basic field tests for alkaloid content in leaf, stem and root tissue

[Frut]

thanks Darklight,

please let me ride the illusionary wave of 'this is not a brain grinded to dust, eyes wasted by computer, phd type study' just a little longer

thanks for the lead, again I am pointed to TLC, I am really going to have to try and understand this TLC procedure it seems complicated at the moment

regards

Frut

I hear you, the fact I an opening a Phd worth of work. Sweet Lord I hope not!

Mwahahahaha- you so are.

Guys how about the old UV light

There was a wealth of info by a bloke named Fractal here about alkaloid field tests using the UV light and TLC. The search engine can't seem to find them, maybe they went missing in an earlier upgrade? If anyone has copies of the posts or can find them on the SE, put the link here. The info in question was detailed but the process facile once you had all the gear

cheers

Darklight

Ah- here's some of it- keep looking for more

http://www.shaman-australis.com/forum/index.php?showtopic=2621&st=0&p=21647&fromsearch=1entry21647

 

[reshroomED]

Can't seem to post a link for some reason.

Think the thread you're thinking of is http://www.shaman-au...er&fromsearch=1 "Refractometers"

ed

[reshroomED]

I am really going to have to try and understand this TLC procedure it seems complicated at the moment

regards

Frut

 

TLC's pretty simple as long as you have a bit of basic chem sample prep/procedure knowledge.

I've a neat little TLC basic tutorial in .djvu format that I can host or email you if you'd like.

ed

[Frut]

Yes please reshroommed,

Any help with getting a basic understanding of TLC at this stage is appreciated.

I have dejavu loaded I think though

email on my profile is best

regards

Frut

[Frut]

Mods How do I get rid of this warn status under my name? I dont want people to think I am a risk to communicate with

[Maurice]

If its alks you want,

just get your own breeding program going.

[Frut]

Thanks Maurice,

Alks are one of a interesting range of options with Sida IMO

Alks was what I thought I could start with as a simpler measurable characterictic of the wild population

Before entertaining any plan to selectively breed more alks or any other property, I would like to start by selecting the best strain in the natural population around me

this is why I am fussing over the simplest possible field test, to test as many plants as possible in situ and take as few samples back to the lab for further analysis as possible, time is the essence. as it is a hobby, not a phd

Maurice, I take it you think genetics is more important than environment when it comes to Sida and Alks,

could you tell me why you take this position?

regards

Frut

[Chiral]

Here's what I have to come to understand or believe if you wish to understand alk content on a species.

Count plants in given patch, take 1/3 of plants, weigh total plant matter, process/blend up plant material, A/B extract, solvent pull, salt out and evaporate.

Dry out and weigh final salts to arrive at alk figure in %...

So say 30 plants weigh 30 kilos your final salt extract weighs 3gms so your figure is 0.03% alk content..assuming my dodgy maths are correct then we can with utter confidence conclude that this will indeed be quite an accurate projection of alk content per plant across the whole patch of plants in that area.

this is how I see it in my mind, I can't actually envisage getting figures any more accurate than that honestly, due to subtle variations across the soil content or placement of plant in regards to being in more shade than another for longer periods per day etc, these variations are almost beyond human capabilities to quantify with any real accuracy, that's why averaging is far more an acceptable way to gather or quantify this type of data.

The only other way with any really super accurate data is to very carefully farm the plants from seed, all plants in identical conditions, test to see if potency is carried over when cross breeding or is maintained or reduced after constant cloning, and test for potency variation due to climatic changes, seasons, drought conditions, full sun or partial shade and test for increased potency due to increase or decrease when providing extra nutrients into the growing medium.

[Maurice]

Thanks Maurice,

Alks are one of a interesting range of options with Sida IMO

Alks was what I thought I could start with as a simpler measurable characterictic of the wild population

Before entertaining any plan to selectively breed more alks or any other property, I would like to start by selecting the best strain in the natural population around me

this is why I am fussing over the simplest possible field test, to test as many plants as possible in situ and take as few samples back to the lab for further analysis as possible, time is the essence. as it is a hobby, not a phd

Maurice, I take it you think genetics is more important than environment when it comes to Sida and Alks,

could you tell me why you take this position?

regards

Frut

 

All I said was that bioassay on a safe (non-toxic) plant (Sida) will give you the best and quickest results for your needs.

You should just do it, until you find the plant you want to breed from!

I know nothing about Sida, but its the same for all plants, you want best growing conditions and best genetics.

[Alchemica]

For the identification of the alkaloid, all you might need is a test reagent. It's not like you need to go and get a saliva analysis test kit, depending on the alkaloid, Marquis or Mandelin reagent might be enough (you can buy online in Australia for testing pills via Enlighten or through some herbal high retailers etc.). These will give a colour reaction to certain alkaloids (you need a concentrated enough solution to test, preferably without chlorophyll) but simple extraction of the basified plant material with chloroform and then testing the chloroform solution might suffice. I posted a link on reagent tests earlier on in my first post.

[Once you've had the chance to read up on the TLC basics]

If you want to quantify how much of an alkaloid you have, TLC is still a pretty clever way to go about it. You can compare samples of known alkaloid concentration (after visualisation) with your unknowns after you've run the TLC. The distance moved by the alkaloid spot vs the solvent front will give you a rough idea of what the alkaloid is, while the intensity of the developed spot can be related to the amount of alkaloid (if you are careful with the loadings of solvent/alkaloid you spot on to the plate). Using a scanner or digital camera, you can then import your TLC plate (with different intensity spots corresponding to different alkaloid concentrations) and use software to input the known concentrations and from there work out the unknowns. My previous post has some other tips.

Reagents: http://media.enlighten.org.au/literature/testkits.pdf

Computer-Aided Thin Layer Chromatography

Identification of Pharmaceuticals via Computer-Aided Thin Layer Chromatography

Macherone, Anthony J., Jr.; Siek, Theodore J.

J. Chem. Educ. 77, 366-367 (2000)

Abstract

In toxicology laboratories, thin-layer chromatography (TLC) provides a quick and accurate method for qualitative identification of unknown biotoxins that may be present in a fluid or tissue sample. These varied samples are presented to the toxicologist for analysis on a routine basis and also during emergency situations or after a death. The ability of TLC to identify or rule out hundreds of compounds in a single analytical run makes it amenable to emergency toxicology and forensic chemistry. However, the ability to decipher the various reactions and elicit meaningful results from the raw data can take years of experience. It would be useful therefore, for analysts to obtain a firm background in TLC while still in academic training. This analytical laboratory experiment demonstrates the methodology of TLC in its relation to toxicology and forensic chemistry. It is easily adapted for high school seniors or undergraduates and employs experimental techniques associated with TLC and post-lab data analysis with concomitant introduction of concepts. The procedures are designed to introduce the student to the concepts, mechanisms, methodology, and reactions of TLC while building skills in record keeping, data analysis, and deductive reasoning.

____ ___ __ _

Rapid, Simple Quantitation in Thin-Layer Chromatography Using a Flatbed Scanner

Johnson, Mitchell E.

J. Chem. Educ. 77, 368-372 (2000)

IGORPro 5 Software http://www.wavemetri...ro/IgorPro.html

Abstract

A standard flatbed scanner is shown to be a viable tool for quantitative thin-layer chromatography (TLC) plate analysis. Simply scanning a visibly stained TLC plate into a computer substitutes for much more expensive plate readers. With common image analysis software, "elution" profiles can be obtained. The resulting "chromatograms" can be analyzed in the same manner as other chromatograms. Iodine-stained cholesterol and cholesteryl esters are shown to yield nonlinear calibration curves, but the overall sensitivity is excellent for such a simple method. Detection limits are submicrogram for heavily stained spots. Spot intensity, and therefore detection limit, depends strongly on the amount of time the spot is exposed to iodine. Reproducibility is excellent for spots deposited by aspiration from a glass micropipet. Peak area and peak height relative standard deviations (RSDs) were generally below 5%, and retention factor precision was as low as 0.8% RSD.

TLC BASICS:

http://orgchem.color...rt/TLC/TLC.html

http://orgchem.color...Cprocedure.html

Iodine visualisation of TLC plate:

The staining of a TLC plate with iodine vapor is among the oldest methods for the visualization of organic compounds. It is based upon the observation that iodine has a high affinity for both unsaturated and aromatic compounds.

Preparation

An chamber may be assembled as follows: To 100 mL wide mouth jar (with cap) is added a piece of filter paper and few crystals of iodine. Iodine has a high vapor pressure for a solid and the chamber will rapidly become saturated with iodine vapor. Insert your TLC plate and allow it to remain within the chamber until it develops a light brown color over the entire plate. Commonly, if your compound has an affinity for iodine, it will appear as a dark brown spot on a lighter brown background. Carefully remove the TLC plate at this point and gently circle the spots with a dull pencil. The iodine will not remain on the TLC plate for long periods of time so circling these spots is necessary if one wishes to refer to these TLC's at a later date.

I'll try and put the pdfs up if I can, otherwise you should be able to search and find what you are looking for.

Edited December 30, 2009 by Alchemica