TITLE: Cryomiller SOP
AUTHORS: Lorenzo Lucherini

[DESCRIPTION]
Standard of procedure for cryomiller Retsch at the Soft Materials Laboratory (EPFL)

[STEPS]
SECTION: Refill the N2 dewar
4. Use two wrenches to unscrew the N2 pipe from the dewar. Firmly push downwards both wrenches at the same time.
 

SECTION: Refill the N2 dewar
5. Place the dewar in the lift and block the wheels. Pull the red strap in front of the door of the lift and send the lift to floor 0. 

SECTION: Refill the N2 dewar
6. Hand-tight the butterfly screw of the N2 line to the inlet valve of the dewar.
 
 

7. Open the inlet and the outlet valve of the dewar.
 

SECTION: Before milling - setup the cryomiller
14. Screw the N2 line to the dewar. Hand-tight the N2 line by using two wrenches. Do not over-tight the bolts.
 

SECTION: Before milling - setup the cryomiller
15. Open the inlet valve of the dewar.
SECTION: Know your instrument
1.  

SECTION: Before milling - setup the cryomiller
17. Switch on the cryomiller.
SECTION: Setup the cryomilling program
22. Have you already used the cryomiller?
SECTION: Setup the cryomiller
23. If you already have an established milling protocol, you can save the parameters in a free memory bank (for example P3, in the picture).
 

SECTION: Prepare the sample
18. The milling jar volume is 50 mL. The sample to be milled should occupy maximum about 1/3 of the total jar volume. For efficient milling, introduce 10 mL to 20 mL of sample in the milling jar.
SECTION: Prepare the sample
19. If your material can be prepared in smaller pieces, cut it or break it to facilitate the milling process.
Example: Materials like paper, plastic films, and hydrogels can be cut into small pieces by scissors.

SECTION: Choose the right milling ball
21. Tip: use a milling ball whose diameter is about 3x the size of the biggest particle composing your sample.

8. Set the main switch (red) to 1. Then, press the green button. The green button lights up and the line of the N2 reservoir is now open.
 

9. Turn the tap counterclockwise to start filling up the dewar. 
 

10. Monitor the dewar as it get refilled. The dewar is full when you see some liquid nitrogen coming out of the outlet valve of the dewar (see video).
 

12. Put on your cryogenic gloves. Close the tap. Then, close the inlet and the outlet valve of the dewar.
13. Unscrew the butterfly screw from the dewar. 
SECTION: Before milling - setup the cryomiller
16. Build up pressure inside the dewar by slowly opening the pressure build-up valve. 

Let the pressure rise to 1 bar, then close the pressure build-up valve. Wait for the pressure to drop below 0.5 bar (~ 10 seconds). Then, slowly open again the pressure build-up valve until you reach 1 bar. Repeat this process for 3 times until the pressure stabilizes around 1 bar.

Note: reaching a stable pressure value may require some hours, due to the pressure feedback system of the dewar. Monitor the internal pressure of the dewar during your first cryomilling cycle.

Tip: refill the dewar at least 2 h before using the cryomiller, to allow pressure to stabilize. 
 

SECTION: Filling the milling jar
20. Tip: 1/3 of the volume filled with the sample, 1/3 of milling balls, 1/3 of free space. 
SECTION: Setup the cryomilling program
23. To run a quick and dirty test, use the "--" program: this is a blank memory slot that allows you to try a quick test without saving the milling parameters.
 

11. When the dewar is full, set the main switch (red) to 0. This step closes the electronic valve between the main reservoir and the filling line.
SECTION: Refill the N2 dewar
3. Tip: Refill the dewar at least 2 h before using the cryomiller. Ideally, plan to refill the dewar the day before using the cryomiller.
Refilling in advance allows the pressure inside the dewar to stabilize, avoiding issue during the cryomilling cycles
SECTION: Know your instrument
2. 
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