Dear colleagues,
Our lab is testing a new freeze-dryer's performance, but we are not sure if we've been using it correctly.
It's a benchtop type, with a transparent drum manifold, operable at -74oC and pressure ~20 mTorr.
We've tested water and 200 mM ammonium bicarbonate, respectively, in 1ml (in a microfuge tube) and 10ml format (in a falcon tube). Before the freeze-dry, we've pre-frozen the test samples (i.e. water and ABC) overnight in -80C.
Nevertheless, the result seems not very encouraging:
1a. All samples took a long time to dry (i.e. half a day to overnight), even for the 1ml water or ABC.
1b. For the 10ml ones, the water and ABC have already melted in the midway (except their cores); guess this may affect the sublimation process?
2. Before freeze-dry, we've wrapped the tube tightly in parafilm (with a few tiny holes punched through the film).
When we apply the vacuum, yet, the frozen water or ABC were just 'sucked up', only blocked by the parafilm --> this makes them hard to freeze-dry on the tube wall.
3. After freeze-dry, we also reckon that some freeze-dried powder (e.g. ABC) have spilled out, concerning the risk of contamination.
We would appreciate it a lot if you could please advise and share your experience.
Would you also have any idea - on the relationship between freeze-drying time vs. sample volume and type?
P.S. Is water or ABC too 'aquatic' to freeze-dry?
Thank you very much.
Hi Kay,
we had the same issues for our freeze-dryer. If you put 10 ml in a 15 ml falcon tube, the volume/surface area ratio is worse as a worse can be. I strongly recommend you to not exceed 2 ml when using 10 ml Falcon tubes (Btw: Falcon tube is the brand name, use 'microcentrifuge' or 'centrifuge' tube for Eppendorf vials and Falcon tubes for publication ;). Better go for 50 ml tubes and add max. 5-7.5 ml to it.
Do not freeze the tubes by just freezing them: Use a dry ice/ethanol bath and move the tubes just as you would do for titration in a rotary movement of the hand so that the surface area increases and the sample disperses throughout the wall. I attached a hand drawing of what I mean. The best is clearly to use a round-bottom flask where the sample solution is frozen at the inner wall during rotation while freezing (the 'normal' way how the freeze-dryer is used in the lab).
Do not use parafilm with small holes, the sublimation is problematic in that case and we had melted samples, too. We try to avoid any parafilm or aluminum foil on top of the samples, which is usually no problem if the volume added is below 30-50% of the total volume of the vial or flask and the sample is frozen as thin layer throughout the wall. If you like to freeze-dry 1 ml sample volumes, take two 2 mL microcentrifuge tubes - one is used to put the sample solution in, the other one is used to cut off the cap. Use a 10G needle (or comparable - resulting hole ~ 1 mm in diameter) to punch 3 holes in the cap (direction: from the inside out not to have residual PP sticking into the sample later on). Close the sample tubes with the punched cap, the intact cap can be later used to close the vial and for labelling after freeze-dry. Freeze the tubes by help of an aluminum rack, placed on dry ice and freeze-dry with the rack, this ensures a low increase in temperature. It takes at least over night to get the samples dry. For bigger sample volumes, we use 50 ml steriflip tubes (I don't know if available as 10 ml versions). We use the steriflip filter to close the vial. The semipermeable membrane is fine for freeze-drying, do not vent the system too quick not to destroy the membrane. With this, you can prepare sterile samples if needed.
Another thing regarding your freeze-dryer: Do not move the rubber valves at the plastic manifold plate while it is under vacuum! We had such a chamber as well and the weak point of this are the plastic joints welded to the chamber, the connection breaks very quickly and you do not reach sufficient vacuum. You can ask the company for a glass chamber with stainless steel (manifold) plate which is much more robust.
I used to Freeze-dry samples in over 30mL of volume. We just experienced meting in few samples which we had to freeze again and put back into the lyophilizator.
As Marcus mentioned before, although -80ºC should typically work for small volumes, if you use liquid Nitrogen or Dry ice+methanol right before lyophilization will perform much better avoiding melting of the samples.
I do not think there is a linear realtionship between volume and time of lyophilization, it will depends directly on the surface in contact with the " vaccum atmosphere". As larger is the surface as faster samples will be sublimed. For us, typically took around 24hrs to sublime around 30mL.
Also try to do not cover the frozen samples, never paraphilm! For that purposes there is breathable paper that works pretty well.
Even so, you will continue experiencing that some samples will probably melt... so just freeze them again and put back into the instrument.
Dear Marcus and Albert
Thank you very much for your very detailed advises : )
It definitely helps a lot as we are going to start an experiment soon, and glad to know that the instrument is basically alright.
Surely, there are a lot more that we are to learn. Appreciate a lot for your time.
All the best to your research and work!
Hi Kay,
thank you for the message, please keep us updated whether it works better or not. I found a nice paper regarding the theory of freeze-drying process (see link attached) which also deals with the "rate of evaporation in grams per square centimeter". The author states (amongst other things) that "At the lower temperatures required for solutions the vapor pressure is also lower, being 0.29 Torr at -30 °C, and the rate of sublimation is reduced in proportion."
This means, the colder the sample, the slower the sublimation process.
The second link provides nice calculations with real numbers from where you can guess how your samples behave.
Marcus
http://onlinelibrary.wiley.com/doi/10.1111/j.1749-6632.1960.tb49988.x/pdf
http://freezedrying.com/freeze-dryers/general-principles-of-freeze-drying/
Hi Kay,
there you go :)
I added another two article links, hope it works this time.
Marcus
http://onlinelibrary.wiley.com/doi/10.1111/j.1749-6632.1960.tb49988.x/full
http://onlinelibrary.wiley.com/doi/10.1111/j.1749-6632.1965.tb45412.x/full
http://onlinelibrary.wiley.com/doi/10.1111/j.1749-6632.1960.tb49987.x/full
Really? I recognized that I can not access them...
I added some RG Links which should work for sure.
Article The theory and practice of freeze-drying
Article Factors affecting the mechanism of transformation of ice int...
Article Principles of freeze-drying
Article The Principles of Freeze-Drying
Hi Kay,
Everyone has raised lots of excellent points.
Note that parafilm is a potential contaminant. Definitely avoid parafilm when freeze-drying and any other application where it can come into contact with samples. If you are worried about sample loss do as previously advised and use a lid with (clean) holes poked in it. However, as mentioned you really want to maximise the surface area rather than minimise. Be sure to run plenty of blanks (empty tubes) because freeze dryers have been known to lead to contamination
Hi Kay,
your freeze-dryer may already perform well, but there are some important points to mention when you use the unit more frequently.
Be careful when using organic solvent residuals during freeze-drying. You should not only consider your sample, but also the delicate instrument periphery.
The freeze-dryer is equipped with a pressure sensor, which contains a tungsten wire (I don't know exactly how this thing works, but this is needed). Anyway, if you have organic solvents in your sample and you put it on the freeze dryer, this will result in the formation of tungsten carbide - very brittle! Sooner or later the pressure sensor will brake, depends on the amount and duration of treatment with organic solvent vapors. This is the reason why freeze-dryers should be kept under vacuum all the time, even if there is no sample attached for drying. Only for ice removal, the freeze dryer needs to be opened, but afterwards put under vacuum again as soon as possible. This saves the tungsten wire from organic vapours e.g. from the lab. NEVER clean the freeze-dryer with ethanol or related! Organic liquids are even more dangerous to the pressure sensor than the vapor. Use a heat gun set to 60-80°C to dry the internal part of the freeze-dryer instead.
It is always best to remove the organic solvents as effectively as possible to avoid that. There are "chemical resitant" sensors available (cost approx. 600 €, the "normal" ones about 400€ as far as I remember). But still, they can also brake. If you put e.g. 50% AcN solution on the freeze-dryer, this will also dissolve in the pump oil with time and this is not good ans perhaps it can result in explosive hot gases coming from the pump outlet. For this, you absolutely need a freeze-drying unit which freezes the internal cooling loops to -80 °C at least. This ensures that the organic solvents condesate after sublimation. But still, AcN is very volatile and decreases the vacuum which extends the freeze-dry time.
I personally would absolutely not recommend to go for a high AcN concentration for samples you freeze dry frequently, always do an rotary evaporation step before. AcN should be below 0.5 vol-% at least! If you go for a high AcN fraction, the sample will be dry for sure, but the freeze dry unit will brake a lot earlier than you want to.
Marcus
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