Home TLC Thin layer chromatography

[NotGuilty]

Hi everyone. I plan on trying some TLC at home with hash oil. Basically I am looking for some any input/tips you have.

Here is an overview of TLC:

http://en.wikipedia.org/wiki/Thin_layer_chromatography

Here is another link on Grade 4 science using Simple TLC with Chlorophyll and one with inks:

http://www.scienceprojectlab.com/thin-layer-chromatography-for-kids.html

http://www.sciencemadesimple.com/leaves.html#autumn_leaves_science_project

This explains making the plates at home:

http://blog.makezine.com/archive/20...atography_in_the_ki.html?CMP=OTC-0D6B48984890

Here is a quick link on uses of cannabis chromatography:

http://www.springerprotocols.com/Abstract/doi/10.1385/1-59259-030-6:145

I plan use a method similar to the one with pens using plates instead of paper. I plan to try a long soak Iso, QWISO, BHO.

I have a few questions. And plan on trying many different ways. If the results are usable it would be neat to have a 3'x3' "plate", cut out sections and extract the separate compounds.

better hurry up with that camera.

 

[Payaso]

So, many questions....

Using this for potency testing? Looking for contaminants?

Just two so far!

Very interesting indeed.

Keep us posted!

 

[NotGuilty]

Payaso, Potency test yes, not 100% accurate but can get a rough idea? That is IF it even works out. I have a feeling with so many cannabinoids all so similar, It will not look as Clean as the Ink demo. Temp will play a big role here i suspect, by altering viscosities and all that buisness. And I may even need to add a reagent to show results visually.

IF it even works......

 

[ganja din]

Hey notguilty,

Give me an hour or so and ill post how *semi-quantitative* (comparative) TLC is carried out using Fast blue BB reagents. I have researched this for a while ago and have it all figured out.

For true quantitative testing one needs standards of cannabinoids in question. I should have access to standards in the next 6 months and ill share for free

For now, check out "JustTLC" and "JustQuantify" (free online tool).

HTH

 

[NotGuilty]

Thanks ganja din. I appreciate you stopping in. I would love to see this made practical for everyone.


Briefly skimmed over "just TLC", basically a software that does what I had planned. My crude method was going to be to scan the result and figure surface area/volume of the compounds in relation to each other to get a rough idea of the concentrations... using pc scanner and photoshop or something.

 

[ganja din]

Hey notguilty,

Scanning spot density is the best method, one can use Photoshop to do the same, or the free JustQuantify, or the great JustTLC (makers of JustQuantify).

A standard is >98% pure cannabinoid in question. Without a standard one can't tell how much cannabinoid is present in a sample, ex. % THC (by weight). If one does have a standard then they know the quantity (weight) of the cannabinoid in the standard. Thus by comparing spot densities one can calculate the weight of the cannabinoid in sample.

IIRC, JustTLC has been proven as accurate as HPLC if done correctly. There is a independent study on the JustTLC site with evaluates and states JustTLC for use in analytical research.

If one has no standard then one can get a baseline (average) of X samples. Then test samples and save ones which surpass the baseline. Of those, run comparative test to see which has the greatest density spot. The sample with greatest spot density = strongest sample.

Using fast blue BB reagent one can test for many cannabinoids. With use of a reagent we can use spot color as cannabinoid qualification, no need for Rf vales.

Hey! Now I think about it one could very easily use TLC for qualification of hydrocarbons in the sample! This means one can tell if/how pure their sample (ex. BHO) is. For example: one can test to see if there is presence of butane in the sample (qualification). And using a butane standard, one can tell how much butane is present in the sample (quantitication). All one needs to know is the TLC mobile phase and visualization reagent for butane.

OK. Gotta go, working on a method to make Sams secret sift...

 

[BigWillyDee]

read the wiki link and looks very interesting....what exactly will you be using it for...as far as cannabis goes??

 

[BigWillyDee]

nevermind ganja answered them.....

thats pretty cool shit..

 

[ganja din]

Billy,

Read my post above yours one can test bud, BHO, hash, QWISO, Bubblebag hash, etc, etc

 

[ganja din]

Hey notguilty,

I agree everyone should be able to do this. That was my main motivation for that project. There are kits out there like cannafingerprint, or something, but its not worth the money. My process is far superior and less expensive (after initial setup cost)

 

[BigWillyDee]

yeah as soon as i posted your answer was up....

would be great to simplify (if possible) the process into sort of a pocket lab type thing where you could just drop a sample in set the dial to bud, iso, hash, etc and automatically get all the specs

Billy,

Read my post above yours one can test bud, BHO, hash, QWISO, Bubblebag hash, etc, etc

 

[ganja din]

Hey,

Its not that easy. But the method is simple, one doesn't need to be a chemist. All equipment (besides scanner and computer) can fit into a lunch box. The problem for most people is the concepts and terms are alien to them, kinda daunting. But no worries, its really simple process.

 

[NotGuilty]

"Fast Blue B reagent

For detection of cannabinoids, phenols, tanning agents, amines.

Spray with a solution of 0.5g Fast Blue B (tetraazotized di-o-anisidine) in acetone/water (9:1, v/v), always prepared fresh
Then overspray with 0.1M sodium hydroxide solution
Results: Cannabinoids turn dark red/purple in color "

http://www.pharmainfo.net/luckypharmacist/tlc-visualization-reagents-part-3
(5 parts)

I think they are using Silica. My question is about the standards. What media are/will they be? Digital? Physical slides or plates?

And is this http://www.sciencelab.com/page/S/PVAR/10413/SLF1915 what I need?

 

[ganja din]

Hey ng,

Yea that's a method to use fast blue BB, but not ideal for cannabis. No need for acetone/water and spraying is poor application method.

Silica is commonly used as "solid phase". I have done tens of hours of research in TLC and GC, trust me, the method I will present is the best method for us.

Standard of cannabinoid is simply that, a known pure sample of the cannabinoid in question.


Here some links that may help you:

-Quantitative TLC:
www.chromatography-online.org/quant/contents.html


-"Quantitative Chromatographic Analysis"
by Raymond P. W. Scott
http://www.chromatography-online.org/quant/Quantitative Analysis by TLC.html


-Video tutorial for TLC:
http://www.chem.zenkyo.h.kyoto-u.ac.jp/operation_eng/operation_16/operation_16.html

HTH

 

[ganja din]

Hey ng,


Do not use "fast blue B", its toxic and can't be stored eaisly. Use "fast blue BB".

 

[ganja din]

Edit, I made a typo:

Solid phase should be "stationary phase"

 

[ganja din]

NotGuilty wrote:

I have a few questions. And plan on trying many different ways. If the results are usable it would be neat to have a 3'x3' "plate", cut out sections and extract the separate compounds.

That is a method one could use (with much effort) to create a pure standard. But not for smoking. Using HPLC to create standards is much preferred. That is how Sam Skunkman got his THC standard for his GC, he made his himself. I know other people who have done this. I have a great paper detailing how to make standard of THCA-A using HPLC. One can adapt that paper for other cannabinoids if one has an HPLC.

Standards are expensive, and sometimes not what they claim (ex. Sam said Sigma THC standard was only 87% THC, IIRC). That is why he made his own, he could not find a good source. Over 2k for very little is common most of the time. Check out this company to order reference standards. Tho I doubt they will ship to the US without lots of documentation:
www.lipomed.com

BTW, this brings me to Dr. Hornsby (sp?), I have very strong doubts about the quality of his standard, or at least the calibration of his GC. I think his figures are inflated a lot due to poor standard. For example, some guy here claims the Dr. tested his budder (from QWISO IIRC) and it came out like 90+% THC! Haha, yea right! That is proof enough for me. IIRC the icmag member who got the budder tested is the guy who came up with budder, or a friend of the creator of budder.

 

[NotGuilty]

Right, Fast Blue BB Base it says?
http://mil-spec-industries.com/ also offers fast blue BB base. Do I need a license to obtain this or just be able to explain a reasonable use? Toxicity is not really a concern, unless you are suggesting I can recover my isolated Cannabinoids after analyzing.

http://www.sciencelab.com/page/S/PVAR/SLF1114 Here is the Fast Blue BB salt.

I have also seen Alumina used as a stationary phase. Is it better than Silica or not?
ar
EDIT: I am Canadian And yeah i don't think standards would be easy to come by being THC. Is there a usable digital method?

 

[ganja din]

Hey bro,


You want the salt, fast blue bb salt, it shows all the neutral cannabinoids. You don't need a license.

Use silica. Dip the plate, do not spray.

Here is a quick into, this might have typos or slight omissions, I'm tired. If so ill fix it tomorrow:

1. Extract cannabinoids

2. Sample super critical solution with micropipett.

3. Spot plate with micropipett. Two applications per spot can be good. This is the stationary phase.

4. Put plate into 'developing chamber'. This is the mobile phase. In the chamber is the hexane:ether mix (4:1), a little in the bottom. Along one side of the chamber is the filter paper, it is wet with developing solvent (synonymous with mobile phase). The filter paper and solvent are to fill chamber with fumes to facilitate the mobile phase. The developing solution will run up the plate. Dragging invisible lines of cannabinoids with it. Once wet mark reaches top line of plate (0.5" from top of plate) its done. Pull out and allow plate to dry. This can be speed up with very light warm air from hair dryer, but allowing to dry in a vacuum desiccator is much, much more ideal and quicker too

5. Before applying the visualization reagent one should make the silica plate alkaline first, better results with cannabinoids. So mix up as below and quickly dip the plate into the alkaline solvent. Allow plate to dry as before.

6. After plate is dry dip in reagent (1% fast blue BB salt in water). Allow to dry as before.

7. After plate is dry dip in preservation solution. Same as the alkaline solution. Allow plate to dry as before.

8. Scan plate with flatbed scanner and upload to JustQuantify for free spot density scanning and assay. If you place a few different samples on one plate it makes comparisons easier and more accurate.


Done!


You also need (at least):

Chemicals:

Petroleum ether (extract solvent)

Hexane

Ether (mix with hexane as 4:1 hexane:ether as mobile phase)

Fast blue bb salt (visualization reagent - add 1% to water)

0.1M sodium hydroxide (Alkaline solvent and Preservation solvent)


Materials:

Glass jars 1L, 0.5 L, 0.25 L

filter paper

5mg spatula

Microscale

A few eppendorf (1.5 ml each)

Eppendorf holders

Capillary micropipett (3ul)

1ml pipett

Pasture Pipett

50 ml test tubes

5 ml vials

Motor and pestal

Test tube vibrator or ultrasonic vibrator (optional)



US sources of chemicals:

I get fast blue bb salt here:
http://www.sciencelab.com/page/S/PVAR/10413/SLF1114


Carolina chemicals:
(They sell many kinds of kits)
http://www.carolina.com/category/ph...try/chemicals.do?KickerID=int_t_hom_chemicals


Science Lab (same place I get reagent):
http://www.sciencelab.com/page/S/CTGY/10405


Here is a good ebay site:
http://stores.ebay.com/AVOGADROS-LAB-SUPPLY

 

[ganja din]

What do you mean by digital standard? Do you mean a previous scan of a standard? If so that won't work for any amount of accuracy. The biggest reason for inaccuracy in TLC scanning or UV is IIRC the stationary phase (silica) and spotting technique. So one needs to test with the same variables when at all possible. Thus one should spot the standard next to the sample(s) in question on the same plate. If comparing against previous tests then the same type of plate should be used for each and every test.

Worth noting is that it's impossible to accurately compare spots with naked eye due to different shapes of peoples eyes, etc. (That's the short explanation)