I need to lyophilize Fungi that Has been Ethanol Precipitated and dialyzed in water. I am having problems, because it unfreezes (after being frozen in liquid nitrogen) after I connected to the lyophilizer. The lyophilizer is set at 0.030 mBar and -38C. Is this temperature too high? Can anyone give me some ideas how to set the lyophilizer? Thank you
It could be that there is some EtOH or other buffer / formulation elements that are suppressing the freezing point of the sample. It could also be some settings in the system, or it could be a combination of these things.
What is the freezing point of your sample? Simple tests, can you freeze it in a regular lab freezer at -20°C or a ULT at -80°C? Components that can cause issues with freezing are high salt concentrations of the presence of sugars .... or left behind EtOH. It is almost impossible to separate ALL EtOH and water through physical process as the azeotrope - hence I suspect the presence of EtOH is the issue. The EtOH suppresses the freezing point.
At 0.03 milliBar the (boiling = ) sublimation temperature of pure water is -60°C. If the system can continue to hold that pressure during then entire process, then OK.
When you say that the "temperature" of the lyophilizer is set to -38°C - is this is the temperaure of the shelf on which you put your samples, or the temperature of the condenser where the ice collects?
If the temperature of the condenser is ABOVE the sublimation temperture (-38 > -60) then the water vapour will not get trapped in the condenser, and go to the pump, choking the pump and causing the pressure to rise above the set point.
If -38°C is the temperature of the shelf, this should be OK (heat energy comes in at the shelf to help the ice sublime and drive the process, vacuum creates the conditions where it can sublime at low temperature). It may be too agressive, if it is you will likely see one of two things - either you will see the system pressure rise as the process is too fast for the condenser to catch the water vapor - see above note on why the pressure rises, or the product will be pushed out of the sample tubes, which you do not describe happening.
Rob Darrington Thank you for your help. I think you are right. the problem is leftover EtOH.
I can freeze my samples at -20C.
The system I am tying to use is a FreeZone -50C dryer. Doesn't have shelfs. -38C is the temperature of the collector.
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