Chaos and Order - Methods of Purification

[@ThatChemistOld]

Make sure you subscribe to the new channel where all the new videos will be posted! kzbin.info/door/0nEDA3uRd5YuvBfMwQNteg

[@Skunkhunt_42]

This channel has quickly topped my list of "chemtube" here. I work in industry so my focus now is rather narrow (purification is my jam) the chemdraw tips vid was super helpful and many others on topics I have not dealt with in many yrs but are critical to know because most all of this ties together at the end of the day

[@ThatChemistOld]

Thanks!

[@robertlapointe4093]

Nice overview of separation methods. I would add one very old method and one new. "Digestion", is an ancient variation of crystallization which avoids the necessity of completely dissolving poorly soluble materials. Basically a sample of material to be purified (typically a very fine precipitate at this point) is placed in a container with solvent (no where near enough to dissolve the sample) and maintained at a temperature where the sample has some slight solubility. Dissolution kinetics causes the smaller particles (which have proportionally more surface area) to dissolve and the larger particles to grow over time (patience is required, may take hours, or months, depending on solubility). Taken to the extreme, the result is a single crystal suitable for X-ray structure determination. More usually this method is used to convert precipitates which are too fine to filter into easily filterable slurries (for example, in the gravimetric analysis of nickel with dimethylglyoxime). "Nano-filtration" is a relatively new process, which uses membranes (typically cross-linked amorphous polymers on non-woven fibrous supports) as filtration media for separating individual molecules based on size (small molecules flow through the "pores", which are the amorphous regions between cross-links, and larger molecules are retained). These can be used for retaining (concentrating) large molecules or rejecting (collecting) small molecules. I have used them (with less than perfect success) for rejecting product from a reactor, while retaining catalyst. Of course, there can be materials compatibility issues (the first membrane material I tried reacted with the small molecule product, but there are a variety of materials available) and the membranes require holders which can handle a bit of pressure, 10's to 1000's of psi, depending on application. And they can be problematic for rejecting reactive molecules, which can foul the "pores".

[@mytech6779]

Does the digestion require agitation/stirring? I have seen such a setup attempted with highly soluble salts and no agitation and it formed a liquid density gradient and the end result was actually smaller more granular crystals than when it started. It was not maintained at a precise steady temperature, the temp fluctuated with the daily room temp. Though the compound was fairly soluble through the whole temp range.

[@robertlapointe4093]

@@mytech6779 The digestions that I have run were on nearly insoluble materials. One had only very slight convective mixing (NMR sample tube in a wax bath - gave a single crystal after about 3 months). Others were just stirred on a hot plate for a few hours, warm but not boiling, to convert fine precipitates to easily filterable crystalline solids. For a very soluble material, you might want to try going to a poorer solvent and just do a regular recrystallization, possibly with slow cooling to low temperatures. (I have used -78C methanol to crystallize tBu3COH away from tBu2HCOH and -78C THF to separate ((tBu3CO)2H2Ta)2 from ((tBu3CO)2HClTa)2. These compounds were very soluble in hydrocarbon solvents, even at -78C, but the more polar solvents gave good results.)

[@mytech6779]

@@robertlapointe4093 Thanks for the tips. I'm totally an amateur with chemistry, a fair amount of theory but little resources to practice. Mostly focus on inorganic.

[@user-py9cy1sy9u]

Could you give me some pointers or google search keywords for "nano-filtration" so I could learn more? While I personally havent done anything with ABE (acetone butanol ethanol) fermentation its an interest of mine. I think this kind of filtration on molecular level could be used to probably make ABE fermentation commercially viable. While I had this idea for some time now I never knew where to look further. Where to obtain these membranes nor how much they cost.

[@robertlapointe4093]

@@user-py9cy1sy9u For our process, we started out with Evonik's Duramem membranes, which worked well for retaining our catalyst (a porphyrin complex) while letting THF permeate and then moved on to more exotic membranes when we found that the polyimide membrane reacted with our product (b-propiolactone). Since then, this membrane tech must have found a killer app, since they are now offered by retailers, such as Sterlitech. We started with a small (100 mm I think) holder and moved on to small spiral-wound membranes. I think an 8.5" X 11" sheet was a couple of hundred dollars (from which we could punch 6 disks) and the holder was about a thousand dollars. The spiral-wound cartridges were more like $5,000 and the holders about the same. Here is a link to Sterlitech's web-site, with prices for a variety of flat sheet membranes in various pre-cut sizes: www.sterlitech.com/nanofiltration-nf-membrane.html . They could probably provide more up-to-date membrane info than I can. A few pitfalls to steer clear of: the membranes are fairly robust, but can be ruined by finger-prints or even fairly light back-pressure; the holders typically require high "cross-flow" rates, to prevent concentration gradients at the membrane surface, which lead to high osmotic pressure differentials and greatly reduced permeate flow. Hope this helps, I know separations tend to be the limiting factor for economic viability of fermentation based processes.

[@nemeczek67]

So many methods but at the end of the day column chromatography is used almost exclusively. At least in synthetic organic chemistry.

[@ThatChemistOld]

hence why I made this video

[@fsen1999]

And its a damn shame, synthetic organic chemists have forgotten how to do chemistry because they have such a crutch. bring back elegant work ups!!!

[@teyrasiridae4704]

@@fsen1999 In part it's because something like crystallization of more complex compounds can be quite challenging and it's very difficult to find 'tips and tricks' / best practices on purification methods.

[@fsen1999]

@@teyrasiridae4704 I know, im being 50% facetious, when youre making loads of compounds in quick time its just natural, but it is the case that many chemists never get to developing their chemistry to be anything more than rotovap, water, bring, mag sulfate, column, which is a shame.

[@Seorful]

@@fsen1999 I'm happy to develop elegant workups if i dont have to work against time before funding runs out or another deadline has to be met.

[@kalrbaum]

I did an internship at a process development lab of a big chemical Company. During a scale-up I got a compound to >99% purity by trituration while the initial route only got 90% with chromatography! But i also had several grams to work with, where the original had around 100mg. Some things just need this scale to work

[@D3CD95]

These "catalog" videos are so important, very often courses will focus on "we will use that thing" without giving a sense of how broad the topic is. I find it so much easier to think and concieve stuff when you have this kind of "toolbox" in mind. That's real for every science, let it be maths, physics, engineering of who knows what. When you know your tools, you know what to do and it's only when none of your tools work that your have to scratch your head.

[@Aetohatir]

It's crazy how fast the algorithm picked up your channel. Every time I check it's like 1k more. Your daily upload schedule definitely helps.

[@ThatChemistOld]

Thanks!

[@griffinl6057]

I love how the title sounds like an alchemical text as much as an o-chem one lol

[@ThatChemistOld]

Fair

[@basher20]

I subscribe to a bunch of Dungeons and Dragons channels. To me it looked like it was going to be a bunch of Cleric or Druid spells.

[@TheNobody109]

Good video, one minor correction though - HPLC is generaly correctly refered to as high-performance liquid chromatography

[@blubbyblublubasdasd3267]

i always thought it was high price liquid chromatography

[@david3710]

There's never enough of the chemistry content on yt, and you're making some really cool videos! Thanks for your work and I'll surely follow this channel. Goodluck and don't smell anything nasty.

[@ThatChemistOld]

Thanks!

[@nicholastsl]

FYI, as polymer chemist myself, I used dialysis method a couple of times to purify polymers (removing monomers) for post-polymerisation modification later on. It is quite a convenient technique, when it works. XD

[@ThatChemistOld]

awesome!

[@jeffblack8812]

Very thorough list and enjoy your channel. Have been homesick for some good synthetic organic chemistry. Someone already mentioned electrolysis for metals but also want to mention continuos solvent extraction equipment and also have used freeze drying as a way to isolate some specialty potassium carboxylates from water to get around serious foaming when trying to remove water when heating was not an option. Again, nice channel.

[@stevengill1736]

You caught me with the title: chaos theory in chemical engineering. I always liked the concept of parametric pumping ...

[@chemically_literate]

I'd say that recrystallisation is super super important to master as well as dry column vacuum chromatography. Obviously one must have a solid understanding of the basic organic chemistry techniques but these two really help out a lot. Sometimes we dismiss basic techniques as primitive without trying them out properly. Also, ion-exchange chromatography is missing from your presentation. It's quite useful for inorganic chemistry at least. The point about ion traps and ICP is on point as there is little prep chem that can be done under high vacuum but maybe in the foreseeable future things might change.

[@ThatChemistOld]

I think DCVC is overrated

[@Petemate69]

Ran a 5L solvent volume column today so DCVC has me intrigued from an environmental and cost/time perspective

[@NicolasBana]

I think you forgot the most basic method for separating two things based on charge. Put lots of electrons through it ! Copper is purified using very large 99% or so purity copper which is then plated onto pure copper anode to form 99.998% pure copper ! In the field of organic chemistry i don't know about how much it's used but since you spoke about floatation agents in the mining industry, i think it deserve its place on teh list. Discovered this channel recently and even though i abandoned my Ochem studies a couple years ago, i'm still very interested by chemistry and this channel is a godsend when ExplosionsAndFire doesn't get another cubane video in time :D (real talk though, love your channel !)

[@ThatChemistOld]

Good point! This is true!

[@leothecrafter4808]

11:33 A very similar process is actually used to make oligonucleotides, a short (1-100+) chain of nucleotides, very useful in biology for detecting and selectively amplifying DNA using PCR. The first base is immobilized on a controlled pore glass column and then a sequence of reagents is washed over. They are first deprotected, then the next base is added and the protecting groups are oxidized. This is repeated for however many nucleotides you have and then the first nucleotide attached to the CPG is cleaved off. This is done in very dedicated machines and for under 10€ you can have a custom sequence synthesized on demand. This is called the phosphoramidite process, named after the reactive group added to the usually chemically not very active nucleotides.

[@ThatChemistOld]

cool!!

[@slavakuznetsov4966]

Nicely organized! You forgot sublimation. I used it a lot for phenolic compounds. Kinda old school, very simple setup though - "cold finger" (apparatus), some dry ice and sand bath - all that was needed.

[@poonampatel1915]

Thankyou for giving us information... your kind gesture 🙌 made my day...

[@gmcenroe]

Another method used quite a bit in medchem reseaerch labs that have purification groups is supercritical fluid chromatography with CO2. An old method probably not used any more was countercurrent distribution, but that probably employed a lot of glass blowing experts!

[@ThatChemistOld]

yeah I've heard about the hype - I would like to see it at some point; I don't think there are any videos of it on youtube

[@realityChemist]

@@ThatChemistOld I've never done anything with supercritical CO2 elegant enough that I'd call it "chromatography," but normal supercritical CO2 extractions are pretty neat! I used to work for a company that makes aerogels that way, it's a cool process: conceptually fairly simple, but some fairly impressive engineering. I know NileRed has a video making aerogels with SC CO2 extraction if you want to see it. I think the main drawback is that it's not exactly *easy* to do, you need to have pretty good control over your chamber pressure.

[@user-ud3if3gv4y]

Not to forget affinity chromatography for the purification of proteins using e.g. an Hexa-Histidine sequence which can be chelated by nickel or Strep-Tag which binds extremely tight to a streptavidin-like protein

[@Petemate69]

Great overview video. For acidity/basicity ion-exchange chromatography is also a great strategy for seperating complex mixtures since amines stick to an acidic resin and can be eluted with ammonia for example. OH- resin can also be used to seperate anomerically locked carbohydrates into a and b anomers which elute at different times.

[@debanjansen1282]

Excellent... I want to ask one doubt. I heated anthranilic acid with Para substituted ( methyl, bromo, fluro, etc) benzaldehyde in presence of ferric chloride and water used as a solvet, aim to prepare 4H-Quinazolinones. TLC depict the formation of new compounds. After the reaction I filtered the precipitate and disolved in ethanol followed by concentrate by heating (~55 to 60 dig C) under reduced pressure. Than put it in fridge. Next morning I found some precipitate and TLC depict presence of complex mixture .... presence of more than 5 to 6 compounds with trailing ... my ethanol was pure..as I compared by TLC. I don't know what is going on... any idea any one... Requesting you to open a Facebook page for ochem synthesis related events and discussions.. thanks again.. appreciate your initiative.

[@ThatChemistOld]

We actually have these sorts of discussions on the Discord - you are welcome to join and discuss it there :D

[@theolambert6269]

Very nice and exhaustiv video! Just a little precision, SDS-PAGE separates molecules by their mass (SDS is used to negatively charge the molecule so that the remaining separation is only based on their mass) to separate by the charge it would just a PAGE, also called Native-PAGE. There is also other gel techniques like BN-PAGE. You could also add the electrofocusing as a separation technique by the charge/acidity/basicity separation. It can be used as the first step of a 2D gel. Best,

[@ThatChemistOld]

Thanks for the info!

[@jeffblack8812]

One additional purification method used only once was a zone refiner. Used it to purify a solid organic compound for a quantitative nmr analysis.

[@formicacidgd]

I just wanted to say that I really enjoy your content

[@ThatChemistOld]

thank you!

[@janvisagie231]

Kugelrohr is super amazingly great, even for less volatile compounds...I remember distilling nearly all of the 2-alkynyl anilines from a large library of substrates during my msc (min 1g scale). I hooked it up to my vacuum pump on 1*10-3 mbar and distilled over super pure product, generally at 110-130 degrees 🤣...only requirement is prior filtration through a plug of silica to remove most of the left over palladium from the Sonogashira coupling. Edit: Aren't ICP samples among one of the most dilute...I surely wouldn't want to be doing prep work on that🤣

[@gradboften]

There’s one arguably very important separation technique you didn’t include - the separation of water from deuterium oxide via electrolysis, leveraging the kinetic isotope effect. We wouldn’t have deuterated NMR solvents if it weren’t for this method!

[@ThatChemistOld]

True

[@joskistemaker9076]

Great material, well explained. Tiny correction: 1:16 that is a Snyder Column, not a Vigreux.

[@Mutantcy1992]

Important correction since a Snyder column is way better separation per length than a Vigreux

[@ThatChemistOld]

True

[@sasas845]

A question: How much correlates the entropy "work" (for seperation, you need to remove entropy from the system) with difficulty of purification in practice?

[@ThatChemistOld]

That is a great question

[@slimp4644]

But what about zwitterionic compounds of extremely similar pKas? I’m assuming buffers?

[@AlexBesogonov]

I think you overlooked mass spectrometry. It has been used in practice for uranium enrichment in WWII, in macroscopic quantities. Using a device called Calutron.

[@ThatChemistOld]

Yeah but centrifuges are more common, yes?

[@AlexBesogonov]

@@ThatChemistOld Sure. But mass-spectrometry can be used to produce isotopes for elements that don't form easily accessible gaseous compounds. If I remember correctly, it was used to produce pure Ca-46 for some heavy element synthesis.

[@Mutantcy1992]

I think there might be a mistake wrt the centrifuge section. Force does not directly cause a change in solubility. Centrifugation exploits difference in density and size of two components. I'm not sure under what circumstances the force could truly cause a change in solubility, ignoring any heating of the solution that may occur. Perhaps the effect is of suspended solid particles which aren't obvious being driven to the bottom of the tube by increased force. I looked for papers on truly solubility-changing centrifugation and didn't find anything. Do you remember the components in your solution that exhibited this effect?

[@ThatChemistOld]

Sedimentation is a thing that gradually happens - I think this is the correct term

[@Mutantcy1992]

@@ThatChemistOld Ah gotcha, thanks for the response

[@fernandolima4113]

Nice video, just contributing to the algorithm with this comment

[@ThatChemistOld]

Thanks lol

[@chemistryofquestionablequa6252]

I remember being very impressed with the ingenuity of some chemistry students in Poland during the 60s or 70s who were working in a clandestine manner to produce heroin. Even though the motivation was morally questionable at best they used commercially available ion exchange resin to extract morphine from a solution of opium and water. Simple and elegant even though highly illegal. That full process is now known as "the Polish heroin method".

[@Finnnicus]

I feel like this kind of video is really uncommon and useful. Thank you! I always wanted to make a video like this. Have you got a link to a good resource on silver columns tho

[@ThatChemistOld]

I posted it in the #Resources channel in the Discord

[@Seorful]

Seeing as you are doing starte to do videos focused on practical tipps and techniques, do you have experience with growing crystals of organic molecules for xray spectroscopy and would you be willing to do a video about techniques and trick to do so?

[@ThatChemistOld]

I do not have expertise running X-ray - I have experience growing crystals tho, but not hardcore like those glovebox crystal growing people

[@KilgoreTroutAsf]

Very informative video as always. Kudos. I dont know whether this falls within your expertise, but would you consider doing a video surveying common techiques for enantioselective synthesis?

[@ThatChemistOld]

I probably will eventually, but not soon

[@pelegsap]

I'm very happy to have found your channel - your content is very high-quality. I haven't done any chemistry in a long while (after my M.Sc. I switched to software development ¯\_(ツ)_/¯) - and it's nice to be reminded of my formal education and learn new stuff.

[@ThatChemistOld]

Software development is still really important!

[@wendys4306]

Best video for education!

[@electricalychalanged4911]

I guess electrolysis is also a method, too. We actually did use dialysis to seperate out different sized carrier molecules for drug administration in one of my former working groups

[@ThatChemistOld]

Please, If there are any papers discussing this I would be really interested in doing a video about it

[@189643478]

Dialysis is not as common as say gel electrophoresis in biochemistry but it definitely is still used mainly to remove salts or other small molecules from solution. To give one example, sugars or reactive aldehydes react with proteins to form advanced glycation end products (AGEs). To study these AGEs, you can prepare them by incubating sugars/methylglyoxal/other reactive aldehydes with protein in a buffer for a few days/weeks. After this you need to get rid of the excess sugar or whatever glycating agent you used and that is done though dialysis. You mentioned the use of magnetic beads to purify DNA but another very common thing is to use beads that are just heavy enough to centrifuge off. Say you want to isolate a protein from solution, then you can add an antibody to the solution that binds to the protein of interest. You can then use beads that are coated in protein A/G, a substance that strongly binds to the “stalk” of the antibody. Hence, now your protein of interest is attached through the antibody to the bead and you can simply centrifuge it off and wash it several times to remove anything non-specific. This is called immunoprecipitation. You can also do a variant of column chromatography in which the column contains a substance that very specifically bind to whatever you want to purify (affinity chromatography). You can fill the column with a solid phase derivatized with substrate analogues to capture enzymes, lectin to bind polysaccharides, protein A/G to bind antibodies, and also various things that bind genetically engineered fusion proteins such as calmodulin and metal ions. In fact, affinity chromatography is one of the most commonly used ways to purify up DNA/RNA from biological samples. BTW, a funny note. In Dutch we have the synonym “scheikunde” for chemistry literally meaning the art of separation.

[@ThatChemistOld]

It is the art of separation lol

[@doublenikesocks]

Ever heard of protein A chromatography? Protein A is a component of cell walls of s. aureus that binds strongly to antibodies. Monoclonal antibodies are commonly purified using protein A chromatography and eluted using low pH solvent

[@ThatChemistOld]

interesting!

[@chrisboyall8602]

I've just finished my undergrad degree, would you say it is normal to feel like I haven't encountered the concepts, reactions and techniques you talk about in certain videos? When I watch videos of yours or other synthetic channels, I feel like I should know a lot more about the reactions that you talk about. Would you say I need to do more further reading and re-read notes from lectures (which probably wouldn't hurt) or am I being too hard on myself? What were you feeling at the end of your undergrad? Not sure if this makes any sense to you or not, but would appreciate your point of view.

[@ThatChemistOld]

What you are experiencing is totally normal and I felt the exact same thing when I was in group meetings as I entered Grad School

[@chrisboyall8602]

@@ThatChemistOld That really is a relief, thank you for the reply :)

[@bilbobaginutopi2284]

For doing at-home chemistry, what is a good organic solvents to use other than DCM? Since it is a halogenated solvent, how would you get rid of it?

[@ThatChemistOld]

depends on the application - isopropanol and toluene are good, you can buy and distill diethyl ether; butane is an option, xylenes are an option

[@jmisc]

I would like to know more about GC-MS being used to quickly determine reaction kinetics. How to not mess up the instrument, etc.

[@ThatChemistOld]

its kinda a pain to do analytically, cause you have do make standards and do response curves

[@coreymariani2486]

Methods of purification teir list please!

[@dangerdackel]

Well if your compound ever makes it to development you will need kilos of material. Here crystallisation is almost the only viable option unless you have polymorphism. Large scale chromatography is time consuming, expensive and uses vast amounts of materials and energy. So crystallisation is the viable way to go if possible.

[@JustinKoenigSilica]

Hey, great video! please stop moving the mouse so much. it makes me nervous haha also you didn't mention DCVC :(

[@Seorful]

"A column a day, keeps the professor away"

[@ThatChemistOld]

I say “a column a day keeps stagnation at bay”

[@deltasquared7777]

Many time successive methods of isolation/purification are most effective and economical.

[@transkryption]

triple distilled vodka you say...

[@deltasquared7777]

@@transkryption no, successive methods--like to make white lightening, first you gotta filter or drain the corn squeezin's before running the supernatant through the still

[@feelinghealingfrequences7179]

forgot to mention the religious type of purification...pray it pure

[@koukouzee2923]

early gang :)

[@ThatChemistOld]

Best gang

[@jmisc]

HPLC is high pressure liquid chromatography….

[@ThatChemistOld]

high price

[@dlvivlviv]

High Performance Liquid Chromatography.

[@jmisc]

@@dlvivlviv High Pikachu Liquid Chromatography. Please don't take me seriously.. I was kidding, lol.

[@dlvivlviv]

@@jmisc Hi Pie Letter Conference

[@mattflores8911]

Face reveal?

[@ThatChemistOld]

Was there one?

[@ThatChemistOld]

I’ve showed pictures of myself on the discord

[@GigsTaggart]

I really like your videos but the constant twitchy mouse drives me nuts. Please stop that.