Home TLC Thin layer chromatography

[sadpanda]

i wonder what the "Base solution" is?
Fast Blue BB Salt hemi(zinc chloride) salt : http://www.sigmaaldrich.com/catalog/product/sigma/f3378?lang=en <- this is the one im presuming everyone gets
Fast Blue BB Base solution: http://www.sigmaaldrich.com/catalog/product/sigma/917?lang=en

 

[G.O. Joe]

BB base is the BB version of the o-dianisidine I mentioned earlier. No azo dye without the azo part. If you want to do some organic chemistry with it to get BB salt, you'll need the totally inorganic sodium nitrite, concentrated hydrochloric acid, salt, and zinc chloride. And some ice.

 

[sadpanda]

how long does the salt/powder last? for example when you store it in a freezer? as im assuming is best

 

[G.O. Joe]

That depends on how it's used. If you're going to be dipping large plates you might be looking at the larger bottle. If the bottle is going to be opened many times over a long time, it would be a good idea to divide it up into smaller parts, maybe small plastic airtight vials. There might be some expiration date, but that only carries so much weight. My B salt is fine even though the exp. is Aug 2016. It will not be thrown out in the next several years.

 

[sadpanda]

thankyou so much. My heart sunk when i first read about how volatile Fast Blue can be but i really appreciate your reassurance, because this is really important as my friend needs to be sure her strains are high CBD and other than lab analysis and Beam's test i can't think of any other ways. Can't just smoke it to find out like with THC lol. I really love that trick of dividing it up into extra bottles to reduce overall exposure too. Now i guess there's just one last hurdle - getting it delivered! Weight isnt an issue at 1gm lol but i wonder if it'll come frozen, hopefully not too expensive P&H

 

[sadpanda]

AlphaCat comes with "2 microtubes of dye powder (0.06 g). Keep in a dry, dark space."
Cannalytics "put 2ml of water into the spray bottle and add one 20mg scoop of dye powder.. and shake to mix"
None of these test kits seem to mention anything about freezing, so maybe i have been worrying too much about that

 

[sadpanda]

whats the best solvent(s) to use for the capillary action? (the "eluent"?)
Hexane seems to be popular but often in mixes with chloroform or some ether or alcohol or something. This guy gets good looking results from hexane/chloroform https://www.alchimiaweb.com/blogen/make-cannabinoid-analysis/

 

[sadpanda]

i noticed Sirchie's kit (LEO purchase only so i would imagine quite professional and they seem pretty advanced), it has PDFs so i had a look at those, VERY INFORMATIVE!
They/Sirchie use:
Mobile Reagent = 60% chloroform, 40% Hydrocarbons C>=5, C5-6-rich.
Extract Reagent = Hydrocarbons C>=5, C5-6-rich.
Color Capsule = Fast blue B salt. (i wonder what reasons for B instead of BB?)

But then i thought "but _what_ 'hydrocarbons' exactly? that sounds a bit vague!"... but it mentions the CAS number of each, so i looked it up (68476-50-6), and it turns out their "Hydrocarbons C>=5, C5-6-rich" = petroleum ether

So now the question is what's best!? hexane... chloroform... petroleum ether... on their own or together and what ratio? or perhaps Sirchie has already figured out whats best and this is it? but maybe hexane is more affordable/convenient, or... ???

 

[sadpanda]

btw ive got to link this page, its actually from 1974 but seemingly every bit as relevant today!?! It's about cannabinoid testing via TLC, but specifically about the different dyes (Fast Blue B, Fast Blue BB, Fast Corinth V etc) and pros and cons of each ... https://www.unodc.org/unodc/en/data-and-analysis/bulletin/bulletin_1974-01-01_4_page004.html

 

[SkyHighLer]

whats the best solvent(s) to use for the capillary action? (the "eluent"?)
Hexane seems to be popular but often in mixes with chloroform or some ether or alcohol or something. This guy gets good looking results from hexane/chloroform https://www.alchimiaweb.com/blogen/make-cannabinoid-analysis/

Pentane (C-5) has little odor compared to hexane (C-6) and chloroform, it dissolves cannabinoids nearly as fast, and will dissolve less chlorophyl.

4oz of reagent grade pentane can be had for $13.50 (free S&H) from eBay. http://www.ebay.com/itm/Pentane-ACS...792512?hash=item1a0f891800:g:AxwAAOSweW5Veu2v Compare your hexane and chloroform options... and here's the petroleum ether, I suggest the highest grade, 35-60C Bp, http://www.ebay.com/itm/Petroleum-E...422905?hash=item3a826f1bf9:g:4SEAAOSwZAtXJixW

That's the grade PE I keep around for extractions, the higher Bp grades may contain benzene, not good for extractions... ;-) Here's a 60-80C Bp PE for comparison,
http://www.ebay.com/itm/PETROLEUM-E...634093?hash=item19f68d3e6d:g:OhgAAOSwQJhUb48u

https://en.wikipedia.org/wiki/Petroleum_ether

PE is mostly pentane, purchasing 35-60C Bp PE is a common way to score pentane cheaply.

 

[sadpanda]

SkyHighLer - awesome information, thankyou!!!
So does this mean you ONLY use Pentane, for both Extract Reagent + Mobile Reagent?
or do you still use both Chloroform + Pentane for the Mobile?

 

[SkyHighLer]

SkyHighLer - awesome information, thankyou!!!
So does this mean you ONLY use Pentane, for both Extract Reagent + Mobile Reagent?
or do you still use both Chloroform + Pentane for the Mobile?

I'm learning this stuff from you and G.O. Joe, I just knew a little about PE, pentane, and hexane...

Love your spirit!

 

[sadpanda]

My understanding is that pentane is better for hexane in regards to extractions (like BHO etc) as it doesn't have as much odor and doesn't take up as much chlorophyll, but im wondering if, in this specific case of _Thin Layer Chromatography_, if pentane is better, as i don't think i've seen it mentioned yet in any of my cannabinoid TLC searches. I would take an uneducated guess that it's probably at least on par with hexane for this though!

The other thing i'm wondering about is: Do we need both a polar solvent + a non-polar solvent for the mobile phase? or is just one type of solvent all thats needed... and if so does polar/non-polar make a difference?

 

[sadpanda]

btw in regards to petroleum ether (same thing Sirchie's kit uses + chloroform), its also known as petroleum spirit. One product list has different ones with these boiling points:
30-40C, 40-60C, 50-70C, 60-80C, 65-90C, 80-100C, 100-120C
Anyone know which we should be using for TLC? It does say "HPLC grade" alongside one of the 40-60C ones.

 

[sadpanda]

regarding polarity, its interesting that the Sirchie kit only uses one solvent (petroleum ether) for its Extract Reagent, but uses 60% chloroform (non-polar as solvent), and 40% petroleum ether (also non-polar solvent)... but i wonder why not just use 100% chloroform, or 100% petroleum ether?

 

[sadpanda]

some good information explaining why chloroform is mixed with another...
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Neutral lipids or generally storage lipids are extracted with relatively non-polar solvents such as diethyl ether or chloroform but membrane-associated lipids are more polar and require polar solvents such as ethanol or methanol to disrupt hydrogen bondings or electrostatic forces.

To avoid peroxidation of the extracted lipids, during the procedure or later, all solvents should be used peroxide-free. The most convenient is to use "pesticide grade" or HPLC grade" and to buy small quantities at once. Do not stock bottles for a year !! Manufacturers often indicate an expiration date on bottles. To minimize possible deleterious effects due to peroxide formation, pay attention to the expiration dates and keep stocks for no more than 3 months. Furthermore, to avoid contamination of extracted lipids by non-volatile material contained in the large volume of solvent used in the first step of extraction, it is important to use organic solvent containing less than 5 mg dry matter per liter, 1 mg/l is currently proposed.

Diethyl ether, dioxan, isopropyl ether and tetrahydrofuran form peroxides on storage and must be kept in dark bottles or in dark areas. These solvents should be routinely tested to prove that peroxide levels are kept low before use. It must be emphasized that peroxides, even at low levels, may present an explosion hazard after concentration of solvents. Because of its low flammability and its natural origin, ethyl lactate was proposed as a substitute for ethyl acetate in exctracting carotenoids from foods..

Alcohols are good solvents for most lipids, methanol and ethanol being the most popular. Ethanol can be contaminated on storage by aldehydes.

Chloroform is a popular solvent, particularly for lipids of intermediate polarity and when mixed with methanol it becomes a general extraction solvent. It is not very stable, forming phosgene and HCl in air, it should be stored in brown bottles. It is sold after addition of preservatives, ethanol, amylene or cyclohexene. Dichloromethane (or methylene dichloride) is a similar extractant but less oxidizable. Chloroform and dichloromethane are currently stabilized by addition of ethanol (up to 1%) or methyl-2-butene-2 (about 20 mg/l).

Among hydrocarbons, hexane is the most popular but is a good solvent only for lipids of low polarity. Its main use is to extract neutral lipids from mixtures of water with alcohols. A mixture of isomers, called "hexanes", can be used for the same purpose. Hexane can be replaced by petroleum ether which is a mixtures of various hydrocarbons with 5 to 8 carbon atoms. Cyclohexane is sometimes used to store lipid extracts in the cold without danger of evaporation since it freezes at about 6°C. Benzene is no more used since it is now considered as a potent carcinogenic substance, it may be replaced by toluene which, nevertheless, is more difficult to evaporate.

 

[sadpanda]

btw a lot of videos show SPRAYING the dye on, others show quickly (in, out) lying it in a BATH of dye solution (and 'ganja din' earlier on in this thread, seems very knowledgable about TLC, also said its best to bath not spray). Interestingly all the main cannabinoid test kits still use Fast blue B (not BB), and all (except Sirchie) say to SPRAY the dye on. Sirchie uses bath technique. This worries me because with Fast blue B being carcinogenic you really don't want a spray of the stuff in the air! So regardless if i get B or BB (aiming to get BB) i will always use the bath method

 

[sadpanda]

another thing is the final stage where a sodium hydroxide solution is sprayed to stabilize/preserve the dye.

http://chemistry.about.com/od/labrecipes/a/sodiumhydroxidesolutions.htm
Ive read we want "0.1M" solution for that, which according to this guide is 4.0gms sodium hydroxide in 1L of water (distilled i assume). It seems easy and safe enough but there are some important safety guidelines to observe, as mentioned in that link.

Im hoping to skip this stage (and therefore not require any sodium hydroxide) by taking a photo or flatbed scan of the plate before dye deterioration.

Interestingly in one of the posts by ganja din on the second or third page of this thread he mentions using it twice - first immediately after completion of the mobile phase and the plate has dried, to make it alkaline, and then also following the use of the dye, to preserve it. But again im still hoping to skip it altogether, and in the Alpha Cat masterclass video it doesnt seem like they use it.

 

[sadpanda]

If anyone's just taken a quick peek at this thread and immediately got put off by all the chemical names etc, PLEASE DONT.... ITS EXTRAORDINARY HOW SIMPLE THIN LAY CHROMATOGRAPHY CAN BE! and you only need ~3 chems, none of which are too expensive and all relatively easy to obtain - no permits/license required. Do yourselves a favor!!! It'd be great if more people could post TLC plates of their strains

 

[G.O. Joe]

Pentane isn't mentioned because it's included in low boiling petroleum ether.

The solvent pair to use is the one that's available. The UNODC document said petroleum ether/diethyl ether 80/20 and that's the kind of solvent pair you're looking for. The combination evaporates fast and is only slightly polar. And Mechoulam and a lot of others have used it for a long time. Whichever low or high boiling pet. ether/pentane/hexane nonpolar part is fine. Perhaps ethyl acetate and many other things can substitute for the diethyl ether polar part. A nonflammable option is chloroform with just a little bit of methanol. Equal parts of hexane and methylene chloride.