Fractalhead

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.

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.

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.

Hehe. i've heard about that funky rue jelly fungus too. Bizarre.

I forgot to mention, for those of you who don't need all the extra stuff, I can send just the plates and capillaries for $25 inc postage.

Ok, I've put together something of a demo kit to give you all a rough idea of what the basic kits will be like. Here's an image: The starter kits will contain: 1 large beaker 1 small beaker watch glass/beaker cover 10 pyrex test tubes 10 glass pipettes + 2 pipette bulbs 10 glass micro-vials 1 small measuring cylinder 1 large measuring cylinder 10 4 x 10 cm Silica Gel G60 TLC plates (with 254 nm exciteable fluorescent indicator) ~100 glass micro-capillaries (for sample application) This will all come packed in a plastic storage container to keep it all safe on its travels ** Note that some of the stuff that will be in the kit isn't shown in this picture. ** Firstly, there will be more TLC plates (ie. ten 4x10cm plates). You can cut these up as small as you like to make them go further while you practice. If you cut each 4x10 cm plate into five 4x2cm plates (which you could run in the small beaker) you will get 50 runs out of this kit. On each run, you might fit 3 or four samples so you are talking maybe 100-200 samples before you need to order more plates. Of course you may opt for more complete separation by using longer plates in the large beaker. There will also be an extra (larger) measuring cylinder for making up solvent systems and detection reagents. Also, there will be some filter paper for putting inside the development chamber (beaker) so as to saturate the chamber with solvent vapours. The beakers can double as both development chambers for running the TLCs aswell as for preparing solvent systems and detection reagents (they also look cool on your kitchen shelf . You will need to source solvents and things locally as these cannot be sent in the mail. Its amazing what you can do with a bit of acetone, vinegar, metho, ammonia etc. There are a few highly recommended (though not absolutely essential) additional items that you should consider budgeting for when thinking about getting into this: * One would be an aerosol propelled "paint" sprayer. You can get these from the big hardware stores and they basically consist of a glass jar with a aerosol can that screws onto the top and sprays the contents of the jar out as a fine mist. These are about $25 and are perfect for spraying detection reagents evenly onto the plates. * Another thing would be a handheld UV blacklight. With a spectral peak at about 366 nm, these are awesome for showing up all sorts of cool fluorescent compounds without exciting the fluorescent indicator dye that is impregnated into the plates. You can get these lamps from Dickos or Tandy for about 10 or 20 bucks. If you can get your hands on a 254 nm UV light (CAREFUL: DANGEROUS FOR EYES AND SKIN) you can detect UV absorbant compounds as dark spots on the plate against a blue fluorescent background. I've added everything up (including my time and effort) and worked out these kits will cost $60 + $9.50 p/h. I realise its a bit more than I first invisaged but I felt a few extra bits and pieces would go a long way. Those of you who are interested should email me at: [email protected] and we can sort something out. The Ethnowiki TLC Page will evolve rapidly in the near future so people interested in getting into TLC should check there regularly for new info. Finally, a VERY important point I should make: These kits are for conducting LEGAL research on LEGAL plants/substances only. While I am more than happy to offer support and advice with regards to conducting general types of TLC analyses once everyone is up and running... I will NOT reply to any emails or PMs asking me explicitly about analysing illicit substances. [ 11. April 2005, 22:13: Message edited by: Fractalhead ]

There are analytical laboratories scattered all over the place that do contract analysis. Like most things scientific, thorough analysis and unambiguous data (such as that obtainable by GC/MS or LC/MS) is bloody expensive (i would guess $50AU per sample minimum), especially if only a few samples are analysed and if the lab has to spend time preparing/fractionating the samples. While you can get a lot of really useful, accurate information from big fancy machines, these machines cost BIG $$ to buy and maintain and employ people to run them. This is what you will be paying for. If you have a fair idea what you are looking for, you might be better off to just analyse your samples yourself by TLC. This is cheap (less than $1AU per sample), fast and great fun although you may not be able to identify all the compounds in your sample.

Ahhh that's the exact phrase I used to use when trying to make city dwellers question their *own* metropolitan existence!

I was flicking thru the condensed version of Bushfires & Bushtucker called 'Pocket Bushtucker' or something like that by Peter Latz and noticed that under Ipomoea muelleri, it mentioned the consumption of the roots by the Warlpiri and Anmatyerr people and more importantly: the seeds by the Warlpiri. I think this would be a good lead to chase up and find out what preparation methods were involved. If the seeds are roasted or soaked prior to consumption, this might rule out their use as an entheogen. Very interesting. Just how many gnomes have explored this species I wonder?

Maybe teonanacatl can help us out. I know where there is a M. fragrans in Cairns. The only problem is that their is only one tree. I have found nuts on it but not sure whether they will be viable without a male tree around. I think I tried to germinate one or two of these nuts without success. They might have been a bit old though. Teo, PM me if you are willing to keep an eye on the tree and share. Actually, why not try taking some cuttings or even grafting onto insipida rootstock (easy to get). Come to think of it, vegetative propagation from this tree would def be the go since if you grow from seed it can take years before you know whether your plant is male or female (good). If we graft from this tree we'll be sniffing pungent nutmegs much much sooner. Teo, are you up for it? Fuck I wish I was still in FNQ... its a botanical wonderland. [ 31. March 2005, 09:16: Message edited by: Fractalhead ]

I had a really high success rate germinating M. insipida seeds when I lived in FNQ. I don't remember the insipida seeds being anywhere near as pungent as fresh nutmegs tho (geez they are strong!)

smogs, i have a feeling those condensers (at that price) will be the oldschool type (requiring a hose attachment) and not quickfit. Is that right? How much for the quickfit ones?

Yeah, I've started building the TLC section, just a bit of framework. Creating a readily updateable Rf table in HTML format looked like it would be a complete nightmare so i made a link to an Excel spreadsheet (empty at this stage) that you can download and edit yourself. This will have the added advantage of allowing you to 'plot' simulated TLC chromatograms using excels chart function. this will help people recognise patterns on plates. I will gradually add heaps more to this page so people can access virtually all the info they need in one spot. its amazing how hard it is to find good quality info on the web.

Amulte, I haven't worked out how much the kits will be yet but the minimal kits will probably be about $20-30. These will include most of the bare essentials to have a go at the technique and become familiar with it. The rest of the bare essentials will need to be sourced locally. I will also provide the option of a more advanced kit and extra TLC plates.

I'm guessing i'll have the basic kits ready in about two weeks (i'm waiting on a few things).

While everyone is free to figure out their own methods by trial and error, someone may find that for a particular kind of extract or class of compounds that a particular system works really well. If this can be made from common solvents then it makes sense for other people to use this same system as it allows direct comparison between individual plates (and Rf values). However, even when the the same solvent system has apparently been used, slight variations in the qualities/proportions of solvents as well as local conditions and slightly variations in plates can cause general shifts in Rf that affect the Rfs of all the compounds by a similar amount. That's where it is very useful to run a mixture of known Rf reference compounds along side your unknown mixture. This might be as simple as a mixture of common food colouring dyes, as long as they are readily available and show a range of Rf values. The Rf of the unknown is then given as a relative Rf (ie. Rf of unknown/Rf of known). These results are usually much more reproducible between experiments and hence more diagnostic (especially when combined with a positive colour reaction). A database of Rfs, relative Rfs and colour reactions is in the pipeline. I would also like to be able to offer a library of standard compounds/mixtures but this will take some time and obviously won't be practical for all compounds.

Sorry PP, I forgot to answer one of your questions. Standard silica gel TLC plates don't like water since it ruins the silica gel layer so with these sorts of plates you can't use water as the developing solvent. As you point out, many herbs are prepared by steeping in water and one might argue that the most meaningful TLC results for those herbs might come from a water extract applied directly to the plate. However, even a little bit of water in your sample can screw with the separation. You would probably get just as much info about the content of the herb by using an alcohol to extract and applying the alcoholic extract to the plate. This will evaporate easily off the plate and then you can run your plate without a disturbed layer. Alternatively, you can pay a bit extra and get water resistant plates.

Hi PP, Um, yeah, what Auxin said. You can get a nice range of solvents relatively cheaply by using pure solvents from the hardware store or by distilling them out of impure solvents bought from the hardware store. This usually works out significantly cheaper than buying from chem suppliers. At least in Australia, individuals *can* buy most solvents and reagents but for many things you will need to provide photo ID and fill out an end-user declaration which basically states what you will use the chemical for and that you won't be passing it on to some low-life meth-head. Sourcing solvents from common 'household' products is also a good skill to pick up if you intend to fly around a lot with a TLC kit in your backpack since you can't take your solvents on the plane and you will be hard pressed to find a chem-supplier once you get well off the beaten track (where all the coolest plants are). Unfortunately, most of the published solvent systems for the separation of our known compounds/extracts use solvents that are a bit tricky to source from domestic products. Therefore, it is important that new methods are developed using materials that are readily available throughout most of the world or can be brought on board a plane in a safe manner. This can certainly be done.

Wow. That's already more of a response than I was expecting... I think if more people in this community were familiar with the technique and got into the habit of analysing everything they bioassay (including myself), we would all be getting, as T would say, "more scientific mileage" out of our bioassays. It really is quite easy once you have a little practice. TLC plates are a little bit more expensive than chromo paper but the ability to get better separation of complex mixtures is definitely worth the investment. Re price: I think I will try to keep the price of the kits down initially by including in them only what people will need in order to have a go at the technique and see if they like it without too much risk. That way, more people will be able to get into it. I think I should be able to put together a minimal kit for about $20-30 maybe with the option to buy extra plates. There will be a few things you need to source locally but most of these will probably already be around the home or down at the hardware store. You should be able to put together a nice little set up for under $50 including the kit. Sounds like i'd better get to it. Thanks for the support guys. I'll assemble something and report back when I have something specific to offer. BTW Darcy, that's a beautiful quote. [ 19. March 2005, 10:15: Message edited by: Fractalhead ]

The actual plumbing wouldn't have to be expensive as you could probably build this yourself out of bits and pieces from the hardware store (nothing too complicated). A submersible water pump would probably suffice for the cycling of aqueous solutions. I'm beginning to look into various options for ion-exchange materials. Yes, some of the new synthetic resins have extremely high binding capacity, consistent properties and high purity but can get extremely expensive. I think you might be able to get away with the much cheaper option of some kind natural ion-exchange material like sulfonated coal or a zeolite based filter like the stuff used in water purification. Maybe a good option would be to extract the bulk alkaloids on a cheap resin and then fractionate them on a smaller column of high quality resin using a pH gradient. But yeah, like Torsten says, you can reuse these resins heaps of times by just converting them back to their protonated forms by washing with a cheap inorganic acid. I guess this is one example of a situation where we can make an effort to embrace the principles of green chemistry while opening up feasible extraction pathways to a wider range of useful compounds (ie. legal ones).

On a more general note regarding LEGAL alkaloid extractions... I think ion-exchange resin based alkaloid extractions could overcome the challenges encountered with many of the trickier (unstable) compounds. These systems allow the concentration of alkaloids in large volumes of aqueous solution without the need to use proportionally large volumes of nasty (hazardous) organic solvents, and all at low temps and potentially under an inert atmosphere. It is my guess that it is usually in this phase of alkaloid extractions that most of the alkaloid-degradation happens since it is common to boil the material for a long time to extract and then concentrate the aqueous extract down to a manageable volume. This is obviously a big no no for our more sensitive friends. Even the use of lower boiling solvents requires *some* heating. I've seen some great designs for in-field alkaloid extractions of curare alkaloids, quinine, scopolamine and nicotine. In these systems the plant material is put into what is effectively a big tank with a filter at the bottom and (acidic) water percolated down through the plant material like a giant espresso machine. The filtrate containing some dissolved alkaloid salts is pumped through a column of cation exchange resin and when the water comes out the end, all the alkaloids have been adsorbed by the resin and hence the water only contains non-ionic and anionic compounds. This effluent is cycled back over the plant material and more alkaloids are extracted and the cycle goes round and round for a few hours until the plant material is exhausted or the resin becomes saturated. The column is then washed with an alkaline solution like NaOH or NH4OH to freebase the alkaloids which precipitate in the column. All the non cationic crap is then washed out with water and the clean alkaloids eluted with an appropriate organic solvent. Pretty cool, I thought

Yeah I've got that Harborne paper. I would take the reported concentrations with a pinch of salt given the instability of these compounds and the lack of any proper seed age control. Those muelleri seeds could have been quite old. I guess we coul contact the authors. Bioassay reports suggest a higher concentration comparable to woodrose.

Yeah, I've collected M. dissecta in Cairns. Very woodrose like flower in terms of colour scheme. Can't remember any chemical data off hand but it would be worth looking at i'm guessing.

This species occurs in arid regions and has tested positive for ergot alkaloids (never seen a TLC though). I've heard of a few positive bioassays.

You can learn much about the reasons for secondary metabolite accumulation by studying the way in which their biosynthetic pathways are regulated. For instance, some plant cells have pathogen-specific receptors that start signal transduction pathways leading to the up/down regulation of secondary metabolic genes. If your plant cell has sophisticated regulatory mechanisms that link pathogens with secondary metabolites, this is a good indication those secondary metabolites are involved in defense. Alernatively, you might find that some metabolites don't respond to any known pathogen derived elicitors but rather respond to changes in day/night cycle or be regulated in coordination with flower development. This might indicate they are involved in pollination vector attraction or something like that.