Home TLC Thin layer chromatography

[sadpanda]

so happy to hear from you Joe! thought you'd gone missing as soon as i started experiments. Just wanted to thank you especially again for all the help you've given me, it meant i was able to get stuck straight into it when i had all the components to quickly figure it all out, and on this cancer rollercoaster my friend and i don't have time on our side so I can't elaborate enough how much I appreciate your generous and extremely valuable and insightful help, and your patience for putting up with all my newb questions!

Zinc salts generally probably dissolve better in water than NaOH. The B salt dissolves in water easily.

next time i might try doubling the NaOH 1.0M solution from 4gms/L to 8gms/L, then only use half as much, and try dissolving Fast Blue in the other half (pure distilled water) before mixing ... worth a try anyway. Interestingly enough (or you might be thinking "obviously" lol) the Fast Blue did not want to dissolve at all in hexane! It certainly would be both easier and more efficient to get a full dissolve from the BB instead of straining with coffee filter so hopefully that'll work, but if not at least this backup of using coffee filter is still getting me nice reasonably clean results, phew!

You are getting a lot of color for 3 uL. 5 is much paler for me with B but the extract is dilute. Have you tried a more dilute load?

yes im so happy with the colors!! "Not only will we let you see all your cannabinoids, we'll make it stunningly BEAUTIFUL for you too!" - Mother Nature last Friday, lol. I know Chimera prefers UV, but gimme pretty colorful dots any day! I did read that BB is more vibrant than B though so maybe thats why, but other than that single brief mention i've never seen any written comparisons between the two sadly. Would you say vibrancy is the only main functional difference? The scanner does seem to give a slight color boost but very mild. And remember the very first experiment I did was to determine how much (how little as it turns out) to use, so I'm pretty sure I'm not using too much, and if you can tell by how long i had to wait for free shipping i'm a cheapskate and FBBB isn't cheap so don't want to use any more than needed, lol Pretty much all my scans so far are either 2uL or 3uL, with just one or two 4uL's (ie. when only 3 lanes on full plate). In the case of that very latest run where i did the 3hr decarb each lane = 3uL, each 1.5ml eppendorf had exactly 50mg bud sample (half of what the commercial kits recommend), and exactly 1.0mL hexane so each tube looks about 2/3 full.

Having high-performance reversed-phase plates would make for good separations, yes. This is about the same as HPLC, which normally uses such fancy materials.

I'm so happy at the separation we're still able to get from our non-fancy mixes though, maybe won't hold up for HP but easily good enough for home hacks like me, phew! and it was cool to see how even other solvents like acetone and isopropyl alcohol were relatively useless by comparison, gave me more appreciation of having the right solvent for the job.

500. It was never presented by the person that posted it as the time and temperature that you need to have buds in your oven. The graphs show certain general trends and knowing what's going on is what's important. GW Pharma has their advice on large quantities and the different cannabinoids and that's all posted here a long time ago. The different acids have different stabilities and Mechoulam says THCA-B melts at 184.

so the $64,000 question (might be a bit more in 2016) - how long and at what temp do you prefer to decarb buds?

ps. Yes ive decided one more final night of experiments tonight!
#1) high THCV strain. Ive done recent tests with a high-CBG strain but want to try a high-THCV one (lanes 5 and 1 respectively).
#2) test if leaving the mobile phase to go into 'overtime' (for 5-10 mins after the solvent front has already reached the top) will result in better use of the limited plate space and provide better separation - or will it just make a mess of it all once the plate is fully waterlogged!? cant wait to find out!
#3) i want to try Fast Blue just in pure distilled water, no NaOH. Will it dissolve better!? will the plate still develop as well and vibrant and 'fix' the dye as well? another user said he started with NaOH but now doesnt bother, so thats a good sign at least that it should be ok!
All that and more in tonights episode of How Freaking Awesome Is Science(!)

 

[sadpanda]

Experiment: Pushing the solvent front into overtime!

The hexane:diethyl solution doesn't make as good use of the limited space on the plate as chloroform does, so I wondered ...
Q. When the solvent front reaches the top of the plate what would happen if we leave it a bit longer? an extra 4 and 10 minutes for example
A. in terms of readability, it seems not a lot unfortunately! the cannabinoids have hardly moved much further up, and it seems being waterlogged longer just clogs things up and starts to lose definition (i guess at this stage we're just trying to squeeze more people into a building that's already full to capacity, getting new people in by pushing some people out the windows but maintaining roughly the same number!):

I also tried the Fast Blue BB in just pure distilled water, but it didn't appear to dissolve any easier. After that i ended up making half of the soluton 1.0M NaOH, so half as weak as usual, because i wanted to ensure it was preserved to show my friend. Adding the NaOH to the pure distilled water + Fast Blue mix changed its yellow color to slightly darker almost immediately due to some reaction.

My final final experiment before i hand the kit over to my friend later this week - 20-minute-interval decarboxylation over 4hrs with high-THCV strain, coming up next! ...

 

[BigWillie]

Nice TLC plates, buddy.

I am doing them with 70:30 Heptane - Ethyl Acetate. Works for what I need, I use the same mix for the column.

 

[BigWillie]

Experiment: Pushing the solvent front into overtime!

I also tried the Fast Blue BB in just pure distilled water, but it didn't appear to dissolve any easier.

Man, my BB Salt just disappears with Costco water...(that's the secret ingredient) and away I go stainin' stuff. Maybe, you got a bad lot?

 

[BigWillie]

Sad,

39 more posts and I will be able to share my work photos, about column stuff but still interesting.

 

[sadpanda]

i put it into several bottles when i got it so as to minimise how much i need to open them so maybe i left the first one out of the freezer too long i dont know, but it's from Japan and imported here by a reputable lab and i'm getting great results from it
[FONT=Arial, Helvetica, sans-serif]
[/FONT]> 70:30 Heptane - Ethyl Acetate.
cool! always good to have more options i have neither of those two though. im wondering if i should switch over to Pentane when my Hexane runs out, seems to be slightly less toxic and possibly even slightly better results

in regards to uploading photos i just post to imgup.net and use [ img ] tags, or you could just post the url if img tags dont work but they should

 

[G.O. Joe]

so the $64,000 question (might be a bit more in 2016) - how long and at what temp do you prefer to decarb buds?

The first stage is total dryness and the clock doesn't start until then. Coincidentally last Friday 3 ditchweeds were selected for seed and after deseeding the bud hay was heated at 120 for 75 minutes. Distilled water only was used with the FBB and the main spot showed immediately. It was not as brilliant as yours so there was NaOH overspray but that didn't seem to help a whole bunch. It's 0.1 not 1 M.

It was KOH actually and furthermore neither weights nor volumes were measured. You can dissolve the dye in a vial of something and add IPA extract to see what happens.

Not all that fast blue shows at low Rf is necessarily the acids, until proven.

 

[sadpanda]

75 mins for 120C! thats awesome to hear because that's the ballpark my TLC decarb runs are pointing at also hehe (btw yes i've only been using dry samples - not crumbly dust perfectly dry, but ready-to-smoke dry)

after tonights final decarb run i'll be taking all the decarb scans into JustTLC to take all the Volume metrics as it sees them and plot a graph, should be easier to read than blurry dots, and for the life of me i can't tell the difference between the 15% and 20% dots on the Alpha-Cat chart! obviously im not testing %, but JustTLC can still put a volumetric number on each spot, and its neighbor spots are all relative and made under the same conditions... ie. i wont know if one spot is 5% THC, but i should be able to determine if one spot has 5% more volume in its THC spot than its neighbor as they're all in same ratio, so hopefully the accuracy in that sense will be ok! can't wait to see the graphs later tonight

Not all that fast blue shows at low Rf is necessarily the acids, until proven.

i wondered about that! the commercial kits just say "Acids {" with a big long curly bracket - i wish they'd put an "etc" in there or something

Time to develop plates...

 

[sadpanda]

Experiment: Decarboxylation of high-THCV strain over 4hrs @ 125C

Well i was not expecting this! I'm pretty sure i did everything by the book with this run, i was very careful to be precise with everything and even had fresh eluent, yet there are strange anomalies in the result such as fluctuations!

And when the THCV level fluctuated up, the THC level seemed to go down!? then the THCV level drops down again and THC goes back up, as if to roughly suggest "more THCV = less THC"? And there's exactly one hour between each fluctuation. How odd but interesting... didn't see that effect with the high-CBG strain a few posts ago

here's the high-CBG (yellowy-orange) one again for comparison:

 

[sadpanda]

btw does anyone have any idea how early you can test a growing plant for CBD and CBD:THC ratio? (i never had access to any growing plant matter anyway so wasn't an option to test)
Somebody asked in another thread and it got me wondering, and he suggested he got ok results from 6-week-young veglings. I would've guessed that maybe 1 week into flowering, the "window" at least would be open by then, but ive got no idea how different the CBD:THC ratio will be two months later come harvest.
Anyone have at least a slight idea!? cos i have no idea but find it a fascinating Q

 

[OXOSSI]

1.) Decrease the load by 50 %
2.) Run it 2-3 times, when the eluent reaches the top of the plate, dry it and do it again
3.) Good TLC mixtures to use Hex:Et.Ac (20-40) Hex:EtOH (10%) HexCM (Varies) Hex-ether Hex-DXM-Ether.

 

[TerpChild]

Just read this thread.. Sadpanda holy cow bro, thank you so much for all of your hard work!

 

[TerpChild]

Also big thanks to Joe and Chimera! This was an awesome discourse and very educational.

 

[PhenoMenal]

hello, sadpanda here! yes, sadpanda = PhenoMenal.
Yes, I am guilty of using a female personality and avatar, but I was banned (for reporting a security vuln), and desperate -- a family member was diagnosed with a terminal form of cancer. Things are at their dire ends now though - we will find out by next Friday if medical science has exhausted all options to save my (family member); if it fails then he will only have 2-3 weeks left to live.

5 years ago i was banned by Skippy when i made the apparent mistake of reporting to icmag that I'd found a security vulnerability with their chat client (IP address leak), so I was never able to post as myself again. Extra frustrating because I always got on well with Gypsy on chat, but never had any correspondence with Skippy. So I created the sadpanda account. And now in 2017/18 i'm getting all these PM's saying Gypsy has unbanned me, so it's all a bit weird and i'm still trying to find my feet again. 5 year ban just for trying to help at a forum I've been at since 2006 - ~7 years before i was banned by Skippy ("who?what?huh!?"). Very frustrating on so many levels ... and 5 years ban for trying to help? Sorry but there's no other way to say it - it was insane.

Anyway going back, we needed CBD ... but how the hell can you tell if your buds have CBD in them?

So, how can you detect CBD? There are basically only three practical ways...
1) send into a lab (not cheap, and only avail in some countries)
2) Beam's test, but that's just a yes/no test
3) TLC - Thin Layer Chromatography ...

I came across this icmag thread, where SOME OF THE PIECES OF THE PUZZLE HAD BEEN PUT FORWARD ...

And thanks to G.O. Joe I was able to fill in the rest of the puzzle pieces - and start contributing to the results also

Eventually I'd figured out the entire process - all required components and their inner processes etc, but I lost my sadpanda icmag login details and my PhenoMenal login was still banned by Skippy, so I ended up posting [here]

TLC is a beautiful and powerful tool that is within reach of most home users ... they just don't know it yet.

 

[PhenoMenal]

btw I have samples taken from veg and early flowering for both Dinamed and CBD Therapy so i'll be doing their TLC's very soon, will be interesting to see how much CBD they show being so early on! ♥experiments
The first Dinamed i did showed to be 1:1 CBD:THC but it was very indica-dom, whereas Dinamed is supposed to be sativa-dom... anyway my 2nd grow of her is sativa-dom, so fingers crossed! will post results in the next couple weeks

 

[p0opstlnksal0t]

hello, sadpanda here! yes, sadpanda = PhenoMenal.
Yes, I am guilty of using a female personality and avatar, but I was banned (for reporting a security vuln), and desperate -- a family member was diagnosed with a terminal form of cancer. Things are at their dire ends now though - we will find out by next Friday if medical science has exhausted all options to save my (family member); if it fails then he will only have 2-3 weeks left to live.

5 years ago i was banned by Skippy when i made the apparent mistake of reporting to icmag that I'd found a security vulnerability with their chat client (IP address leak), so I was never able to post as myself again. Extra frustrating because I always got on well with Gypsy on chat, but never had any correspondence with Skippy. So I created the sadpanda account. And now in 2017/18 i'm getting all these PM's saying Gypsy has unbanned me, so it's all a bit weird and i'm still trying to find my feet again. 5 year ban just for trying to help at a forum I've been at since 2006 - ~7 years before i was banned by Skippy ("who?what?huh!?"). Very frustrating on so many levels ... and 5 years ban for trying to help? Sorry but there's no other way to say it - it was insane.

Anyway going back, we needed CBD ... but how the hell can you tell if your buds have CBD in them?

So, how can you detect CBD? There are basically only three practical ways...
1) send into a lab (not cheap, and only avail in some countries)
2) Beam's test, but that's just a yes/no test
3) TLC - Thin Layer Chromatography ...

I came across this icmag thread, where SOME OF THE PIECES OF THE PUZZLE HAD BEEN PUT FORWARD ...

And thanks to G.O. Joe I was able to fill in the rest of the puzzle pieces - and start contributing to the results also

Eventually I'd figured out the entire process - all required components and their inner processes etc, but I lost my sadpanda icmag login details and my PhenoMenal login was still banned by Skippy, so I ended up posting [here]

TLC is a beautiful and powerful tool that is within reach of most home users ... they just don't know it yet.

Have any of you found a chart/overlay for the plates like what alphacat's kit uses to get a "not too accurate" quantitative reading of the terpenes?

im just now looking at starting up TLC for myself and also charging a small fee for local Care Givers to get cheap tests done. The ability to actually quantify (Alpha-Cat says their tests are quantifiable to 1.0% accuracy) each terpene makeup would be an astronimical benefit as opposed to just seeing each terpenes ratio to the others. I know the only way this could be possible is if each input material is minutely measured to exacting precision. the solvent ratios, dye, mixed media, flower, etc... all have to be perfectly proportioned and measured so that the end resulting dot sizes are quantifiable in the end. I would either need to utilize Alpha-Cats charts for measurement or use some type of software to scan the plates and measure the resulting profiles and create a database of ranges. I would then need to pay to send in a few flowers that ive tested at home to a lab that is accurately quantifying these terpenes so that i could build this online database to use the data points to extrapolate my at home TLC data points.

 

[dybert]

btw how awesome would it be if breeders made TLC plate scans available of their strains, would help to give a lot more to go by than just the current "This strain flowers in xx weeks, has a nice fruity smell, great high" descriptions

View Image
"6 of 8 Fruity Jack phenos displaying high CBD"

I have scans and results of thousands of plates.

 

[PhenoMenal]

p0opstlnksal0t,
my advice is don't bother trying to get % from home TLC, there are too many variables that all need to be exactly 100% just to get semi-accurate readings. TLC is a qualitative test, not quantitative. You really need to send it to a lab if you want %'s you can rely on.

It's still a beautiful tool for revealing both the EXISTENCE and RATIO and APPROXIMATE QUANTITY and overall FINGERPRINT of cannabinoids though!!!♥ just not accurate %.

You mentioned selling it as a service, but I feel you would be doing a disservice to your clients by using TLC to provide %'s because you could be off by as much as 5-10%, which is a huge error margin considering strains top out at ~30% THC. If people are willing to pay for such a test they generally want accurate results, and TLC's margin of error simply cannot accommodate that.

For example, in the AlphaCat image you posted for example, look at the 12% and 17% blobs:

... almost identical to the naked eye yet 5% difference. I would encourage you to provide a scan of the plates to clients, rather than %'s, because it really is afterall a VISUAL test (hence the need for the special Blue B/BB dye!), and one that doesn't provide concrete numbers.

I have scans and results of thousands of plates.

sweet!!! where!?

 

[PhenoMenal]

btw somebody recently asked me "it would be good if TLC could tell me if it was a male or female", but no chromatography (not even state of the art) can answer that question as it is a question about genetics, not chemical composition. Phylos is one company i know of that has such a genetics test

 

[Ringodoggie]

Not to take the thread off topic but home DNA extraction is definitely possible as well. And, DNA can be seen with a standard optical microscope. All that needs to be seen is the Y chromosome.

So, sexing at home via DNA is definitely within the scope of most people.