Recovering samples that have evaporated

[UBC Forestry SIF]

Hi All,

We had a visiting scientist who prepared samples long before my GC was ready to receive them. She had extracted plant jasmonic acid and salicylic acid using published derivatization techniques, and diluted the the extract in hexanes before sealing the plastic microcentrifuge tubes and freezing. Before the GC went down, I managed to run one test sample, which I further diluted in hexanes to 1:20, and got a nice chromatogram. Now months later with the GC fixed, she has left and most of the hexanes have evaporated in the freezer (some vials appear completely evaporated).

I tried to redissolve the analyte in the empty tubes by adding the exact amount of hexanes (so I don't alter the original concentration too much). Some of the dried tubes, when redissolved, have what appears to be a little gelatinous bead that doesn't dissolve at the tube bottom, despite vortexing or waiting a while. I then made a 1:20 dilution to put on the GC as usual. Regardless if the sample had a little bead or not, peaks are not showing up. Small peaks appear when I make a 1:10 dilution.

Has anyone experienced something similar? I thought only the hexanes were evaporating, but could certain analytes also escape a sealed tube, along with the hexanes? Perhaps the compounds have broken down over time? The visiting scientist is long gone, but is awaiting the data. Since I cannot reproduce her experiment, I am trying to salvage what is left in the tubes.

Thanks,
Alice

[bisnettrj2]

I'm no expert, but if the hexane evaporated in the freezer, then that means the hexane had a chance to escape the vial - and water had a chance to enter. Your acids are probably in the water (the gelatinous beads?), which aren't miscible in the hexane. I would probably start over at this point, unless you had novel samples as starting points. Maybe you can try to add a dually-miscible solvent, like acetone, to try to reconstitute the two layers? Just a guess, I'm probably wrong, but at this point even a broken watch is right twice a day, right?

[prepcolumn]

Hi All,


Has anyone experienced something similar? I thought only the hexanes were evaporating, but could certain analytes also escape a sealed tube, along with the hexanes? Perhaps the compounds have broken down over time? The visiting scientist is long gone, but is awaiting the data. Since I cannot reproduce her experiment, I am trying to salvage what is left in the tubes.

Thanks,
Alice

Alice
Derivatization (possibly silylation) for GC is generally performed to make the molecule more evaporative and this makes the molecules to be analyzed with GC. That's why, it is probable that your analyte also evaporated with hexane.

[UBC Forestry SIF]

Good point about the derivatization. To clarify, the JA-SA protocol mentions vapour-phase extraction, methylation and methanolysis. This protocol is one not usually run in our lab, so I am not familiar with it. Usually our extracted fatty acid samples are derivatized. My mistake.

[Don_Hilton]

What material is the plastic microcentrifuge tube? If it is polypropylene, I would wonder about long exposure of hexane to the plastic...

[RJH007]

Viewing your problem I have a couple of thoughts
1. The sample you tried to reconstitute - did you continue the evaporation to absolute dryness before adding the hexane again? If you didn't then residual water or hexane - sample polymers could form this gelatinous pellet. This would never redissolve in the new hexane and hence you would be subject to a significant decrease in recovery. You can see this quite often when you don't neutralise plasma prior to extraction with hexane, if there is too little hexane it goes gelatinous and you get no recovery at all.
2. Evaporating to dryness or freeze drying the sample could get you back to original state whereby you could reconstitute in hexane or dichloromethane. The biggest problem would be to ensure you have 100% recovery and without an internal standard in the original solution that is going to be difficult. You could try adding an internal standard to hexane and add a know quantity to each tube prior to evaporation but this would only get you around half your problem.

hope some of this helps

best regards

RJH

[UBC Forestry SIF]

I assume the microcentrifuge tube plastic is the typical polypropylene. I am unable to source where these tubes came from, so cannot check.

When I reconstituted the sample, I chose tubes that looked visually empty. Perhaps there was some residue at the bottom tip I did not see, hence the formation of the jelly bead.

RJH007 brings up a good point about the internal standard. I had stressed the importance of this to the visiting scientist who prepared the samples. Unfortunately she was not aware of this aspect, despite the protocol saying an internal standard needs to be added. (there was a big language barrier). Anyhow, I have removed the Parafilm from all vials and am allowing all vials to go to complete dryness inside the freezer. However, as you say, even if I could recover some of the analyte into solution, who knows how much is left. Doesn't make for very accurate concentration calculations.

Thanks all for your input!

[RJH007]

I do have one last though, if you add an internal standard to the tube and then allow it to evaporate naturally in the freezer you do not change you sample at all. However, after you reconstitute your samples in a known volume it should be possible to transfer the solution to a clean vial and you can then repeat the reconstitution on the original vial so you have two samples. Ideally the first would contain 100% sample plus 100% internal standard, the second would be clean of everything.

We do not live in an ideal world so the first would not be 100% but more like 85-95% or even lower (30 - 50% worst case), this remaining sample should therefore show up in the second (or third if its a really bad recovery). Since you added an internal standard it should be possible to get a concentration of both solutions and get a summation for the total.

This is a compromise and you would be making some significant assumptions on recovers but at this time I cannot think of any other way round this problem

RJH

[Peter Apps]

Sorry, but it is hard to think of a worse combination than plastic tubes sealed with parafilm to store solutions in hexane. Parafilm does a reasonable job as a barrier to water vapour, but it very permeable to organic solvents, and especially hydrocarbons. Given long enough, even higher MW compounds like your analytes will diffuse through it. In addition, the wax in the parafilm will contaminate the samples to such an extent that you will have to use a very selective detector - like an MS - to make your analyte peaks stand out from the forest of contaminant peaks.

Peter

[debpilot]

Jasmonic acid is volatile (will evaporate easily), as is its (sic) methyl ester derivative.
http://www.samuelfurse.com/2012/02/when ... onic-acid/

[ontherun]

Just to point out that I have seen the formation of gelatinous white-ish stuff (sometimes separate grains are visible) with pure mixes of hydrocarbons in hexane (“pure” as in straight from the manufacturer). That happens after transferring the mix to vials (from the original ampoule) and storing the vials at ~0 Celsius over one day. It may have to do with temperature. The gelatinous matter normally disappears if the vial is left at room temperature for a few hours.