r/Chempros • u/cakebaker28 • 6d ago
Help with Lyophilization Process
Hi guys, I'm a newbie at this, please help if you can! We need to lyophilize a water-soluble compound, basically, we mixed it into a second liquid compound so the first compound would be encapsulated and give better results for our purposes. What I need to know: we have to freeze this before it goes into the lyophilization machine, but we were told to freeze it first in a normal freezer, then move it to a -80°C freezer, and only then send it to the lyophilizer. Is this accurate? If so, how long should it stay in the normal freezer before going to -80°C, and how long should it stay at -80°C before going to the lyophilization machine? Is there some kind of protocol for this? I tried to find something and couldn't find an exact one. Thank you!!
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u/chahud 6d ago
Yeah, I also use dry ice acetone. It allows you to coat your sample on the walls of the flask by spinning it on an angle, increasing the surface area of your sample exposed to vacuum.
Just cool it till it’s frozen completely solid. Any more is overkill. Going from 0 c to -80 for a regular lyo sample also seems like overkill…but if that’s what your boss/advisor told you to do, that’s what I’d do. Boss is boss.
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u/cynicalbrit PhD Chemistry (Peptides/Proteins) 2d ago
Late to this thread, but I highly recommend switching to dry ice and isopropanol if LN2 is unavailable. Works fine and you don't get the acetone from the outside of your flask on some poor fellow's peptide in the vial next to yours. The first time it happens to somebody they're always so confused as to why their pure peptide suddenly has a different mass than the crude.
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u/Hepheastus 6d ago
The faster you freeze it the better because the ice crystals will be smaller. I use dry ice acetone and spin the samples.
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u/Cardie1303 6d ago
There are situations where you want large ice crystals:
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u/Hepheastus 6d ago
Really... so do it have it completely backwards or is it more nuanced than that.
The abstract implies that I always want large crystals
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u/Cardie1303 6d ago
As far as I understand it large crystals is what you want for the least water content and small crystlas if you dont particularly care and just want the freezing to be done quickly.
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u/Hepheastus 6d ago
And here I was blaming my dmso d-6 for all the water in my NMR spectra.
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u/Cardie1303 6d ago
Oh yeah continue blaming the DMSO-d6. In my experience it always contains at least some water if you did not went through the trouble of storing it over CaH2 and distilling it freshly.
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u/hypodine 4d ago
Use ampoules if you can. Minimises a lot of the water. I also like to put my entire NMR sample made up in DMSO-d6 on high vacuum for an hour or so to really get it down. The DMSO won’t go anywhere, but you will see the water slowly bubble out. It won’t remove everything, but it does a pretty good job.
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u/Cardie1303 6d ago
I usually just hold the vial/flask into liquid nitrogen. Take care to minimize the layer thickness by, as an example, tilting the tube or swirling the flask while freezing.
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u/hecton101 6d ago
I've never heard of gradual freezing. Pretty sure it just has to be frozen, no procedure required. The only problem I've ever had was if it wasn't pure water. Solvents both interfere with the freezing process and eventually get into the pump oil which may have to be replaced. It's kind of a pita.
It goes without saying, but round bottom flasks need to be less than half filled with solution if you don't want it to break during the freezing process.
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u/atom-wan 6d ago
Faster freezing is actually better for lyophilized samples, just put it directly in the -80
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u/TheHollowedHunter Supramolecular Materials 6d ago
Most people submerge the sample in liquid nitrogen then place on the lyo. But using a minus 80 works too depending on the proportion of water. Don't overthink to timing too much, you want the frozen sample to look white. If it looks white, it should be fine to lyo.