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Fractalhead, March 18, 2005 in Ethnobotany
oh goody :D
I have some TLC questions if anyone here has the answers?
Do the solvents used have to be miscible? I've seen solvent systems recommended which simply won't dissolve in each other. This kinda looks weird to me, I thought you'd want them mixed all nice and evenly no matter what proportions they were in, not to mention the difficulties of dispensing small amounts of separated solvent systems equally over several smaller jars
How long is too long in terms of developing time? I tried some plates the other day from a paper, that took 1/2 hr to develop. The compounds all ended up at the top of the TLC strip in a horizontal blur when they'd started as a nice strip. Personally I reckon the solvent system was wrong and the compound dissolved entirely in the top part of the separated solvents, and was able to stay as a band cos the long developing time meant it was able to dissolve because it was so slow. Am I wrong?
What was the type of plate you used at EB3 Fractal, and do you have the details on it? Aluminium backed something or other special wasn't it?
I've got the hang of the TLC basics now, but I still have a lot of messing around to do with solvent systems and documentation etc.
Umm...Dipolar doesn't really mean anything.
A molecule with a net dipole (variation in electron density across it's atoms) is said to be polar.
Acetone is less polar than ethanol and more polar than hydrocarbons.
I don't know what you mean by "metabolic structure". "Metabolic" means to do with the synthesis and degradation of molecules in a biological system. It's got nothing to do with polarity.
Ahh shit, yeah i ment molecular structure, sorry dude i was way off there.
as for polar/di/non its used when refering to solvents. it is to signify the shape of the molecule and the solvent action. there is a dipolar catagory for solvents and acetone is in it. its writen on this 20 gallon drum of pure acetone. if anyone have a friend who works in a steel mill and they need solvents ask your friend but do not store them at your house as your insurance will be void. :D
EDIT - Dipolar Aprotic Solvents are what they are called. do a google man. here is one good resource for solvent info http://www.usm.maine.edu/~newton/Chy251_25...s/Solvents.html
[ 05. April 2005, 10:34: Message edited by: Amulte ]
The relevant distinction they are explaining in that reference is between protic solvents (eg alcohols) and aprotic solvents (eg ketones).
The terms polar and dipolar are reffering to the same thing, ie the presence of an electrical dipole, as opposed to non-polar solvents which have either little or no dipoles, or no overall molecular dipole (as in CO2 which has two dipoles which cancel out as they point in opposite directions).
What that ref calls "dipolar aprotic solvents" are also called "polar aprotic solvents".
AH HA! thanks dude, had an discussion about this with a mate about 2 months ago. i owe you a beer man cuz you just won me six! lol. i think ill just go back to uni and do chemistry again. too bloody long ago now. sorry for confusion and thanks for pointing my right. i was almost there but too many people tell me differnet. back to uni/tafe 4 me!
DL I can answer your Q's:
Do the solvents have to be miscible?
Yes. The final solvent system should be one homogeneous liquid. Otherwise, in scientific terms: you'll get all kinds of funky shit happening.
How long is too long for development time?
Well it depends on the length of the plate and varies from solvent to solvent. 1/2 an hour is a perfectly reasonable run time for a slow solvent and a long plate (say ~10 cm). If the development chamber is not saturated with solvent vapours, the solvent front tends to move a lot slower and the Rfs are a lot higher (ie. things travel further up the plate). In addition to keeping a nice sealing lid on top of the jar/beaker, the way to get a saturated atmosphere is to place a piece of filter paper around the inside of one half of the beaker/jar such that it draws up and becomes saturated in solvent system. It then releases and disperses solvent vapours into the upper chamber atmosphere. Give the beaker a bit of a shake to get the paper all wet. You can lean the plate against the other side of beaker. This will make things move a bit quicker.
If all your spots end up at the top of the plate, your solvent system is too polar and hence, competes too well with the polar silica gel for adsorption of the compounds. Try a less polar solvent. I will put up a list of solvents and their dielectric constants (polarity measurements) shortly as a guide to help people choose solvents.
The plates I used at EB3 were Silica Gel G60 Aluminium backed plates with F254 fluorescent indicator and 0.25 mm thick layer. This is the most common type of plate and is very versatile. The choice of backing material depends on the kinds of detection reagents/methods you want to use. If you want to use high temperatures to "char" your compounds with or without a detection reagent you will need the aluminum backed plates. However, if you don't want to use high temps but do want to use corrosive acidic detection reagents etc. then you need the plastic backed plates as the aluminium will react and screw your plate up. The kits will come with 5 of each so people can try everything out.
Hope this helps. Keep at it, DL.
Thanks fractal, I thought they had to be miscible, I was wondering how it would work otherwise, and obviously it didn't.
Hey, that's a nice trick - haven't heard of that before. It'd be good especially for long runs, or if working in a hot/windy environment where you were losing solvent.
I've updated the ethnowiki TLC section with a Table of solvent parameters including dielectric constants. Dielectric constants can be used as a rough guide in choosing solvents to try when you need to adjust the polarity of your solvent system up or down but are not the be-all and end-all of solvent system design. Optimising a solvent system is more of an art than a science but there are strategies you can employ to minimize the time spent on trial and error. I will expand on these as time goes on.
Next, I'll make a grid showing miscibilities of various solvents in one another.
got mine today and it come in a tackle box, i feel like a CSI lol just waiting for someone to call me and tell they need somthing anilysed
As a way of giving back and helping to promote the conservation of the knowledge we benefit so greatly from, I've decided that for each TLC kit sold, I will donate $5 to the Amazon Conservation Team.
Headed by Mark Plotkin (author of 'Tales of a Shaman's Apprentice') these guys have some really cool projects going on to help conserve shamanic knowledge.
went to dick smiths today to grab a UV light all they had were those security one, didnt have an abrosbance on them:( any one know if they are any good???
Fractalhead i would be keen to purchase one but not for a month or so yet, is this cool?
[ 24. April 2005, 03:53: Message edited by: hebrew ]
also to make up detection reagents could i use metho instead of the 96% ethanol, the stuff here is 96% ethanol 4% somthing else :D lol. and i had no luck finding and aerosol paint sprayer so it a normal fine mist from a spray bottle ok.
[ 24. April 2005, 07:59: Message edited by: teonanacatl ]
The emission spectrum (which is what i think you mean by 'absorbance', teo) of all your common fluorescent tube blacklights has a characteristic peak at a wavelength of around 365-366 nm. This type of UV is often just called longwave UV as opposed to 254 nm UV which is referred to as shortwave UV. Unlike 254 nm UV, 366 nm UV will not excite the fluorescent indicator that is impregnated into the TLC plate layer but will cause many fluorescent compounds (many indoles) to glow.
Hebrew, you can order whenever you like. Unless it all dies in the arse, this should be an ongoing thing and the kits should be available for months (if not years) to come.
Teo, regarding the use of white methylated spirits to replace 96% ethanol in the making up of vanillin and ehrlichs detection reagents- I've been meaning to try this out so I can add it as a hint on the ethnowiki. I am 99% sure this would work fine but until I try it myself I would be reluctant to advise it in case you waste your reagent - of course you can try it if you want I will do it in the next week or so and report back.
You can get away with a pump action sprayer if needs be. As long as it is not too wasteful and creates a nice fine even mist. I've even seen a pipette used to pipette the reagent onto the surface of the plate and it worked acceptably.
just wondering if using metho worked fractal???
Sorry I've had a lot going on and haven't had a chance to do it quite yet. I will do my best to get around to it this weekend.
any luck fractal :D
Hi guys. I've finally got my shit together and tested out the use of methylated spirits for the purposes of making up Ehrlichs and Vanillin detection reagents for TLC. Just as I suspected, metho works perfectly.
These reagents are extremely sensitive (ie. you can detect minute amounts of compound with very small amounts of reagent) so you should be able to make the 500 mg of each that come with the kits go a long, long way. It is best to make up these solutions fresh each day since they don't keep very well.
I was introduced to a really cool method for using these recently:
1. In a CLEAN vial or test tube, dissolve a matchhead or so of the reagent in ~200-500 ul of metho and add a 2-3 drops of conc. HCl (hardware store grade is fine). Mix.
2. On a CLEAN surface (eg. a watchglass) saturate a piece of filter paper (that has been cut to a size slightly smaller than the TLC plate to be detected) with a few drops of the reagent solution so it is moist but not dripping all over the place.
3. After the TLC run, let the solvent system evaporate off the silica and then carefully push the plate silica face down onto the saturated filter paper. It is easiest to place one end down first and then smooth the rest of the plate down by running your finger down the plate. Make sure the plate surface is in even contact with the filter paper and within a few seconds, remove the plate from the filter paper in the reverse fashion (try not to damage the layer by sliding it all over the filter paper).
4. IMMEDIATELY hold the plate over a heat source such as an electric hotplate or stove (better to use tweezers or forceps or pliars etc. rather than touch the plate and reagent with fingers). It is important to do this immediately in order to evaporate the ethanol and prevent diffusion of your spots. The heat will speed up the colour reaction. The spots should appear within a few seconds. You aren't trying to char the plates so don't put the plate on the stove but hold it up above the heat source. You'll know when its warm enough
So there you go. Sorry for taking so long, but I've been in limbo for the past couple of months. Looking forward to hearing some more feedback. Please et us know how you go!
yahoo now i can start
How about we start making some stock solutions of those things we would like to identify. Then it will not matter if we are using different solvents, as we can compare our extract to a known stock solution. If our points match we know what we have.
Different solvents will carry different substances in different ways, meaning there will be no absolute correlation between different solvents and the distance the solvent carries our extracts.
With a stock solution of a knoewn strength it will be easier to make a quantitative estimation based on how many times you apply your extract in comparison to the stock solution
Now, are there any TLC packs still available?
Thats an excellent idea foolsbreath and something I have been trying to get around to for some time now. Which standards do you have in mind? Maybe we should start something along the lines of the Australasian Free Standard Ring (AFStR)... I certainly don'e have time to prepare every standard people might like. These standards don't have to be pure compounds or solutions of pure compounds but may be well characterised mixtures. As long as we have some kind of benchmark. Looks like I need to start offering some small volumetric flasks and some volumetric capillaries...
There are a few challenges with this standards based approach however. Firstly is the instability of many of the cooler compounds. Secondly is the legality of many of the cooler compounds. These things make to trade or possession of some standards a little tricky. In these cases, we may have little choice but to use relative Rfs and colour reactions for identification.However these problems only cover the minority of potential standards. Bring it on... I will offer a few standards as soon as I can - GC/MS verified where possible
BTW... I can definitely put you together a kit if you want one. Just email me to line something up.
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