[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]

Originally Posted by sadpanda

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]

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-...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 - https://phys.org/news/2015-01-total-p...s-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"
https://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: https://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) ...
https://www.abc.net.au/news/2014-05-2...othing/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.