[sadpanda]

so Joe just my FINAL QUESTION i promise!!! (THANKYOU for your time and generosity answering all my previous questions, which has allowed me to get to a position where sometime later this week I can start doing experiments and sharing results, im getting excited!)

For convenience sake is it ok if i add the NaOH in with the Fast Blue bath, or do they need to be two separate baths?

[G.O. Joe]

That's part of the directions to follow, see visualization. The basic directions for TLC, GC, and HPLC are in the UN guide in full - hence the title Recommended methods for the identification and analysis of cannabis and cannabis products. If your tap water is as alkaline as mine, you may find that sufficient - but try it according to the directions first except maybe doing the dipping and the BB salt.

[sadpanda]

ok cool yes the UN visualization doc recommends two methods, one with methanol, but the one i'll use is "50mg (Fast Blue B) in 20ml of NaOH (0.1 N)" - although i'll have to figure out myself if 50mg B == 50mg BB (not sure how exactly though! so i'll just have to experiment with different levels). And it seems "0.1M" and "0.1N" are the same, at least in regards to sodium hydroxide solution... that is, a ratio of 4.0 grams to 1.0 litres water (i'll obviously be cutting that down quite a lot as i only need ~20ml for the bath ... hope it's not too much hassle crushing the NaOH pellets!). It's good to know tap water is generally fine to use but I figured i'd play it safe by using distilled water as it's cheap and i figured the less unwanted crap/molecules getting in the way the better, especially for somebody like me lol. (I'm guessing there wouldn't be much of a problem anyway due to the specificity of Fast Blue, but i might as well play it safe!) Anyway it's Monday now at last and I'm expecting most of the few remaining parts to arrive this week! I do have the main parts so I could start TLCing now, but until i get some glass measuring syringes (ebay says arrival 12th-16th ie this week) to know exact quantities, plus lightproof freezer container for Fast Blue storage, working with it will just be a waste as i'll get no solid data. Patience will be a virtue!

Planned experiments:
1) with and without NaOH.
2) varying ratios of hexane:diethyl-ether, to help confirm the suggested 4:1 as best.
3) varying ratios of Fast Blue to determine optimal.
4) non-cannabis tests:
4a) I have a strawberry which i managed to not eat, now frozen: in 2014 (using a technique they say was developed in 2011) scientists determined, thanks to Fast Blue, that strawberries have a lot more phenolics than previously thought so itll just make for an interesting organic non-cannabis test. Just don't ask me what any of the resulting dye spots are... "hey check out all the THC in this strawberry!".
4b) Also I think it'd be cool to try some random leaf from a non-cannabis plant, a tree leaf or something.
4c) piss test!? You know you want me to. I'm pretty sure i don't want me to. Fast Blue is used to detect the THC metabolite THC-COOH in urine. THC-COOH = 11-nor-delta(9) tetrahydrocannabinol-9-carboxylic acid.
5) leaving the developed plates out for various number of days for scans to measure the deterioration of the dye (my current understanding is it fades 'in a few weeks', which is why i was hoping to avoid NaOH if it was only being used as a preserver, but clearly the reading states alkalinity is important with Fast Blue). Will try with and without NaOH.
6) figure out best temperature and duration for on-plate decarboxylation in oven

All these experiments to try, at my personal expense, because despite RTFM'ing for a full month there's still nothing concrete, with so many differing measurements. So that's where we are ... this amazing and relatively inexpensive technique to determine THC:CBD ratios, yet hardly anybody is doing it because there's just not enough specific information available! :( hopefully we can change that and open some doors. And yes, it surprises me that most breeders don't seem to use it.

anyway i can do all these tests because it's easy to cut the alfoil TLC plates for individual runs - we don't need the more-expensive glass plates for this (and i dont have anything to cut glass ones with either), another money-saving and convenience-improving tip thankyou again

Watch this space! The band at Experiment City are already doing sound checks and should be jamming by the weekend

[sadpanda]

actually rather than trying to make 25ml of sodium-hydroxide:water 0.1N solution each time, it seems easiest (and more accurate and consistent) to simply make 1L worth and then keep that stored aside for easy use whenever its needed.

The SDS just suggests storage as "Keep container tightly closed in a dry and well-ventilated place" and is "Stable under recommended storage conditions", and Sigma sells 0.1N premade solutions, so pre-making 1L and storing it aside seems fine

https://www.instructables.com/answers...ide-container/

Sodium hydroxide should NOT be stored in glass. Do not use polyethylene terephthalate (aka PET or PETE) type plastic either which is probably the most common household plastic (water bottles, etc).

I use high-density polyethylene (aka HDPE) or polypropylene (aka PP) which are somewhat common around the house. Other plastics work too but you will have to look at its chemical resistance chart, not all plastics will work. The sodium hydroxide I have came in a #2 HDPE bottle.

Certain metal containers are OK but I tend to avoid metal because there are so many different compositions and chemicals that can't be stored in or even near any kind of metal container. As mentioned, use an airtight container because it is extremely hygroscopic.

and https://www.ineos.com/globalassets/in...ance-guide.pdf (chemical resistance chart for HDPE) for sodium hydroxide concentrate gives it the maximum "Satisfactory" rating (the only other ratings are Some Attack, and Unsatisfactory).

Im pretty sure cola etc drinks come in clear PET bottles in which case we can't use those, but i just checked in my fridge and ive got a 2L iced coffee flavored milk, as well as orange juice, and checking the base they both say HDPE, with a 2 in the recycling logo, so it sounds perfect no need to buy off ebay, ill just rinse it out with tap water and then a little bit of distilled water and then a little bit of high quality isopropyl alcohol that ive already got nice and easy to pour too with handle

[sadpanda]

Something else i've seen a lot on TLC plate images is a CURVE on the outer runs, resulting in a "° o o °" kinda shape rather than the expected flat "o o o o", yet they still have the same Origin, so the Rf values are seemingly affected too:


It seems this is because of using jars that don't have flat bottoms. Not only do jars without flat bottoms result in that above effect, it also means you need more eluent than you would if the base was flat, and that also means having to use a bit more eluent (the eluent wont even be touching the base of the plate until its ~3mm deep), although the latter isn't so much of a problem as i think we can retain and reuse the leftover.

But anyway i just invested $2 in a 50pcs bag of 4mm (smallest, probably best) stainless steel ball bearings, hopefully sitting a ring or two worth of those around the outer base will result in less eluent + a more desirable flatter result (but hopefully without a 'wavey' effect from the balls). Interesting experiment anyway ... and of course I will keep my eye out for elusive flat bottom jars (have to be the right dimensions too of course), but hey... $2.

[Chimera]

Go to Lowes's or Home Depot and find a large glass brick used in construction. Take it to your local glass cutting shop and have them carefully cut the top off of it- voila a perfect developing vessel. I saw them in use at Arno Hazekamp's lab in Leiden, a nice little money saver tip. Shopping via science catalogs is worse than buying at grow stores, they think everyone is spending grant money so prices are always high high high!

[sadpanda]

a 'glass brick' so one of these?

and then take it somewhere and pay them to cut it for me? i dunno that sounds a lot more effort and expense than just buying a flat bottom jar or using a filler like ball bearings, and no airtight seal either

[sadpanda]

Another experiment i'll have to do is try to figure out whats best for on-plate oven decarboxylation of 2µl extract dots ... (takes less time and won't stink up the place like decarbing buds does)

Montana Biotech - "Heat plates in a 300°F/149°C preheated oven for ~3.5 minutes. Remove from heat and let cool."

Alpha Cat - "Heat the plate for 40 seconds in the oven at 100°C/212°F"

Alchimia - "we place the plates in a Pyrex tray which we put in the oven at 120-140°C/248-284°F during 5-10 minutes."

Journal of Chromatography 1990 tested the "Effect of heating time and temperature on the THC content of an n-hexane marihuana extract after heating on the glass surface in an open reactor", with a graph demonstrating 252°F/122°C for 27 minutes is optimal (but doesn't say it was on plates, and doesn't give quantity)

Cannalytics - "Heat the spots by holding the tip of the smallest possible flame of a cigarette lighter on the glass side of the TLC plate for 10 seconds. Hold the plate at a 30 degree angle to avoid blackening."

I want to over-decarb at least once anyway to hopefully see the THC spot degrading into the CBN spot

Also some interesting notes about it in this patent https://www.google.com/patents/US20070077660

In an embodiment, the sample may be placed on a little piece of aluminum foil or on a small dish or in a crucible, etc. Then the sample can be heated in e.g. an oven for about 2-8 minutes, preferably about 3-6 minutes at about 80-200° C., preferably about 120-180° C. By trial and error (i.e. varying heating temperature, heating time; and developing the TLC plate 11 and checking the result), the appropriate conditions can be found. If e.g. different reddish spots appears between the CBN spot and the natural smear of acids on the fingerprint (the not heated version) the temperature was too high and/or the heating period too long. Heating for about 4 minutes at about 150° C. provided good results.

Alternatively, in a specific embodiment, the method of the invention further includes:
spotting on the TLC plate an amount of the extraction liquid with the extracted cannabinoid(s), followed by a heating of the plate.

Referring to FIG. 5, after providing one or more spots 12 to the plate 11, as described above, TLC plate 11 can be heated. The same heating conditions as described above can be used, e.g. 4 minutes in an oven at about 150° C. Alternatively, the TLC plate 11 may be carefully heated with a candle or lighter 21. To this end, the plate 11 may be seized between thumb and index-finger or with a tweezers, etc, preferably such that only the edges of the plate 11 are touched. The spots 12 are carefully heated, about 2.5-4 cm (h4) over the flame of candle or lighter 21 for about 1-3 minutes, at both sides of the plate 11 (silica side/glass side). Also for this method applies that some trial and error may be necessary to find the right heating conditions. Good results were obtained with a distance h4 of about 3 cm and a heating time of 1½ minutes. Heating is usually performed either directly after weighing the cannabis sample 2 or after spotting the extraction liquid with the extracted cannabinoid(s) 7 on the TLC plate 11. Hence, for determining the cannabinoid content in the cannabis according to the method of the invention, until development, only one of the optional heating steps as mentioned above is chosen.

[G.O. Joe]

Silica is acidic. This is a much less popular graph from the same report. Aluminas are more reactive. Anyone in the cannabis analysis business who doesn't know this, maybe they're not a better source of information than forensic chemists.

[sadpanda]

awesome pic Joe, thankyou!!! Im quite perplexed by it though! ...

As soon as the material gets heated all its Delta9 THC starts to degrade!? rather than increase!? its just Delta8 increasing?? its almost as if it's saying "if you want delta-9 THC don't decarboxylate at all, not even for a second" !?

and i thought the THC would degrade into CBN, yet the CBN level constantly decreases???!?

now i'm even more confused/not sure how many mins to go for... is the chart suggesting 10 mins @ 145C is best for highest THC? but... highest D8 THC, lowest D9 THC!?