Home TLC Thin layer chromatography

dongle · May 11, 2018

Looking at your plates again, I'm wondering if the stationary phase coating might be too thick.

Can't wait til delivery time tomorrow.

PhenoMenal · May 11, 2018

yeah i can't wait for tomorrow, woohoo!

dongle · May 11, 2018

PhenoMenal said:
i just use tweezers, gripping from the bottom of the plate (below the origin points), lay it in, and then gently push it down until the FBBB liquid comes over the top of the plate too, so i know the full plate has good contact. I like to give it at least 3 seconds but try to get it out by 5. Not sure what would happen if you leave it in for several minutes, probably lose a lot from the plate, but might have to try that sometime.

Ive been using only about 50mL dye solution in like a 8"x6" ish pyrex dish which doesn't create a deep enough pool to lay flat, so what I'm doing is tilting the dish towards the 6" edge so I have a 1"-ish pool at the edge, and i quickly dunk it into the bath for probably 0.75 - 1 second and pull out. The stationary phase with water soluble binder really wants to loosen up / dissolve almost immediately so i have to make it quick

PhenoMenal said:
* not sure if itll make much difference, but you might wanna cut a little bit off the bottom of your plates, just to the point where the silica is in full cover so that the capillary action gets an even start across the full width of the plate

Yeah probably won't make a difference but it's good practice in general
The solvent front line isn't perfectly straight but i haven't had any issues with that really so far i dont think

I did once tilt the development chamber a couple times once and made the line go zig zag

PhenoMenal · May 11, 2018

u should get a smaller pyrex dish! im only using 25ml dye solution, thats enough for 3 (5x10cm) plates. It'll only cost about $10-30 but halve the amount of sodium hydroxide and FBBB youre going through, and no need for any funky tipping etc so it'll pay for itself

btw i just came across an interesting quote about the edge effect from [link] (i was just wondering about the basic process of making plates)

... it is not necessary to use filter paper for the lining of the developing chamber, a paper towel is quite satisfactory and is much cheaper.

sweet! i'll have to try that. nice cheap and accessible hack if it works. just gotta get a nice clean paper towel though that wont interfere with the eluent lol. Or a cut-to-jar-size sheet of paper maybe. For some reason im too cheap to buy filter paper. And for that reason, don't wanna use too much either as it'll absorb more.

dongle · May 11, 2018

Filter paper is standard issue equipment around here

But yeah definitely need a smaller dipping tray. I was looking around for a smaller container but couldn't find one lying around

Also gonna break down the fbbb into some eppendorfs and the chloroform into pipetted amber vials

dongle · May 11, 2018

Jeeeeeeeezus, it never ends

The package isn't even here yet but I'm already disappointed.... it didn't specify binder on the Amazon ad, so i asked the seller a question and finally now responded that the binder is gypsum FFS I might have to return the package before it even arrives

The annoying thing is that my mailbox is about half an hour drive away from work, so just to check it out today, I have to drive an hour + wait 5 minutes in line to sign for the package, just to find out that it is indeed useless 99%

The seller was like "yeah it's clearly gypsum, can't you see the letter "G" in the product ID indicating gypsum?"

I mean, for fucks sake, why not just clearly specify gypsum in the product description??? Can't you tell by the presence of a vowel in my name that I didn't want a gypsum binder TLC plate, motherfucker???

PhenoMenal · May 11, 2018

doh

hopefully a bit better luck with the pro plates

Ringodoggie · May 11, 2018

I have been watching this with high hopes. I would love to be able to make my own plates. Eliminating the need to buy plates (and dye) would be a giant plus.

Did I see where someone used the 'black light' style plates (vs FBB dye plates) and was not pleased? Maybe they were gray scale rather than color?

Sorry to interrupt.

dongle · May 11, 2018

Ringodoggie, I was told by someone on my IG account that the 254nm fluorescent plates can work to identify spots (by Rf value Im assuming), but obviously you won't have the advantage of "color coded" spots (which isnt all *that* useful IMO)

dongle · May 12, 2018

The pro-made aluminum backed plates are nowhere near as fragile as my DIY ones, day & night difference. Much smoother, even coating. The gypsum binder isn't strong ebough to allow writing on it with a pencil (it will scrape off). It doesnt wash off anywhere near as fast as the DIY either.

One thing I noticed for today while setting up my development chamber, is that it took a long ass time for the filter paper to soak up the solvent. (Last time I walked away to take care of something else for over an hour and came back to it ready.) Then I watched the AlphaCAT youtube video again, and found they did not use a filter paper development chamber equilibration.

Since the filter paper is almost ready already, I will time one plate with it and one without.

Results within the next hour or so.

Jack El Frutero · May 12, 2018

Hi,

I'm learning a ton from that tread, thank you'll very much!

Quick question: I got a 100g jar of "Fast Blue BB 98% pure", manufactured by Across Organics. For some reason it does not dissolve in RO water at all. Methanol dissolves it completely, but after I add 0.1M NaOH solution to it something falls out of the solution and whole thing become like a gray-brown paste. It does not reveal any colors on TLC plates, while under 255nm UV light I can see trail and sepatate dots (I use plates with F254 UV dye).
SDS for that FBBB:
https://www.fishersci.com/store/msd...endorId=VN00032119&countryCode=US&language=en

What do I do wrong?

I;d really apreciate any help.

Thank youю

dongle · May 12, 2018

I faintly recall there are two forms of FBBB, one is an azo-something and the other is slightly different. I forgot which is which and whether both are supposed to work or only 1 works for this purpose

Jack El Frutero · May 12, 2018

Thank you, dongle.

dongle · May 12, 2018

Got there. I'll type up details and post more pictures later. An avalanche of work is about to pile down on me so I have to wrap this up real quick and get ready so I don't get buried for a month.

Jack El Frutero · May 12, 2018

It looks like I got wrong FBBB:

4'-Amino-2',5'-diethoxybenzanilide Formula: C17H20N2O3 Formula weight: 300.35 CAS Number: 120-00-3

while actually I need this:
Sort

: 4-Benzamido-2,5-diethoxybenzene diazonium chloride hemi zinc salt,
Formula: C34H36Cl4N6O6Zn Formula weight: 831.89 CAS Number: 5486-84-0

Oh, well...

dongle · May 12, 2018

Yes my bottle reads "hemi zinc blah blah". I almost got the wrong one as well, I was tempted to go with the higher purity one just in case but realized G.O. Joe talked about how they're different and mentioned to get one kind (however didn't say not to get the other kind).

Ringodoggie · May 12, 2018

Hey guys. Would love to see links to your sources for the correct dye next time you buy it so the rest of us don't buy the wrong stuff.

Thanks

dongle · May 12, 2018

Wet-ish plate, I think. Better separation than last run.

Spot sizes. The left spot is probably up to 5-10x as concentrated as the center and right. Center and right were same sample as first single spot run.

PhenoMenal · May 12, 2018

dongle,
nice! the brakes are off now!
Good to be on the pro plates if only to eliminate other issues that your plates mightve been giving you ... maybe then you'll be able to suss out the procedure for homemade plates to work? But maybe your "just buy pro-plates" recipe is still the best for homemade plates lol.

Jack,
doh unlucky!

doesn't help that they call them both "Fast Blue BB" eh

The one i got is made by Wako (FUJIFILM Wako Pure Chemical Corporation), its 5 gm (which is HEAPS in this context!), its number thingy is "062-05463", and simply labelled "Fast Blue BB Salt", and CAS number is "5486-84-0", which yes is the one you said you need to get.

It's the same stuff that dongle has now, but made by a different company, and mines a bit clumpy now whereas dongles is still nice and powdery, tho i still get results as good and vibrant as the day i got it. If you google "062-05463" you'll see a few references. I just contacted a couple of labs in my country to see if they could get it for me (give them the CAS number!) and the first one i contacted said yes, and asked if i wanted 5gm, 25gm or 100gm. I'm glad i went with 5gm lol, which is a vial the size of a thumb. Now look back at the tiny plastic micro-scoop that I use lol... it'll last aaages. Store in your freezer.

dongle · May 12, 2018

Ringodoggie said:
Hey guys. Would love to see links to your sources for the correct dye next time you buy it so the rest of us don't buy the wrong stuff.

Gotta do your homework, can't rely on anyone but yourself unless you pay them.

I didnt read the full thread and skimmed over what I felt were important details, and caught the post from G.O. Joe and I think others did too.

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