week to see what effect it would have, and at least in my test the result wasn't positive at all. The results were pretty preliminary - I just looked for the yellow of the IS/SS mix in the final concentrate, and didn't quantitate anything on the GCMS - so it might be worth trying it again, but still. I've been running at a fairly low heater power setting this whole time, since I observed a positive change when I did that some months ago, but I have also been occasionally testing higher settings on head-to-head challenges to make sure that some combination of factors didn't flip the script on me and make high heat better. I haven't found that goldilocks zone yet, but I hope I do soon.

Thanks for your help! I'm going to test out some of those ideas, and if the results are especially noteworthy I'll come back to this and update you.
One problem solved, thank you James! It was residual hydrocarbons present in the sodium sulfate powder. After I rinsed it with DCM, let dry under the hood and then fully dried overnight in a 120 C oven, the background disappeared completely. I did that with the glass wool too, but by itself I couldn't see any substantial difference over the control group I did nothing special to.

I got some technical guidance from the manufacturer of my concentrators that said they now recommend vacuum pressures that substantially differ from those given in the manual for their equipment, so I hope that combined with these changes will get me where I need to be. We'll see!
ScientiaPotentiaEst wrote:
One problem solved, thank you James! It was residual hydrocarbons present in the sodium sulfate powder. After I rinsed it with DCM, let dry under the hood and then fully dried overnight in a 120 C oven, the background disappeared completely. I did that with the glass wool too, but by itself I couldn't see any substantial difference over the control group I did nothing special to.

I got some technical guidance from the manufacturer of my concentrators that said they now recommend vacuum pressures that substantially differ from those given in the manual for their equipment, so I hope that combined with these changes will get me where I need to be. We'll see!


We have had a lot of success using the N-Evap water bath with the needles for nitrogen to concentrate the samples, it seems to do it gently enough not to loose too much recovery.
The past is there to guide us into the future, not to dwell in.
5 posts Page 1 of 1

Who is online

In total there is 1 user online :: 0 registered, 0 hidden and 1 guest (based on users active over the past 5 minutes)
Most users ever online was 1117 on Mon Jan 31, 2022 2:50 pm

Users browsing this forum: No registered users and 1 guest

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food & Beverage, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry