Chromatography Forum

"As you can see from this chromatogram I am missing the larger peaks that were present in the chromatograms yesterday (mostly fatty acids), but the later plant sterols still appear. The peaks that appear around 26 minutes in this chromatogram can not be identified as linoleic or stearic acid despite being in the same place as previous chromatograms. I'm at a bit of a loss here, perhaps I need to increase desorption time and temperature?"

I wonder if the plant sterols are external on the mites because they were walking on plant materials?

For temperatures, you can try both higher and lower temps just to see how they work. You may also find at what temperature the exoskeleton material begins to break down by detecting release of compounds you would believe are internal to the mite.

Hi Joe

Looking good.

With the thermal desorption I think that you probably have big enough peaks to work with - if you expand the vertical axis you'll see more components than you can wave a stick at. Before you go further with it you need to condense the mite vapour and make sure that the predators go for the condensate.

Rather than thermally desorbing the adsorbent you tumble with try extracting it with solvent - I don't think that the materials are likely to be very thermally stable. You could try something inert like diatomaceous earth if you wanted to do thermal desortion.

I am really interested to hear what happens when you let the predaotrs loose on those TLC plates.

Peter

Hi Joe

Several cross over threads

I will post out a 50cm length of surgical grade tubing but not until next week just to slow you up - enough for 50 experiments

Cut into 1cm lengths. place into a beaker in a GC oven at approximately 200 °C for several hours to condition and remove already absorbed atmospheric volatiles

Remove and place in sealed containers to avoid contamination. I used 2ml septum sealed vials. Use tweezers to handle them.

Place one in your mites.

Desorb by dropping into your thermal desorption unit or injection port. Use another as a blank rum to .distinguish between residual tubing artifacts.

Regards

Ralph

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Enjoy your ice cream

You have nailed the application technique on the TLC - it looks great

The tubing can be reused many times. Alternatively it can also be solvent extracted with approximately 200uL of solvent rather than thermally desorbed.

You would need to run an extraction of the tubing as is as a blank extraction control to compare

Regards

Ralph

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You have clearly nailed the application technique on the TLC in a very short space of time- it looks great

The tubing can be reused many times. Alternatively it can also be solvent extracted with approximately 200uL of solvent rather than thermally desorbed.

You would need to run an extraction of the tubing as is as a blank extraction control to compare

Most solvents are compatible apart from THF

You will find that the tubing swells with Chloroform based mixes but will regain its shape after evaporation.

The important thing is to do a blank control on a blank piece of tube with the solvent of choice

Yes. I seem to recall that to my cost tenax will dissolve in chloroform

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Keep your plates in the fridge until its time for

"Smithers - release the mites"



Regards

Ralph

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Hi Joe

I posted the tubing today.

I prefer to use it in thermal desorption mode.

The tumbling SPE or diatomaceous earth suggestions sound better for solvent extraction.

I recall conditioning the tubing in an empty 1/4" glass GC column - several of the pieces placed in and left with a small flow of helium at high temp to remove volatiles and reduce siloxanes. You could use your thermal desorber for this stage.

Below - The top thermal desorption trace is one that I ran to compare a poorly conditioned tube with that of a better conditioned tube (lower trace). The major peaks are siloxanes



If you must try solvent extraction try 75/25 methanol/chloroform. CHCl3 on its own seems to just get totally absorbed and swell the tubing. Try testing a few pieces first to get the right solvent mix.

Sometimes it is helpful to think of PDMS as being a very thick low polarity liquid/solvent.

It would be worth preconditioning the tubes with a triple extraction using that solvent. Say 200uL for each extraction using one of those vials that you used to show us a picture of the extracted mites. Try running each of those 3 conditioning extracts in reverse order to see if the background siloxane peaks are reduced to an acceptable level

This is a comparison trace of a sample (top) vs blank (bottom) of a solvent extracted tube that I used in the analysis by exposure to fungal volatiles



In the case of a negative result on your plates

This would be interesting in itself.

In my opinion, every negative result is actually positive in that it directs you towards the correct result.

You said originally that an extract had exhibited biological activity.

1. Even if the TLC solvent system had not migrated and separated your applied spot then one would have expected some activity around that spot .

2. or even you had just applied it and not taken it through the procedure. as a control

Unfortunately I have no experience of how you confirmed the biological activity of your extract.

The implication from 1 and 2 could be that the active compound(s) are too volatile and are being lost in the TLC procedure

Regards

Ralph

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Hi Joe

A combination of chemicals is entirely possible. Also, if you get attraction in an olfactometer the attractant must be airborne, so it might have evaporated off the plates. Ticks are attracted by prey volatiles, and then squalene which is just barely volatile acts as an arrestant that stops them moving. If your plates had lost the volatile attractant the predators would only get arrested (!?) if they stumbled across a patch of arrestant. How fast do they move and how long did you let them wander around ?

I would be tempted to cut the TLC plates into strips about an inch wide, run them and then led the predators loose - with less area they would be more likely to walk onto an arrestant spot.

Also it is worth seeing what they do with the spot loaded at the origin before the plate is developed.

Also they might need an air current, or a concentation gradient to track "upwind" to a prey.

Peter

Hi Joe

Don't worry, when you receive the tubing you should find that it will fit in your unit - it is a narrow OD tubing. For us dinosaurs it is 1/8th inch OD= 3mm I selected it to fit in equipment like this Even narrower OD tubing is available If it is a problem just cut it lengthwise

Peter wrote
Also it is worth seeing what they do with the spot loaded at the origin before the plate is developed.

I agree and I posted this thought earlier

see

2. or even you had just applied it and not taken it through the procedure. as a control


I still like his SPE idea

The chromatograms you have posted are really helpful, although there doesn't seem to be much difference between the solvent blank and the actual sample one? Yes - That is why we always run a control

I really like your enthusiasm but just take pause, re-read and digest all of the previous comments

The images that you are posting are really interesting

Kind regards

Ralph

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