Home TLC Thin layer chromatography

[dongle]

Got myself a bottle of chloroform (anhydrous, >=99%, contains 0.5-1% ethanol as stabilizer) and some fast blue BB salts (>= 80% dye content) from Sigma Aldrich, but not getting the stunning results I've seen posted.

So far using a fairly fine silica gel 60 on a microscope slide with roughly 25% calcium sulfate hemihydrate as binder. The extract I'm testing is >80% THC distillate dissolved in roughly 1.5 parts pentane.

 

[dongle]

Hit submit too soon.

Here's the chloroform I'm working with:



And my plates. My dip technique is getting better each try:

 

[dongle]

Basically, not getting the "separation" of the different cannabinoids, but then again a HPLC analysis shows >90% D9 THC, ~1.5% CBG, <LOQ CBN and ND on all others. I will try to run a different sample with a wider range of detected cannabinoids next time to see how much difference it makes.

 

[dongle]

Ok so here's a run of some crude BHO in roughly 1:1 pentane.

 

[dongle]

@hashschwifty on IG informed me that my sample is too concentrated and I'm overloading the plate. Makes sense. Out of plates for the day and don't feel like baking any more tonight, so when I pick up tomorrow I'll dilute down much more and apply a smaller drop.

 

[PhenoMenal]

dongle, i use a micro-pipette device with 1uL tips, and I only use 2 or 3 drops from that per TLC lane. (1mL being 1000uL, we're talking less than a bees dick). Definitely don't wanna be using an eyedropper which probably starts at about 1mL lol
https://i.imgur.com/FiYv4kj.jpg

I've never made my own plates, I don't know if your plates are causing any problems or not, but the plates i use are 5cm x 10cm silica-coated aluminium plates (no fluorescence indicator), they're reasonably affordable, and much more affordable than the glass plates, plus you can cut them with scissors. Overloading seems to be your main issue though.

the other thing is make sure youve given the samples enough time in the eppendorf tube for the solvent to extract the cannabinoids, though that's probably not your current problem.

btw, just to be clear, you're using chloroform for the extraction phase, and chloroform also for the mobile eluent phase? (no problems with that, somebody else here also does that with success, i'm just wondering)

But as you now know, TLC is pretty easy! it's just a matter of dialing in those parameters

 

[dongle]

Nice to see you again, I remember you when I used to actively post here as a grower.

LOL @ bee's dick.

Where did you buy your plates? IIRC, you were using them in "landscape" form, for 5cm height vs 10cm, presumably because >5cm was unnecessary?

I've been heating standard 3mL plastic transfer pipettes gently with a heat gun and stretching out the tip to make a narrow microcapillary-like tube at the tip. Most likely a lot more than 1ul.

 

[dongle]

What type and thickness of aluminum? Thicker than heavy duty foil? How's the finish, smooth/polished or ...?

From what I can gather off AlphaCAT's instructions, a 10:1 v/w dilution is done (1mL solvent:100mg material)

My extract was roughly 1.5:1 and much more than 1ul, so makes sense.

I won't rule out that my DIY plates might not be optimal, but right now I'm convinced I've overloaded the plate with too much analyte

 

[dongle]

I was using pentane for the extraction phase (because i already had some dissolved at that ratio) and chloroform for developing the plates. Id venture to guess that the solvent the sample is dissplved in is hardly important especially since ive been drying out the spot gently with either convection oven or heat gun

 

[dongle]

If my memory serves me correctly, aren't you an Aussie and therefore should be awake and have replied to my post already?

 

[PhenoMenal]

no i always stand the plates in the jar in portrait mode, as you can see with all my plate scans. The 5cm width is just enough for 5 lanes but i usually do 4, and the 10cm height is comfortable room for good separation, while still being able to fit in a small jar for quick efficient mobile phase and decent chamber saturation without too much eluent (i only use 12mL).

the plates i use, no theyre not like aluminium foil, theyre a bit thicker, like a business card (needs to be firm enough to stand up on its own), but still easily cut with a pair of regular scissors. Cant remember the exact price but I got a box of 100 of them, it works out somewhere between $0.50 and $1 a plate, and i enjoy the confidence I get from professionally-made plates! If I made my own i have a feeling theyd just be a series of hills and valleys rather than a perfect plain. It'll be interesting to see how your home-made plates perform when you get good separation going

and therefore should be awake and have replied to my post already?

or maybe i was busy working!?

 

[dongle]

So about the thickness of disposable aluminum pans, pie plates, etc?

 

[dongle]

Tomorrow morning I will try following more or less the AlphaCAT instructions, which I should have done in the first place but got impatient.

I will also look for longer backing material, since the 5-6cm microscope slides seem like they might not be enough.

Thanks for dropping everything and coming to answer my post. As you should; forget work!

 

[PhenoMenal]

dongle, i just checked, the ones im using are German-made by Merck, "TLC Silica gel 60", aluminium, 5 x 10 cm. It's a small box of 50 plates, and cost ~US$75 when i bought them a year or two ago, so $1.50/plate. It's not the "F₂₅₄" or whatever version - that's a fluorescence indicator which we don't need when using visual dyes like Fast Blue, though im guessing theyd still be fine.

I'm sure there are cheaper plates, and I know there's also insanely expensive plates, but I've been very happy with these, they're professionally well made, and I think $1.50 for 4-5 tests isn't too bad. The 10 x 5cm is one of the standard sizes and there are several other manufacturers making basically the same plate.

Maybe homemade ones are perfectly fine for this though, i have no idea, I think you're the first person in the thread making their own so it is very interesting!

I will also look for longer backing material, since the 5-6cm microscope slides seem like they might not be enough

yes, 5cm is too short for good separation, very squishy. 10cm seems ideal and very practical, im very happy with it. you can also get 20cm if you want, but would need a larger developing jar and a bit more eluent

btw, be aware of different quantities when using Fast Blue B or BB ... it seems B needs a fair bit more than BB. I think all the commercial ones use B not BB, because B is kept refrigerated whereas BB is kept in the freezer.

Tomorrow morning I will try following more or less the AlphaCAT instructions

See also the TLC tutorial in my sig. site seems down at the moment, but here's googles [webcache]

 

[dongle]

So I've been reading about the different types of binders used in TLC plates, and realized that I was using the weakest binder available (gypsum) whuch explains a lot.

That information had me putting some $15+$4 (next day prime shipping) polymeric binders on Amazon, and scrambling to try to figure out how to find the right backing.

Then i finally came to my senses and realized that buying a $70 box of 10x20cm will get me 96pcs of 5cm x 10cm plates for less than $1/plate and 0 headaches.

Glad I found out about the different binders; otherwise I would have spent hours tomorrow looking for proper backing to my shitty gypsum binder which probably would have failed at one point or another, either flaking off during development or dipping.

 

[dongle]

About $0.15 - $0.18 per spot, depending on whether you use 4 or 5 lanes per plate. Hard to beat considering DIY plates with microscope slides only gets you 1 spot per plate and only 5cm of separation space for $0.07 on just the glass alone not to mention prep time, medium, and binder.

Definitely a win.

I'm glad you quit work to come answer my post. Just like you should have.

 

[dongle]

Oops microscope slide is 7.5cm not 5cm

 

[dongle]

I got the kind with the 254nm fluorescence indicator, only because i had no choice in that price range.

I wish i could edit posts

 

[dongle]

Roughly how long does it take for your 10cm plates to develop in the chamber?

It takes about a minute or minute and a half for chloroform to climb up most of a 75mm glass plate. I'd guess I was utilizing maybe about 6cm of that space. I'd expect a 10cm plate to take a bit more than 2x as long as 6cm to develop, since the climb rate drops as height increase, in my experience.

The AlphaCAT video says 25 minutes or something but that seems overly long

 

[dongle]

What are your thoughts on a using something like a methanolic solution (9:1 meth:water?) of BB and using it as a cannabinoid presence indicator on alkane (heptane or pentane) solutions? In the same way a liquid pH indicator test would be used.

Would that work? I just want the solution to turn color if it contains cannabinoids...