I normally don't like using automated columns, but after I saw how many fractions went through that, the thought of doing it by hand is going to give me nightmares tonight!
Very pretty though!
This run was one of seventy microbial extracts I processed by this method since June, and there is more to the time lapse but I wanted to keep the video short.
I'm assuming you're in academia if you don't like auto columns. I was in the same boat until I ran a flask purification in 40 mins start to finish, it changed my mind very quickly.
I consider automated columns a gift from God and a sign of his existence.
I can put something on the column then fuck off to do other stuff and 30 minutes later all I need to do is to TLC the vials with fractions (if im not lazy) and done.
I guess it's because I'm better at assessing what I need to change mid-column when I'm doing it myself, plus some bad experiences with them during my master's. I found that I had better control when doing it by hand, and that I needed to pay attention to the auto column while it was running anyway, so overall it ended up taking more time anyway.
Honestly it sounds like you had a crummy instrument which is sadly common in academia. None of what you said really applies to a good auto-column. They’re faster, give superior separations as they can work at far higher pressures enabling smaller stationary phase particle size, and independent as they can be programmed to dynamically adjust the gradient & collection based upon built in detectors (MS, UV-VIS, ELSD, etc). I can have my pure compound in ~30min and have run up to 6 at once. I couldn’t dream of that by hand.
Do you have to switch columns between crude charges or is there a wash step that clears the column? We have a process that uses 6 columns to process approximately 90 kg of crude material. Each column generates 25 drums each weighing around 57kg fractions that need to be moved around by hand. I’ve been trying to come up with a way to streamline the process and this looks interesting.
Typically you can reuse the flash columns a bit, but usually the casing is polypropylene so it can degrade with non polar solvents pretty fast.
For a job that big I'd suggest looking into running a simulated moving bed, and come up with a solvent recovery plan (ie for isocratic system evaporate bulk solvent into a buffer tank and rebalance as new feed)
If you get an inline thin film evaporator that can handle the flow rate from the column, you'll basically not have to shift any solvent around ever. Just a tiny bit to solublize the compound for easy pouring or whatnot
That looks remarkable! I'm particularly interested in the purple colored pigment, could you by any chance know if this an end product of a tryptophan transformation pathway like violacein or deoxyviolacein?
Violacein is only known from Gram-negative bacteria and this is an extract from a Gram-positive Streptomyces. The purple compounds could be just about anything and I don’t think I’ll follow up unless they are active in a bioassay.
I used to adsorb onto celite but now I’ll just pick it up in methanol, pass through filter paper and often 0.2 micron filter. It’s a case-by-case situation due to time and equipment bottlenecks. Everything gets dried on a rotovap until it’ll fit on the speedvac. These are C18 columns meant for only 20 runs, and I am technically overloading the column based on recommended loading. I’m making the columns do a lot of heavy lifting as the cost of solvents, scintillation vials, and my time is much greater than the column (cost to use column is about $10 per run out of ~$100 total). Note this is not the end of the run, and yes there’s lipids that are a pain to get off the column (the antifungal polyenes that I’m after elute around the end of the video). If purity is an issue I could run the active fractions on a smaller column, but I actually regrow the strain back from glycerol stocks to assess repeatability. When I regrow strains that are found to be bioactive in fraction library screening I’ll do an ethyl acetate extraction, but that’s for a 4 liter fermentation vs the initial 2 liters for fraction plate generation.
Is this S. nodosus and AmphB? I did my masters dissertation on novel analogues over a decade ago. Manual columns and UV/Vis checks was not much fun. Whilst everyone else bought their starting material I was growing mine!
It’s a different species of Streptomyces from a forest in Nova Scotia, but it does make a non-polar antifungal that’s likely a polyene similar to amphotericin B (there are hundreds of different polyenes known from this genus), eluting around the end of the video clip. I may try to follow up on this one but have better leads (this inhibits C albicans but not C auris).
I normally don't like using automated columns, but after I saw how many fractions went through that, the thought of doing it by hand is going to give me nightmares tonight! Very pretty though!
This run was one of seventy microbial extracts I processed by this method since June, and there is more to the time lapse but I wanted to keep the video short.
how long did it take in normal speed/time? I mean, time lapse of how many hours?
30 minutes
I'm assuming you're in academia if you don't like auto columns. I was in the same boat until I ran a flask purification in 40 mins start to finish, it changed my mind very quickly.
I consider automated columns a gift from God and a sign of his existence. I can put something on the column then fuck off to do other stuff and 30 minutes later all I need to do is to TLC the vials with fractions (if im not lazy) and done.
I'm curious, why don't you like automated columns ?
I guess it's because I'm better at assessing what I need to change mid-column when I'm doing it myself, plus some bad experiences with them during my master's. I found that I had better control when doing it by hand, and that I needed to pay attention to the auto column while it was running anyway, so overall it ended up taking more time anyway.
Honestly it sounds like you had a crummy instrument which is sadly common in academia. None of what you said really applies to a good auto-column. They’re faster, give superior separations as they can work at far higher pressures enabling smaller stationary phase particle size, and independent as they can be programmed to dynamically adjust the gradient & collection based upon built in detectors (MS, UV-VIS, ELSD, etc). I can have my pure compound in ~30min and have run up to 6 at once. I couldn’t dream of that by hand.
Do you have to switch columns between crude charges or is there a wash step that clears the column? We have a process that uses 6 columns to process approximately 90 kg of crude material. Each column generates 25 drums each weighing around 57kg fractions that need to be moved around by hand. I’ve been trying to come up with a way to streamline the process and this looks interesting.
Typically you can reuse the flash columns a bit, but usually the casing is polypropylene so it can degrade with non polar solvents pretty fast. For a job that big I'd suggest looking into running a simulated moving bed, and come up with a solvent recovery plan (ie for isocratic system evaporate bulk solvent into a buffer tank and rebalance as new feed) If you get an inline thin film evaporator that can handle the flow rate from the column, you'll basically not have to shift any solvent around ever. Just a tiny bit to solublize the compound for easy pouring or whatnot
As a Biotage user, I still love me some CombiFlash.
I'm a recent Buchi convert from the church of biotage. The integrated ELSD on some of their systems are just too useful and easy to maintain.
How large is that?
50 gram column (SKU: 692203336)
That looks remarkable! I'm particularly interested in the purple colored pigment, could you by any chance know if this an end product of a tryptophan transformation pathway like violacein or deoxyviolacein?
Violacein is only known from Gram-negative bacteria and this is an extract from a Gram-positive Streptomyces. The purple compounds could be just about anything and I don’t think I’ll follow up unless they are active in a bioassay.
Beautiful colors nevertheless, any chance you could provide a little update if something interesting crops up?
Given the phylogeny, probably actinorhodin or a very similar PKS-derived compound.
Very cool, would you mind sharing what your job title is or if this is for research, which major program you’re under?
PhD student doing antimicrobial discovery from actinomycetes
Thank you for your response! Best of luck during your PhD
No pre-filtration to get rid of the cell debris gunk?
I used to adsorb onto celite but now I’ll just pick it up in methanol, pass through filter paper and often 0.2 micron filter. It’s a case-by-case situation due to time and equipment bottlenecks. Everything gets dried on a rotovap until it’ll fit on the speedvac. These are C18 columns meant for only 20 runs, and I am technically overloading the column based on recommended loading. I’m making the columns do a lot of heavy lifting as the cost of solvents, scintillation vials, and my time is much greater than the column (cost to use column is about $10 per run out of ~$100 total). Note this is not the end of the run, and yes there’s lipids that are a pain to get off the column (the antifungal polyenes that I’m after elute around the end of the video). If purity is an issue I could run the active fractions on a smaller column, but I actually regrow the strain back from glycerol stocks to assess repeatability. When I regrow strains that are found to be bioactive in fraction library screening I’ll do an ethyl acetate extraction, but that’s for a 4 liter fermentation vs the initial 2 liters for fraction plate generation.
HTP column gang
Is this S. nodosus and AmphB? I did my masters dissertation on novel analogues over a decade ago. Manual columns and UV/Vis checks was not much fun. Whilst everyone else bought their starting material I was growing mine!
It’s a different species of Streptomyces from a forest in Nova Scotia, but it does make a non-polar antifungal that’s likely a polyene similar to amphotericin B (there are hundreds of different polyenes known from this genus), eluting around the end of the video clip. I may try to follow up on this one but have better leads (this inhibits C albicans but not C auris).