Where to purchase a solid sampler?

[Ento_Joe]

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[GOM]

Hi Joe

I think that chloroform/methanol or DCM/methanol would be a better extraction solvent. Hexane will only extract the non-polars.

Ii still think that the chromatography can be sorted

@ Peter

Thank you- your comment is very much appreciated, since I am still worried that the idea is too off the wall.

Regards

Ralph

[Ento_Joe]

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[GOM]

Hi Joe

In answer to your questions

1. 50/50 is the usual mix for DCM/MeOH

2. A silica plate would be a good start

3.. An interesting paper - I need to read it more thoroughly.

4. You have demonstrated that hexane appears to be extracting biologically active compounds ( which is interesting in itself) - perhaps start with that as your developing solvent on the plate. Then later try the above more polar extraction mix together with that extraction solvent as an developing solvent to compare

5. 10 minutes extraction - you have the luxury of time - try 1 and 2 hours to compare

6. i still think that we can sort out the GC

I still think that it is an off the wall idea but would love to know if it works

Regards

Ralp

[Ento_Joe]

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[GOM]

Hi Joe

Cool - looking forward to your results - impressively fast response

The TLC is only suitable for low/non volatile compounds

I still think that there is scope for improving your GC

With regards to visualising the plates we can worry about that only if you see any positive results without treatment. Afterwards we can look at UV or charring with sulphuric acid for visualisation when we have established the Rf values

Cheers

Ralph

[Peter Apps]

Ralph, that TLC plan is brilliant - antibiotic assays upscaled from microbes to mites !

Joe - why not do the SPME with live mites - they will stir themselves and rub their cuticles on the fibre.

If the peaks from the extract are too small you could concentrate it by evaporation.

Peter

I agree with you Peter, Ralph's TLC plan is a stroke of genius!

I thought if I undertook the SPME analysis whilst the mites were alive I could end up with VOCs that don't originate from the cuticle being adsorbed onto the fibre - i.e. it would be difficult to determine the source of the chemicals?

I have previously evaportaed my samples to dryness under nitrogen and resuspended them in smaller volumes of solvent, which had no effect on peak size. I shall dig out one of my chromatograms, but it will have to be tomorrow now as my lab is locked up for the evening.

Cheers,

Joe

Concentration of the sample having no effect on peak size may be a worry - it implies that the peaks you are seeing do not come from the extract, or far less likely that the hexane is saturated. Try the solvent mixtures suggested already by Ralph.

To develop the TLC plate you just let a bunch of predators run around on it and take a photo when they have arrested on the active spots - equivalent to the bioautography methods used for antimicrobial discovery; http://www.rsi2004.lubelskie.pl/doc/sty ... _E_art.pdf
and Choma and Jesionek Chromatography 2015, 2, 225-238 doi:10.3390/chromatography2020225
which were the first two hits on a Google of "TLC antibiotic assay auto".

I wish that I could get my large carnivore study species to run around on TLC plates !

Peter

[Ento_Joe]

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[Peter Apps]

Hi Joe

If you are still seeing hexane after 5 min there may be a problem with the setup or method - which is very likely to be fixable.

I work on scent communication in African wild dogs, lions, leopards, spotted hyaenas and cheetahs, with most of the current focus on wild dogs and leopards. Also some spin off results from small carnivores. The plan is to replicate scent marks to make artificial territorial boundaries to stop carnivores moving from protected areas into livestock areas, where they fall foul of lethal controls by livestock owners. http://www.bpctrust.org/bioboundary-project.asp

Peter

[Ento_Joe]

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[rb6banjo]

This has been a fascinating exchange. I read it twice but I can't seem to find the information regarding what happened in the SPME analysis of the multitude of mites. Did you see anything?

Joe, early on you mention that your instrument has a thermal desorption system attached to it. Did you ever try to merely do a direct thermal extraction of some of your mites using that? Thermal extraction is taking your solid sample, putting it in a tube, and using an external heat source to somewhat selectively (because you can control the temperature of the tube) desorb/extract volatiles and semi-volatiles out of the solid sample and get them on the GC column that way.

[Ento_Joe]

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[GOM]

Hi Joe

Sorry for the delay - I have been out all day strawberry picking and eating ice cream at the local farm with my grandson.

I do have a few other questions:

1. Would a chloroform:methanol solvent mixture also be in a 50:50 ratio?
2. How much of the extract do I need to add to a TLC plate?
3. Should I precondition TLC plates prior to use?
4. How long do you leave the plate in the developing solvent?
5. I use the same solvents for developing TLC plates as I do for extraction? i.e. hexane for hexane extracts, chloroform:methanol for chloroform:methanol extracts, etc.

1. usually yes - doesn't have to be but is what people often choose
2. Depends on the plate and the sample concentration. Typically from what I recall 50 - 200uL range. The crucial thing is to apply in small aliquots and allow each application to dry before adding the next. The prevents spot diffusion and keeps it to a tight spot or band.
3. Not sure
4. Until it approaches the top of the plate- but not all the way. Need to mark the solvent front point when removing from the chamber.
5, Not necessarily - but hexane is a good starting one - it may need to be modified slightly if the components aren't moving. Actually, thinking further, migration or separation of the dark green colour may give an indication of having a suitable developing solvent

See ( amongst many others) for "practical TLC" this link for school practical

http://www.saps.org.uk/secondary/teachi ... c-pigments

Some useful tips and and a possible source of plates. Have you considered using paper?

Regards

Ralph

[Ento_Joe]

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[GOM]

Hi Joe

Thank you - that is very kind of you.

It would also be nice if you acknowledge other members who have contributed, which I suspect that you will anyway

We look forward to hearing more on your experiments in this fascinating study.

Regards

Ralph