Home TLC Thin layer chromatography

[SkyHighLer]

The DE's fine, if I was going to come out with a kit, I'd compare hexane/DE to pentane/DE, like I said less smell and toxicity danger, and the cost difference is minimal.

These are distilled products you know... like distilling the solvent, ethanol from the solvent, water. ;-) In the case of hydrocarbons, you start with Lucifer herself, crude, and start refining....... don't get me going on about Crude, see GW's first Signature line.

So, understanding how the quality of a hydrocarbon solvent can be determined even by the layman by going to a table or two is a valuable lesson indeed, much love.

From the group mind, it's playing from random shuffle at Pandora,

https://www.youtube.com/watch?v=NdiRhzTsSnk

 

[sadpanda]

I'm going to have to leave it up to somebody with chemistry experience to trial other solvent mixes like pentane - I consider myself very fortunate to at least have that "we tried these 6 mixes for cannabinoid separation, here's the best one" paper, and hexane & diethyl ether seems relatively newbie-friendly enough BUT I will be also trying 3:1 and 5:1 mixes (possibly more) of these two to help confirm that 4:1 is indeed the way to go I'll scan and upload those in a week or two when i'm able to start. Also looking forward to uploading some scans to show different levels of Fast Blue.

But just to confirm, you think the hexane and diethyl ether that I bought will be ok for this TLC??? (although yes next time I will try to get HPLC grade... is 95% ok or has to be 99% for this?)

 

[SkyHighLer]

Your solvents are just fine for your purpose.

Recall one of the six mixes was with low Bp pet ether, which is predominantly pentane, the rest being hexane, and since you're combining it with the ethereal dissolving power of diethyl ether, I bet the difference between pentane/DE and hexane/DE is mute.

Also, since you've DE coming, you need to understand that just sitting there it forms explosive peroxides. Do a search for basic handling and storage,

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

 

[sadpanda]

*phew!!!* THANKYOU very very much for your expertise and help with this, I'm soooo relieved I didn't just waste money on these two! It's good also for others to know that pentane is also another option then - can never have too many options! (although my problem was just finding 1 good one, lol). This one used nothing but chloroform but still got great results. Do you think chloroform might be better than hexane:diethyl ? I'm almost tempted to get a 100ml bottle to try, but then i've already spent a fair bit on this lol

I'm getting excited now, not long until i can start sciencing the shit out of this and help further demystify the process i also have a Bluebird Botanicals CBD oil as well as a CBD oil syringe from another company so they should make interesting spot samples to go along with my bud samples of half dozen or so misc high-THC hydro strains, plus help provide additional confidence in identifying the CBD & THC spots (not that there should be any problems with those two though!)

 

[subrovka]

Max. limits of impurities:
30% Colour (Saybolt)
0.09% Benzene
0.001% Non-volatile matter
0.001% Sulfur
0.2% Aromatics

Likewise the Diethyl Ether, is "Minimum assay 99.5%", with Max 10% Color (APHA), and then a list of about 30 other things with really low like 0.02 - 0.000001 values.

So is this ok for TLC??? *holds breath* (and if i do have to buy again, i can get HPLC grade, is 95% ok or does it have to be 99%?)

hi, saybolt colour is just the definition of the colour of petroleum/(saturated) hydrocarbons. it ranges from -16 (very dark, much impurities) to +30 (very clear, less impurities)

 

[sadpanda]

subrovka, THANKYOU for filling in another piece of the puzzle!!! And im happy to report that mine is Saybolt +30 so that sounds best for this TLC (on the bottle it's actually listed under Maximum Impurities % so i misread it as having 30% of this 'saybolt color chemical' added, lol!) You have yourself a great weekend too!!!

 

[sadpanda]

What is Fast Blue BB dye, anyway?

What is Fast Blue BB dye, anyway?

As I wait for my Fast Blue BB salt and a couple other final components to arrive, I wonder ... what exactly is this special dye "Fast Blue BB", anyway!?!?

Apart from it reacting to cannabinoids and strawberry phenolics, all I knew was that BB was created due to carcinogenicity concerns with B. (BB however is still also a suspected carcinogen and instead of aerial spraying like some kits suggest we should shallow bathe regardless of B or BB). While B and BB can seemingly be 'interchanged' (it was afterall made as something of a replacement) as per for example cannabinoid detection I personally do not know or understand the differences between B and BB, although BB should be stored frozen while B kept cool or refrigerated, and BB stains apparently take ~10-25 secs to appear vs B's ~5, and BB's "final intensity of response is better, being more vivid", as described in this excellent article which also explains the search for a Fast Blue B replacement and why BB was chosen - https://www.unodc.org/unodc/en/data-and-analysis/bulletin/bulletin_1974-01-01_4_page004.html

Anyway it turns out that while Fast Blue BB has been around for quite a while (1974 it seems, ~5 years after Merck introduced Fast Blue B for use in detecting cannabinoids) its use with fruit and vegetable phenolics is as recent as 2011?! or maybe im not interpreting that right. Anyway while I couldn't find an "About Fast Blue BB" page this article makes a pretty good second

This is from 2015 - http://phys.org/news/2015-01-total-phenolics-fruits-veggies.html

New tests count total phenolics in fruits and veggies

Agricultural Research Service investigators have a long history of designing and developing reliable analytical methods for measuring nutrients and other compounds in foods. ARS scientists have now devised new analytical methods for detecting and measuring concentrations of phytochemicals called "polyphenols" in plant materials.

The class of health-promoting compounds is found in certain foods and beverages and is also referred to as "phenolics." At the ARS Eastern Regional Research Center (ERRC) in Wyndmoor, Pennsylvania, scientists first reported on the new test and used it on a variety of samples of beverages, such as teas and juices; grains, such as rice and quinoa; and flaxseed.

In the laboratory, the scientists used the new method to measure the amount of phenolics in the various food samples by mixing them with Fast Blue BB diazonium salt. Under alkaline conditions, diazonium salt specifically couples with phenolics to form stable complexes that can be directly measured.

When compared to results using a traditional method to assess phenolic concentrations, the new Fast Blue BB method assessed higher amounts of total phenolics in most of the beverage and grain samples tested, and lower amounts in flaxseed and some juice blends tested. The results suggested the traditional method does not assess or pick up all phenolics present in samples tested and inadvertently measures other compounds besides phenolics.

The Fast Blue BB diazonium salt approach is relatively simple, inexpensive, and fast. A study describing the new method was published in the Journal of Agricultural and Food Chemistry in 2011.

A later ARS study conducted at the Henry A. Wallace Beltsville [Maryland] Agricultural Research Center (BARC) further explored assessing total phenolics using Fast Blue BB. This study was performed on strawberries and was headed by plant physiologist Gene Lester with ARS colleagues at BARC and ERRC. They demonstrated that the Fast Blue BB assay provides a higher and more accurate estimate of total phenolics than the traditional assay, called Folin-Ciocalteu, or FC, that has been used for decades. FC uses a reducing reagent that detects phenolics indirectly and lacks the ability to be specific, or screen, for measuring phenolics alone, according to the scientists.

In the study, the researchers used the FC and Fast Blue BB tests to analyze total phenolic concentrations in five different genotypes of strawberry fruit that are commonly grown in the United States. Strawberries are an important source of phytochemicals, in particular phenolics. The team then compared the results of both tests.

The team found that the traditional FC test interacted with vitamin C (ascorbic acid) and sugars that are abundant in strawberry, which alters the accuracy of the assessment of phenolics. That means the FC method picks up other compounds in the plant itself and also in the media used. These additionally measured compounds are referred to as "interfering substances," and they include fructose, glucose, and sucrose and other organic compounds naturally found in the extraction media. There are upwards of 50 interfering substances that impede an accurate measure of the types and amounts of phenolics when using the FC assay method to test for total phenolics.

During the study, the researchers also measured each known FC assay-interfering substance, including vitamin C and the fruit sugars, within the five genotypes of strawberries. They wanted to determine the effect of these interfering substances on the accuracy of the two assays for measuring total phenolics.

The FC method had a significant correlation with vitamin C, meaning it assessed vitamin C as phenolics, and was found to underreport or fail to assess total plant phenolics by nearly threefold. The FC method does not include a step to correct for picking up the interfering substances that are counted among total phenolics, according to the scientists. The researchers concluded that previous studies measuring the phenolics in strawberry fruit by use of the FC assay have greatly underestimated the amount of the fruit's total phenolic concentrations.

Significantly, the new Fast Blue BB method had no interaction with vitamin C or with the other interfering substances. "Because Fast Blue BB is a direct assay, it only targets phenolic compounds, whereas Folin-Ciocalteu is an indirect assay—you can get false positives, primarily by picking up ascorbic acid and other substances," says Lester.

The study was published in the Journal of Food Composition and Analysis in 2012.

Fast Blue BB Goes Green

While the Fast Blue BB method as originally developed at the ERRC was aimed to measure phenolics only in plant tissue that does not contain chlorophyll, the BARC researchers reasoned that the test should also work with green vegetables if modified. Lester led a study to gauge how summer and winter weather conditions affect levels of specific nutrients and compounds, including phenolics, in spinach. For the study, he and colleagues modified the Fast Blue BB assay so it could be used to test green plant material.

"Chlorophyll absorbs at the same wavelength as the Fast Blue BB," says Lester. "We developed a simple, rapid, chlorophyll-removal step that eliminates this confounding factor in the original assay so that the method could be used to assess green plant tissues."

The scientists used the modified method to gauge the amount of total phenolics in different spinach cultivars grown under different production conditions. "We showed the Fast Blue BB can now be used universally to accurately assess total phenolics for all fruit and vegetable plant tissues," says Lester. The study was published in the Journal of Agricultural and Food Chemistry in 2013.

and here is that "study describing the new method published in the Journal of Agricultural and Food Chemistry in 2011"
http://pubs.acs.org/doi/abs/10.1021/jf103711c

Simple and Rapid Method for the Analysis of Phenolic Compounds in Beverages and Grains
Marjorie B. Medina*

Abstract: A new method for the detection of phenolics in food systems was developed. This method is based on interactions of phenolics with Fast Blue BB diazonium salt in alkali pH, forming azo complexes, with the absorbance measured at 420 nm after 60 min. The linear regression correlations (R2) of gallic acid calibration standards were >0.99. The phenolic content (gallic acid equivalent) of samples analyzed yielded higher ratios (1.7−6.6) of the total phenolics by Fast Blue BB to Folin−Ciocalteu methods in most beverages and grain samples, but in flaxseed and some juice blends, the ratios were <1. The lower ratios suggest the presence of non-phenolic reducing constituents measured with the Folin−Ciocalteu method as “total phenolics”. This method is simple and inexpensive and can be used to rapidly assess the total phenolics of foods and beverages.

 

[G.O. Joe]

These are the azo dyes I was talking about and visible spectrophotometry has come up in the past. The water-soluble phenols they're looking at however are very different from THC and the isolation procedure will not work on pot. That doesn't say anything about BB or it's history and neither does this - FBB might work just as well.

The characteristic colors of FBB with cannabinoids indicate a higher wavelength than the 420 nm used there, but obviously the wavelengths to use will be self-evident upon careful examination. Either way a cheap 340-1000 nm visible spectrophotometer is the instrument to use although someone is trying to sell the model I have for $1500 on ebay.

The method would work if the specific products are stable and absorb in a predictable way. Calibration if possible would require purified standards and it's probably been looked into by many people who haven't said so.

There are many ways to spray a plate with FBB and not be exposed to anything. Outside, or inside a bag would be easy enough. FBB with cannabis TLC goes back to at least 1964: http://dx.doi.org/10.1016/S0021-9673(01)95077-0

 

[sadpanda]

more awesome info, thanks!!!!

btw that made me do a more general search, just for "azo dyes" and i came across this 2014 one about the Australian government considering banning some azo dyes (which the EU did in 2003) ...
http://www.abc.net.au/news/2014-05-...cinogenic-dyes-more-found-in-clothing/5482040
so hopefully Fast Blue B/BB won't be affected there! and if they are hopefully it'll only apply to clothes manufacturing etc and still allow easy purchase for this TLC type of thing. When first learning about this TLC cannabinoids test it was such a relief to learn that B and BB didn't seem to be on any lists or it would've been game over straight away and we'd be blind about THC & CBD ratios i would only have Beam's CBD test then. Anyway hopefully im worrying over nothing.

There are many ways to spray a plate with FBB and not be exposed to anything. Outside, or inside a bag would be easy enough.

ahh! i like the bag idea. I think we'll be sticking with the bath method though, as cancer is already a problem! but i've also got the impression from several other ppl that the bath method gives a more even wash and better results anyway?

btw I came across a page a while back about how to NEUTRALIZE the rest of the Fast Blue bath so it can be disposed of safely ie it said it can then be thrown down the sink or drain or toilet or something, but for the life of me i haven't been able to find it again

 

[G.O. Joe]

The azo dye is the reaction product with a phenol such as cannabinoids. Spraying is very popular. FBBB is a diazonium and FBB is a tetrazonium salt. The reactions are typical of diazonium salts like reacting with phenols, and replacement of N=N by OH by boiling in water or pouring down the drain.

Many less specific developers have been tried out. Gaoni and Mechoulam used potassium permanganate in aq. cupric acetate. Hazekamp used fluorescent plates with UV, and anisaldehyde in sulfuric acid.

 

[sadpanda]

Many less specific developers have been tried out. Gaoni and Mechoulam used potassium permanganate in aq. cupric acetate. Hazekamp used fluorescent plates with UV, and anisaldehyde in sulfuric acid.

I HAVE TO TAKE MY HAT OFF to all the researchers who've helped push the boundaries of science of this extraordinary plant. And not just to those who made massive breakthroughs, but everyone who contributed their own small piece of the puzzle!

Thanks to all their groundwork, here in 2016 somebody like my friend and i - fans of science but far from scientists, just people fighting cancer from hospital and home - have a tool to help determine THC:CBD ratios in plants\oils\etc to allow what we need - complete independent control over dosages of both THC and CBD.

I mean ..... WOW. There really should be a few expletives in there but there doesn't exist a word with enough punch.

 

[sadpanda]

How long do i decarboxylate these tiny 100mg samples (both dried and fresh), and at what oven temperature? and I think I'll have to wrap them in aluminium foil so that the oven fan doesn't blow them around, which may affect the time required a bit?
Thankyou! ps. i now have Fast Blue BB salt in my freezer!!! havent opened it yet, just waiting on a couple more parts for measuring and then I can get started

 

[SkyHighLer]

It's Labor Day, probably not in the States!?

Glad to know you could get what you need where ever you are sadpanda.

 

[sadpanda]

yes im one of those rare 96% who don't live in the US hehe Happy Labor Day!!! hope you all have a great day with friends and fam, and maybe even a great meal too hehe

Glad to know you could get what you need where ever you are

anybody should be able to get BB or at least B anyway, as neither or on any special lists, and it's a legitimate useful dye for phenolics so it's not 'cannabis-specific', just need to do a bit of googling, and I think even if you have no luck with that you can just import it yourself from any of those Chinese or Indian etc labs (mine is from Japan), or contact some labs in your own country and ask them to import it for you, that worked for me and thanks to dry ice, getting frozen BB was still easy and cheap freight.

Here's a happy song i wrote about it, which i like to do a happy dance to (video withheld). I THINK THIS IS TO A 2/4 BEAT BUT I HAVE ABSOLUTELY NO IDEA WHAT 2/4 MEANS. Whatever that Britney Spears "Baby Baby" song uses. The song itself actually goes for 14 minutes as i sing the verse 28 times.

---
I'VE GOT A FEW GRAMS OF FAST BLUE

Iiiiiii've got a few graaaams, a few graaams of Fast Blue BB...
Oh yeeeeahhh, a few graaaams, a few graaaaams baaabyyyy...
Gonna staaaaain meee, gonna staaaaain me some cannabinoiiids...
It's such an amaaazing woooord, nothing rhyyyyymes wiiiiith... cannabinoiiids...
I'll be determining the ratioooooo, the ratiooooo of THC to CBD...
And withhh that capabilityyyyy, we can dose the medicine down to a teeeaaa...
Oh cancerrrrr.... all our weaponsss will beee deployyyeddd....
And with cannabinoooids on our siiide, you're gonna be absolutely destroyyyed

---
(no it actually doesn't need workshopping, i've already done that!)

 

[SkyHighLer]

Jim Dandy whalin' nicely...

For you youngins

(Lord Have Mercy On My Soul, Black Oak Arkansas)

https://youtu.be/fCgs8KMU-nI

 

[sadpanda]

I'm still not sure if i should get the (sodium?potassium?) hydroxide, as I'm a bit confused as to whether it's required or not. Some texts seem to suggest it's used as a 'fix' to preserve the dye (apparently it degrades quickly over a few weeks), but others seem to suggest its used to make the result more vivid. As i'll be flatbed-scanning my plates I have no need to keep the plates, so if it's just for fixing/preserving the dye then it's an extra expense and stage I'd like to skip.

btw i came across this interesting pic at http://jbcs.sbq.org.br/default.asp?ed=239

That image, as well as the text about it, seems to suggest 1) the quantity or volume of FBBB, and 2) time elapsed, both play a role in the resulting color. Well, I will be experimenting with differing quantities of FBBB, and I'll also try to do some scans of the plates after 1hr, 12hrs, 24hrs, 48hrs, 1wk etc etc to observe color changes. I should have enough components to start before next weekend (all the main parts are here, just waiting on a couple of minor essentials like measuring and storage) so i look forward to sharing the experiment results then

 

[sadpanda]

my other concern is "alkalining the plate first"??? I don't think any of the commercial cannabinoid test kits do any alkalining or whatever. But when googling for Fast Blue BB i came across this quote:

I tried finding out what chemicals they use for the extraction solvent, developing solvent (mobile phase), and alkalining solvent. An important issue is the extraction solvent. It wise to use one which develops neutral cannbinoids only. And high quality stationary phase (ie. silica plate) is very important for accuracy. All I know is they use the standard visualization reagent "fast blue BB". However, the plate should be alkined before, or during application of the reagent. Also, there should be a final step past the fast blue BB; that is a preservation solvent (0.1M sodium hydroxide; same thing for alkalining solution). I don't think they use the last step

or does the hexane or diethyl ether im using provide that alkalining?

 

[G.O. Joe]

More people have added base than have not so you might as well. Azo couplings in general are favored by base, but strong base will cause other reactions, so that 0.1M solution is specified. Diethylamine has been popular for cannabinoid analysis, and also LSD manufacture so you may wish to avoid it. Exposing the plate to ammonia fumes while wet with the fast blue might do something very similar.

One of your posts addresses many of your questions, and has all the directions you need:
https://www.icmag.com/ic/showpost.php?p=7582761

 

[sadpanda]

ok *sigh* I will reluctantly add NaOH at least it's easy to get locally so i'll certainly have it within the week, and it's cheap enough, so i shouldn't whinge too much lol. btw it seems from that doc that bicarbonate of soda might also be ok to use - they use it in the "presumptive color tests", but use NaOH for TLC. I'll of course play it safe and just get some NaOH.

btw i just noticed this in that UN doc ... basically answers my Q (at least i think it does) as to why my eluent is hexane + diethyl ether, and not just hexane ...

Since cannabinoids are easily soluble in most organic solvents, methanol, petroleum ether, n-hexane, toluene, chloroform and solvent combinations, such as methanol:chloroform (9:1) are equally suitable for their extraction. It should, however, be noted, that non-polar solvents such as n-hexane and petroleum ether give a relatively clean extract but will only extract the neutral/free cannabinoids quantitatively, while the other solvents and their combinations give quantitative extractions of the cannabinoid acids as well.

For identification, the simplest clean extraction with petroleum ether is enough, while for the purposes of quantitation and total THC determinations other solvents have to be used.

Although I guess if we only want to do decarboxylated tests maybe we wouldnt need the diethyl ether and could just use the hexane!? but i think i'd always prefer the full spectrum anyway

It's interesting though that in that paper (that tested 6 different eluent solutions and determined 4:1 hexane to diethyl ether is best) they only used hexane as the extraction solvent, not the hexane-ether mix they used in the eluent.

 

[G.O. Joe]

btw it seems from that doc that bicarbonate of soda might also be ok to use

I'm not sure that it can quite do much deprotonation - NaOH is more capable of actually forming something of a salt with the phenol function. Follow the directions.

Although I guess if we only want to do decarboxylated tests maybe we wouldnt need the diethyl ether and could just use the hexane!?

Polar solvent is needed to pull the cannabinoids off the silica, which interacts with the cannabinoids different parts and their different locations in different intensities.