NIST Special Publication 1196

Standard Operation Procedures (SOPs) for the 

Palla VM-KT Vibrating Cryomill: Production of 

Fresh Frozen Standard Reference and Control 

Materials (SRMs/CMs) at the NIST Reference 

Material Production Facility

Amanda J. Moors

Rebecca S. Pugh

Jody R. Evans

Jennifer M. Ness

Debra L. Ellisor

Melannie J. Bachman

This publication is available free of charge at:

http://dx.doi.org/10.6028/NIST.SP.1196
NIST Special Publication 1196

Standard Operation Procedures (SOPs) for the 

Palla VM-KT Vibrating Cryomill: Production of 

Fresh Frozen Standard Reference and Control 

Materials (SRMs/CMs) at the NIST Reference 

Material Production Facility 

Amanda J. Moors

Rebecca S. Pugh

Jody R. Evans

Jennifer M. Ness

Debra L. Ellisor

Melannie J. Bachman

Chemical Sciences Division

Material Measurement Laboratory

This publication is available free of charge at: 

http://dx.doi.org/10.6028/NIST.SP.1196

July 2016

U.S. Department of Commerce 

Penny Pritzker, Secretary

National Institute of Standards and Technology 

Willie May, Under Secretary of Commerce for Standards and Technology and Director 
Certain commercial entities, equipment, or materials may be identified in this

document in order to describe an experimental procedure or concept adequately.

Such identification is not intended to imply recommendation or endorsement by the

National Institute of Standards and Technology, nor is it intended to imply that the

entities, materials, or equipment are necessarily the best available for the purpose. 

National Institute of Standards and Technology Special Publication 1196

Natl. Inst. Stand. Technol. Spec. Publ. 1196, 57 pages (July 2016) 

CODEN: NSPUE2

This publication is available free of charge from: 

http://dx.doi.org/10.6028/NIST.SP.1196
i

Contents

List of Figures................................................................................................................................iii

List of Tables .................................................................................................................................. v

Disclaimer...................................................................................................................................... vi

Standard Reference and Control Material Production .................................................................... 1

Safety .............................................................................................................................................. 1

Oxygen Sensors........................................................................................................................... 2

Personal Protective Equipment ................................................................................................... 2

Safety Data Sheets....................................................................................................................... 2

Palla VM-KT Vibrating Cryomill................................................................................................... 2

Compressed Air Smashing Device ................................................................................................. 5

LN2 Piping System.......................................................................................................................... 6

Cryomill Assembly ......................................................................................................................... 7

Outlet Set-Up............................................................................................................................... 9

Inlet Setup ................................................................................................................................. 14

Cryomilling Assembly with Vibrating Trough Conveyor..................................................... 16

Cryomilling Assembly without Vibrating Trough Conveyor................................................ 20

Preparing Material and Supplies................................................................................................... 21

Palla Cryomill Operation .............................................................................................................. 21

Cryohomogenizing Material ......................................................................................................... 24

Cryomilling with the Vibrating Trough Conveyor ................................................................... 24

Cryomilling without the Vibrating Trough Conveyor .............................................................. 27

Particle Size Measurement............................................................................................................ 28

Disassembling, Thawing, and Clean-up Procedures..................................................................... 29

Labconco Steam Scrubber......................................................................................................... 30

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Cryomilling Overview .................................................................................................................. 33

References..................................................................................................................................... 34

Appendix A................................................................................................................................... 36

Hollings Marine Laboratory Marine ESB Job Hazard Analysis............................................... 37

Appendix B................................................................................................................................... 39

Deconahol Safety Data Sheet.................................................................................................... 46

LabSolutions Powder Detergent Safety Data Sheet.................................................................. 53

LabSolutions Neutralizing Acid Rinse Safety Data Sheet........................................................ 55

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List of Figures

Figure 1. Palla VM-KT Vibrating Cryomill ................................................................................... 3

Figure 2. Palla VM-KT Vibrating Cryomill Electrical Control Panel............................................ 3

Figure 3. Titanium Rods contained inside Material Grinding Chamber ........................................ 4

Figure 4. Teflon Bellow.................................................................................................................. 4

Figure 5. Palla VM-KT Cryomill Material Grinding Chamber and Motor................................... 5

Figure 6. View Inside the Inlet Socket............................................................................................ 5

Figure 7. Compressed Air Defragmenting Device ......................................................................... 6

Figure 8. LN2 Bulk Tank ................................................................................................................ 7

Figure 9. Palla Cryomill Schematic ................................................................................................ 8

Figure 10. Palla Cryomill Large and Small Protective Covers..................................................... 10

Figure 11. Outlet Head.................................................................................................................. 11

Figure 12. Outlet Tube and Temperature Pick-up ........................................................................ 11

Figure 13. Outlet Bellow Upper Metal Band................................................................................ 12

Figure 14. Outlet Bellow Lower Metal Band ............................................................................... 13

Figure 15. Outlet Socket with LN2 Exhaust Hose ........................................................................ 13

Figure 16. Material Chamber End Cap......................................................................................... 14

Figure 17. Inlet Bellow ................................................................................................................. 15

Figure 18. Inlet Socket Attached to Inlet Tube............................................................................. 15

Figure 19. Inlet Socket with Inlet Socket Head Cover ................................................................. 16

Figure 20. Vibrating Trough Conveyor ........................................................................................ 16

Figure 21. Conveyor Support and Mount Platform ...................................................................... 17

Figure 22. Mount Bellow Attachment .......................................................................................... 18

Figure 23. Flexible LN2 Supply Line Attached to Filling Socket................................................. 19

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Figure 24. Cryomill Safety Button................................................................................................ 19

Figure 25. Thyristor Control......................................................................................................... 20

Figure 26. Flexible LN2 Supply Line Attached to Inlet Socket.................................................... 21

Figure 27. LN2 Control Valve Box ............................................................................................... 22

Figure 28. LN2 House Supply Valve ............................................................................................ 22

Figure 29. Cryomill Electrical Control Panel ............................................................................... 23

Figure 30. Cryomilling with the Vibrating Trough Conveyor...................................................... 25

Figure 31. Cryohomogenized Material Flowing from the Outlet Head........................................ 26

Figure 32. Cryomilling material with the Vibrating Trough Conveyor........................................ 27

Figure 33. Cryomilling without the Vibrating Trough Conveyor................................................. 28

Figure 34. Labconco Steam Scrubber........................................................................................... 30

Figure 35. Labconco Steam Scrubber Distilled and Tap Water Lines ......................................... 31

Figure 36. HML Distilled and Tap Water Lines........................................................................... 31

Figure 37. Labconco Steam Scrubber Detergent Cup and Neutralizing Solutions Port............... 32

Figure 38. Labconco Steam Scrubber Display Screen.................................................................. 32

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List of Tables

Table 1. Palla MT-KM Cryomill Schematic Part Numbers ........................................................... 9

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vi

Disclaimer

Certain commercial equipment or instruments are identified in this paper to specify adequately the 

experimental procedures. Such identification does not imply recommendations or endorsement by 

the National Institute of Standards and Technology nor does it imply that the equipment or 

instruments are the best available for the purpose.

______________________________________________________________________________________________________ 

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1

Standard Reference and Control Material Production at the

NIST Marine Environmental Specimen Bank

The National Institute of Standards and Technology (NIST) maintains the Marine Environmental 

Specimen Bank (Marine ESB) at the Hollings Marine Laboratory (HML) in Charleston, South 

Carolina. The Marine ESB is an environmental specimen bank that cryogenically archives

specimens (e.g., marine sediments, fish tissues, mussels, oysters, marine mammal tissues, and bird 

eggs and feathers) collected as part of several monitoring and research programs supported 

primarily by other US government agencies, including, the National Oceanic and Atmospheric 

Administration (NOAA), US Geological Survey (USGS), and US Fish and Wildlife Service 

(USFWS). The Marine ESB is specifically designed to store environmental specimens over long 

periods of time (50 to100 years). A systematic well-designed specimen bank program, is not only 

a valuable component of real-time monitoring and basic research, but it also enables future 

investigators to extend their research into the past (hind casting) and provides for future 

verification of analytical results (quality assurance).

The Marine ESB facility consists of a 73.2 m2(788 sq. ft.) ISO certified Class 5 Cleanroom, a 79.4 

m2(855 sq. ft.) ISO certified Class 7 Reference Material (RM) Production Facility, a 146.3 m2

(1,575 sq. ft.) ISO certified Class 7 Cryogenic Storage Facility, and 46.8 m2(504 sq. ft.) of support 

facilities consisting of ante rooms and office space. Specimen bank and cleanroom protocols for 

projects of the Marine ESB are described in NISTIR 7389 (Pugh et al, 2007). 

In addition to maintaining the Marine ESB in Charleston, NIST also develops and certifies 

Standard Reference Materials (SRMs) and Control Materials (CMs) to support accurate and 

compatible measurements for industry. SRMs and CMs are valuable in industry settings to 

establish instrument calibrations to ensure quality assurance parameters, to validate the accuracy 

of specific measurements, and to support the discovery and development of new measurement 

procedures. The Reference Material Production Facility, housed within the Marine ESB, provides 

the equipment and capabilities to produce SRMs/CMs for certification. These capabilities include

developing lyophilized (freeze-dried) materials using a Millrock Quanta Series Freeze Dryer 

PC/PLC, and cryogenic materials (such as a fresh, frozen powder) using a Palla VM-KT Vibrating 

Cryomill (cryomill). The Standard Operating Procedures (SOPs) for the cryomill are described 

below.

Safety

The Marine ESB has been equipped with several safety features to ensure safe laboratory operating 

procedures. The HML where the Marine ESB is located is a NOAA facility; therefore, all 

personnel must abide by NOAA safety rules and policies. Because NIST and NOAA are both 

federal agencies under the Department of Commerce, many safety policies and procedures are 

similar. The HML Safety Committee has written a Laboratory Chemical Hygiene Plan and 

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Emergency Preparedness Plan that are strictly enforced throughout the entire laboratory. A Job 

Hazard Analysis has also been completed for cryogenic homogenization using the cryomill and 

can be found in Appendix A. The Job Hazard Analysis includes information on work activities 

completed in the Marine ESB and what Personal Protective Equipment (PPE) are required to be 

worn during specific laboratory activities. All personnel who work in the laboratory must read, 

understand, and sign the HML Job Hazard Analysis before conducting work in the laboratory. 

Additionally, the cryogenic processing using the cryomill has been approved through the NIST 

Hazard Review System and can be found at https://mmlweb.nist.gov/safety/.

Oxygen Sensors

The Marine ESB facility (Class 5 Cleanroom, Class 7 RM Production Facility, and Class 7 

Cryogenic Storage Facility) are equipped with oxygen (O2) sensors that are monitored by the 

REES Centron Monitoring System. In the event that O2 is depleted in any of the areas of the Marine 

ESB, an audible alarm will sound, as well as flashing alarm lights, warning individuals of O2 

depletion. 

Personal Protective Equipment

There are two ISO Class 6 ante-rooms (Rooms C102 and D103) that provide access to the Class 5 

Cleanroom and the Class 7 Reference Material (RM) Production Facility through the Freezer 

Room. These anterooms are used by Marine ESB personnel to change into proper ‘clean’ garments 

before entering the clean-air laboratories. Disposable laboratory coveralls, hoods, and shoe covers 

are provided in this room and must be worn at all times while in the clean-air spaces. The material 

used to make these garments is non-woven Tyvek, a highly effective contamination control fabric 

that is resistant to penetration by airborne particles. These garments are processed and packaged 

in a clean environment by the manufacturer and are disposable, but can be worn several times 

before soiling and tears occur. Additionally, hearing and eye protection must be worn at all times 

when operating the cryomill.

Safety Data Sheets 

Several hazardous chemicals are referred to and used during cryohomogenization using the 

cryomill. Refer to Appendix B for applicable SDS documents. 

Palla VM-KT Vibrating Cryomill 

The cryomill is designed to cryogenically homogenize large quantities of frozen material under 

liquid nitrogen (LN2) vapor-phase conditions (-120 °C to -180 °C), Figure 1. The flow of LN2 and 

motor vibration are controlled by an electrical control panel, Figure 2. Titanium rods are rotated 

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within the material grinding chamber (300) when the cryomill motor is on and in motion, Figure 

3. Teflon bellows connect the material grinding chamber (300) and motor to the stationary cryomill 

parts, and allow material to freely flow in and out of the material grinding chamber (300), Figure 

4.

Figure 1. Palla VM-KT Vibrating Cryomill

Figure 2. Palla VM-KT Vibrating Cryomill Electrical Control Panel

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Figure 3. Titanium Rods contained inside Material Grinding Chamber

Figure 4. Teflon Bellow

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Figure 5. Palla VM-KT Cryomill Material Grinding Chamber and Motor

Compressed Air Smashing Device

To ensure a consistent and uniform flow of material through the cryomill, the frozen material must 

be crushed into <2.5 cm diameter pieces in order for the material to pass uniformly into the material 

grinding chamber (300) of the cryomill. Large pieces of material cause an inconsistent flow of 

material and clog the cryomill inlet socket (100), Figure 6, preventing subsequent material from 

being distributed into the material grinding chamber (300). To eliminate this problem, large pieces 

of frozen material are crushed using the Marine ESB compressed air defragmenting device, CAD, 

Figure 7. Details about this procedure have previously been described in Pugh et al., 2007. 

Figure 6. View Inside the Inlet Socket

Outlet

Teflon 

Bellow

Material

Grinding

Chamber

Inlet 

Teflon 

Bellow

Vibration 

Motor

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Figure 7. Compressed Air Defragmenting Device

LN2 Piping System

The cryomill is connected to a LN2 in-line piping system. This system provides a vacuum-jacketed,

insulated pipe along with a cryovent device, the Gordinier Electronics Keep Cold (Model 279) 

thermocouple sensor controller, located in the specimen bank to ensure that LN2 is maintained in 

the piping system and gas is vented out. Liquid nitrogen is brought into the specimen bank through 

an intricate piping system that distributes LN2 to multiple freezer stations. Each freezer station 

consists of a single supply line with either one or two connection valves, totaling forty-one 

connection valves. These valves provide LN2 to 30 LN2 vapor-phase (-150 oC) freezers, 10 ultracold (-80 oC) electric freezers, and cryomill. The LN2 bulk storage tank for the Freezer Room, 

Reference Material Production Facility, and Cleanroom operations is stored behind the HML 

building in a 12 m3 (3,170 gallons) vertical storage tank with a telemetry system to the LN2

commercial supplier (Figure 8). The main distribution lines are equipped with multiple flow valves 

strategically placed to ensure safety. Furthermore, a permanent LN2 vapor vent system was 

installed on the cryomill to safely vent LN2 out of the Reference Material Production Facility. The 

REES Centron Monitoring System: Monitoring, Alarming, and Data Management System is used 

to ensure correct temperatures and LN2 levels are maintained for each freezer. In addition, this 

system monitors room conditions (i.e. temperature and humidity) and percent oxygen levels within 

the Freezer Room, Reference Material Production Facility, and Cleanroom. 

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Figure 8. LN2 Bulk Tank

Cryomill Assembly

Most parts of the cryomill are numbered and locations shown in an illustration developed by the 

cryomill manufacturer, Figure 9.

Table 1 indicates the name and item number assigned to parts of the cryomill. These part names 

and numbers are used in assembly steps. Steps for cryomill assembly are divided into Outlet 

Assembly and Inlet Assembly and are listed below.

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Figure 9. Palla Cryomill Schematic

Photo credit: Technical Machinery Documentation, Vibrating Mill Palla VM-KT
9

Item Number Item

1 Vibrating Body Mount

3 Clamping Yoke

12 Pressure Springs

20 Vibration Motor

100 Inlet Socket

150 Vibrating Trough Conveyor

155 Filling Socket

160 Thyristor Control

170 Mount Cover

180 Temperature Pick-up

200 Inlet & Outlet Bellow

210 Mount Bellow

220 Vibrating Trough Conveyor Inlet Bellow

300 Material Grinding Chamber

310 Material Chamber Outlet Head

320 Material Chamber & Material Chamber Outlet Junction

330 Material Chamber End Cap

400 Outlet Tube

410 Temperature Pick-up

500 Liquid Nitrogen Valve Box

510 Flexible Liquid Nitrogen Supply Line

600 Collection Basin

Table 1. Palla MT-KM Cryomill Schematic Part Numbers

Outlet Set-Up

1. Open both the large and small protective covers of the cryomill, Figure 10.
10

Figure 10. Palla Cryomill Large and Small Protective Covers

2. Place the clean titanium rods inside the clean material grinding chamber (300). Attach the 

material chamber outlet head (310) to the material grinding chamber (300) using the 

provided securing clamps. Tighten the nuts on the securing clamps, ensure that the 6

securing clamps are in a “star pattern” and secure the material chamber outlet head (310)

to the material chamber end cap (330), Figure 11.

Cryomill Large

Protective Cover

Cryomill Small

Protective Cover

Safety Latches
11

Figure 11. Outlet Head

3. Secure the outlet tube (400) to the cryomill body using the 2 securing clamps. Also, tighten

the temperature pick-up (410) to the outlet tube, Figure 12.

Figure 12. Outlet Tube and Temperature Pick-up
12

4. Place a lower metal band onto the lower portion of the outlet bellow (200) and an upper 

metal band onto the upper portion of the outlet bellow. Ensure that the extra metal(s) piece 

of the lower and upper metal bands are facing away from the body of the bellow. Position

the outlet bellow between the material chamber end-cap (330) and the outlet tube (400). 

You will need to compress the bellow to position it properly. Expand the bellow until it 

overlaps the grooves on both the material chamber outlet head (310) and the outlet tube 

(400). Tighten the screws on the metal bands of each bellow to secure it in place. See

Figure 13and Figure 14.

Figure 13. Outlet Bellow Upper Metal Band

Extra metal piece facing away 

from the body of the bellow
13

Figure 14. Outlet Bellow Lower Metal Band

Attach the outlet socket to the outlet tube (400) using the 4 clamps provided. Ensure that 

the LN2 exhaust hose is secured to the outlet socket using the two metal bands provided, 

5. Figure 15. The LN2 exhaust hose should always be secured to the exhaust pipe installed on 

the cleanroom floor.

Figure 15. Outlet Socket with LN2 Exhaust Hose

Outlet

Tube

Outlet

Socket
14

Inlet Setup

6. Attach the material chamber end cap (330) to the material grinding chamber (300) using 

the provided securing clamps. Tighten the nuts on the securing clamps ensuring that the 6 

securing clamps are in a “star pattern” on the material chamber end cap (330) and material 

grinding chamber (300), Figure 16.

Figure 16. Material Chamber End Cap

7. Close the small protective cover of the cryomill.

8. Place a lower metal band onto the lower portion of the inlet bellow (200) and an upper 

metal band onto the upper portion of the inlet bellow (200). Position the inlet bellow (200)

with the upper and lower metal bands loosely attached between material chamber end-cap 

(330) and the inlet tube. Again, ensure that the extra metal(s) piece of the lower and upper 

metal bands are facing away from the body of the bellow. You may need to compress the 

bellow to position it properly between the between material chamber end-cap (330) and the 

inlet tube. Expand the bellow between the material chamber end cap (330) and the inlet 

socket tube. Tighten the screws on the metals bands to ensure that the bellow will remain 

securely attached, Figure 17.
15

Figure 17. Inlet Bellow

9. Place the inlet socket (100) on top of the inlet tube of the cryomill. Position the LN2 line 

on the inlet socket (100) so that it is pointed away from the mount platform. Secure the 

inlet socket to the Inlet tube using the 4 securing clamps provided, Figure 18.

Figure 18. Inlet Socket Attached to Inlet Tube

If the vibrating trough conveyor is not being used, the initial cooling of the cryomill using LN2 can 

commence at this point. In this case, proceed to the section titled, Cryomilling Setup without 

Vibrating Trough Conveyor. If the vibrating trough conveyor is being used, proceed to step 10.

Inlet 

Socket

Inlet 

Tube

LN2

Line
16

Cryomilling Assembly with Vibrating Trough Conveyor

10. Place the inlet socket head cover on top of the inlet socket (100) and secure it in place using 

the 4 securing clamps, Figure 19.

Figure 19. Inlet Socket with Inlet Socket Head Cover

11. Locate the vibrating trough conveyor (150) and secure the vibrating trough conveyor lid to 

the vibrating trough conveyor base using the 10 securing clamps provided. The securing 

clamps attach in specific places as indicated in Figure 20.

Figure 20. Vibrating Trough Conveyor

Inlet Socket 

Head Cover

Inlet 

Socket
17

12. Attach the conveyor support to the mount platform using the 4 nuts provided, Figure 21.

Figure 21. Conveyor Support and Mount Platform

13. Next, place the vibrating trough conveyor onto the conveyor support. Be cautious when 

lifting the vibrating trough conveyor as it is very heavy! Position the four rubber feet 

on the vibrating trough conveyor into the appropriate spots on the conveyor support.

14. Locate the mount bellow. Place a lower metal band on the base of the mount bellow (210) 

and an upper metal band on the top portion of the mount bellow (210). Ensure that the extra 

metal piece(s) of the metal band is not facing the bellow body. Position the mount bellow

(210), with the metal bands attached, between vibrating trough conveyor (150) and the inlet 

socket head. Tighten the screws on the upper and lower metal bands of the mount bellow

(210) to secure it in place, Figure 22.

Conveyor

Support

Mount 

Platform
18

Figure 22. Mount Bellow Attachment

15. Locate the vibrating trough conveyor inlet bellow (220), Figure 23. Place an upper and 

lower metal band onto the vibrating trough conveyor inlet bellow (220). Ensure that the 

extra metal piece on the metal bands is not facing the bellow body. Place this vibrating 

trough conveyor inlet bellow (220), onto the large opening at the top of the vibrating trough 

conveyor. Do not tighten the metal bands yet; they will be tightened in a later step.

16. Position the filling socket (155) into the large bellow opening. The filling socket will rest

on the conveyor support. There should be a gap between the base of the filling socket (155) 

and bottom of the vibrating trough conveyor (150). This gap allows material to flow from 

the filling socket (155) through the vibrating trough conveyor (150) and into the material 

grinding chamber (300). If a gap is not present, turn the knob on the conveyor support to 

increase the gap. Expand the vibrating trough conveyor inlet bellow (220) sufficiently

between the filling socket and the vibrating trough conveyor. Tighten the metal bands on 

the vibrating trough conveyor inlet bellow (220) to ensure that the bellow will remain 

securely attached.

17. Place the mount cover with the flexible LN2 supply line (170 and 510) onto the filling 

socket (155). Loosely secure this assembly onto the filling socket using 2-3 securing 

clamps, Figure 23.

Mount

Bellow
19

Figure 23. Flexible LN2 Supply Line Attached to Filling Socket

18. Check that all securing clamps on the cryomill are tightened.

19. Close the large protective cover and secure the safety latches on both the large and small 

protective covers, Figure 10 . When the large protective cover is closed, the cryomill safety 

button is depressed allowing the cryomill to be operational, Figure 24. The cryomill will 

not operate with the large protective cover open. 

Figure 24. Cryomill Safety Button

Mount Cover 

with Flexible LN2

Supply Line

Filling 

Socket

Mount 

Bellow
20

20. Plug the electrical line from the vibrating trough conveyor (150) into the thyristor control 

(160) attached to the outside of the cryomill. The thyristor control (160) controls the 

vibration speed of the trough conveyor which controls the material flowing into the 

material grinding chamber (300), Figure 25.

Figure 25. Thyristor Control

21. The cryomill is now assembled with the vibrating trough conveyor (150) and ready to be 

cooled using LN2.

Cryomilling Assembly without Vibrating Trough Conveyor

Depending on the material size to be cryohomogenized, the vibrating trough conveyor (150) may

not be required for use. In the event that the vibrating trough conveyor (150) is not being used to 

distribute material into the cryomill and the material will be manually added to the material 

grinding chamber (300), the following steps should be performed. 

1. Secure the inlet socket (100) to the inlet tube using the 4 securing clamps provided. 

2. Place the flexible LN2 supply line attached to the mount cover (510 and 170) onto the inlet 

socket (100). The cryomill is now assembled without the vibrating trough conveyor (150)

and ready to be cooled using LN2, Figure 26.
21

Figure 26. Flexible LN2 Supply Line Attached to Inlet Socket

Preparing Material and Supplies

Material chosen for cryogenic homogenization utilizing the cryomill must be frozen before 

cryogenic homogenization. The frozen material must then be crushed into <2.5 cm diameter pieces 

in order to flow uniformly into the material grinding chamber (300) of the cryomill.

Additionally, collection basins are needed and should be pre-cooled prior to being used to collect 

homogenized material as it flows from the outlet head of the cryomill. Collection basins may be 

stainless steel buckets, stainless steel baskets lined with Teflon bags, or plastic basins. Depending 

on the material being cryohomogenized and the type of analysis being conducted, it is 

advantageous to have several collection basins available for use to eliminate certain contamination 

issues.

Palla Cryomill Operation

After the cryomill is successfully assembled, it is cooled using the LN2 supply from the NIST LN2 

bulk tank. Below are steps to begin cooling and operating the cryomill.

1. Ensure that all nuts and securing clamps are tightened. Close both cryomill protective 

covers and secure the safety latches. There are three safety latch points that must be secured 

before proceeding with the cryomill process, Figure 10.

2. The LN2 control valve box (500) controls the flow rate of LN2 into the cryomill, Figure 27. 

Ensure that the black knob is turned to the right (closed) prior to operation and during setup.
22

Figure 27. LN2 Control Valve Box

3. Place the flexible LN2 supply line attached to the mount cover (170 and 510) onto the 

desired resting place, either the filling socket (155) or the inlet socket (100). In the event 

that the vibrating trough conveyor is being used, secure the LN2 hose to the filling socket 

(155), Figure 23. In the event that the vibrating trough conveyor is not being used, secure 

the LN2 hose to the inlet socket (100), Figure 26.

4. Turn the LN2 supply on by turning the knob that is closest to the floor on the house piping 

system, Figure 28. Opening this valve allows LN2 to flow to the LN2 control box.

Figure 28. LN2 House Supply Valve

LN2 valve 

to cryomill
23

5. Next, turn the black knob on the LN2 valve box (500) at least 5 rotations counterclockwise,

Figure 27. This allows LN2 to flow from the LN2 control box, through the LN2 supply line, 

and into the cryomill.

6. Next, on the electrical control panel, turn the power to the cryomill ON. In Figure 29, turn 

the large red knob clockwise so the “I” is visible in the knob view window. It will be 

necessary to wait several seconds for the control panel to run machine diagnostics.

Figure 29. Cryomill Electrical Control Panel

7. Place a collection receptacle at the base of the cryomill underneath the outlet socket to 

catch any residual LN2 that may flow out once LN2 is flowing into the cryomill. Any 

residual LN2 vapor will be exhausted through the exhaust hose.

8. Turn the nitrogen release key to “I”. This key starts, and stops, the flow of LN2 into the 

cryomill. Liquid nitrogen should begin to flow from house supply line, to the LN2 valve 

box (500), through the flexible LN2 supply line (510), then through the material grinding 

chamber (300). If LN2 does not begin to flow, make sure that the house LN2 valve, Figure 

28, is open and the flow rate valve on the LN2 control valve box (500), Figure 27, is opened. 

9. A preprogrammed temperature setting for LN2 (-190 °C) has been set using the electrical 

control panel temperature settings. To program a different set temperature, see the Palla 

cryomill technical documentation for instruction. 
24

10. Allow several hours for the cryomill to reach the desired pre-set temperature. Upon 

reaching the desired temperature, the LN2 control valve box will begin “clicking.” This 

clicking is an LN2 valve in the LN2 control box opening and closing controlling the flow 

of LN2 into the cryomill, thereby maintaining the set temperature of -190 °C.

11. After the desired temperature has been reached and maintained for at least 30 minutes, the 

LN2 key can be turned off to the “O” position. 

12. With the LN2 key in the off position, depress the white button labeled “unbalance motor 

on.” The vibration motor (20) will start and the titanium rods will begin rotating. Allow 

the motor to run for 20 to 30 seconds. The temperature inside of the material grinding 

chamber (300) will dramatically increase, or warm. The increase in temperature is due to 

the titanium rods initial rotation. CAUTION- This step causes the titanium rods inside the 

material grinding chamber (300) to rotate and without material flowing through, is very 

loud. Participants in the immediate cryomilling vicinity should wear ear/hearing protection.

NOTE - If the vibration motor does not start, make sure that the large and small protective 

covers are closed. 

13. Depress the red button labeled “unbalance motor off.” The vibration motor will stop, 

therefore stopping the titanium rods from moving. 

14. Turn the nitrogen release key to “I” to begin the flow of LN2 until the desired temperature 

is reached again. After reaching desired temperature a second time, the cryomill is ready 

to be used. 

Cryohomogenizing Material

After the cryomill has reached and maintained the desired temperature, the pre-frozen material can 

be distributed into the material grinding chamber (300). The following steps describe how to 

distribute material into the cryomill with and without the vibrating trough conveyor (150). 

Cryomilling with the Vibrating Trough Conveyor

1. Ensure the LN2 key is in the off position and remove the mount cover (170) with the 

flexible LN2 supply line (510) from the filling socket (155) and place it below the cryomill 

base out of the way. Place a pre-cooled collection basin (600) below the outlet socket to 

collect cryohomogenized material as it flows out of the cryomill.

2. Depress the white button labeled “unbalance motor on.” The vibration motor (20) will start 

and the titanium rods will begin rotating.

3. Turn the black knob on the thyristor control (160) to the desired vibration speed. 
25

4. While standing at the top of the 4-step safety ladder, begin evenly and continuously 

distributing material into the filling socket (155), Figure 30, using a stainless steel scoop. 

The material will flow through the vibrating trough conveyor, through the material grinding 

chamber (300), and out through the outlet head, Figure 31.

Figure 30. Cryomilling with the Vibrating Trough Conveyor
26

Figure 31. Cryohomogenized Material Flowing from the Outlet Head

5. Once a collection basin is full of frozen cryohomogenized material, it should be placed 

quickly into either an LN2 freezer, or the LN2 cryocart, and a new collection basin placed 

under the outlet head in its place to continue catching material. 

6. Repeat step 4 until all material has passed through the cryomill, or the cryomill temperature 

reaches -80°C. See Figure 32.

7. When the cryomill reaches -80°C, it will be necessary to depress the “unbalance motor off” 

and cool the cryomill again. 

8. Place the mount cover (170) onto the filling socket and secure it using a securing clamp.

9. Turn the LN2 key to the on position to allow LN2 to begin flowing through the cryomill 

again. Allow the LN2 to flow until the desired temperature is reached again. Repeat any 

necessary steps to complete homogenization. It is not recommended to have LN2 flowing 

into the cryomill while the vibration motor is on. 
27

Figure 32. Cryomilling material with the Vibrating Trough Conveyor

Cryomilling without the Vibrating Trough Conveyor

1. With the LN2 key in the off position, remove the mount cover (170) with the flexible LN2

supply line (510) from the inlet socket (100) and place it below the cryomill base out of 

the way. Place a pre-cooled collection basin (600) below the outlet socket to collect 

cryohomogenized material as it flows out of the cryomill.

2. Depress the white button labeled “unbalance motor on.” The vibration motor will start and 

the titanium rods will begin rotating.

3. While standing at the top of the 4-step safety ladder, begin to continuously distribute 

material into the Inlet Socket (100), Figure 33, using a stainless steel scoop. The material 

will flow directly into the material grinding chamber (300) and out through the outlet head, 

Figure 31.

Collecting 

SRM after 

cryomilling

Filling stainless 

steel buckets with 

material ready for 

cryomilling

Distributing 

crushed material 

into cryomill
28

Figure 33. Cryomilling without the Vibrating Trough Conveyor

4. Once a collection basin is full of frozen powder material, it should be placed quickly into 

either an LN2 freezer or the LN2 cryocart and a new collection basin placed under the outlet 

head in its place to continue catching material. 

5. Repeat step 3 until all material has passed through the cryomill, or the cryomill temperature 

reaches -80 °C. 

6. When the cryomill reaches -80 °C, it will be necessary to depress the “unbalance motor 

off” and cool the cryomill again. 

7. Place the mount cover (170) onto the inlet socket and secure it using a securing clamp, 

Figure 26.

8. Turn the LN2 key to the on position to allow LN2 to begin flowing through the cryomill 

again. Allow the LN2 to flow until the desired temperature is reached again. Repeat any 

necessary steps to complete homogenization. It is not recommended to have LN2 flowing 

into the cryomill while the vibration motor is on. 

Particle Size Measurement

Once cryomilling has been completed, the particle size distribution of the material is measured 

using the Malvern Mastersizer 3000. The instrument uses laser diffraction to determine the sizes 

of particles within the range of 0.01 to 3500 μm in diameter. As particles pass through the 

instrument, scatter produced by two lasers gives the measure of particle size. See ROA 646.06-14-

309 for additional instructions.

Inlet 

socket
29

Disassembling, Thawing, and Clean-up Procedures

Upon completion of cryomilling, all parts of the cryomill and RM Production facility must be 

thoroughly cleaned following the steps below, as well as the cleanroom maintenance steps 

described in NISTIR 7389. 

1. Turn the cryomill off by depressing the red button labeled “unbalance motor OFF.” The 

motor and titanium rod movement will stop.

2. Turn the LN2 house supply valve off, Figure 28. Turn the LN2 control valve box knob , 

Figure 27, clockwise to close the valve and stop any additional flow of LN2 into the 

cryomill.

3. Open the large protective cover of the cryomill to allow thawing to begin. Using a socket 

wrench, loosen the nuts on the securing clamps attached to the outlet head. The nuts on the 

securing clamps may still be frozen and may take several minutes to thaw before they can 

be loosened.

4. Meanwhile, the inlet set-up may be broken down. All pieces secured to the top of the small 

protective cover must be removed before the cover can be opened. 

5. Remove the mount cover with flexible LN2 supply line (170 and 510) from either the filling 

socket (155) or the inlet socket (100), and place it on the floor. Disassemble the remainder 

of the cryomill inlet set-up, place all items in the sink, and thoroughly rinse items with 

warm water. After rinsing, wipe the items with Deconahol and a Texwipe, and place the 

cleaned cryomill parts aside to dry on absorbent mats.

6. After all parts, including the conveyor support, have been detached from the small 

protective cover, open the protective cover to thaw and clean the material grinding chamber

(300). Do not remove the mount platform from the small protective cover; it is permanently 

attached, Figure 21.

7. Loosen and remove thawed securing clamps located on the material chamber end cap (330)

and outlet head and rinse if necessary. Remove titanium rods from the material grinding 

chamber (300) and rinse with warm water. Thaw and clean the material grinding chamber

(300) with warm water. After drying, wipe the material grinding chamber (300) with a 

Texwipe and Deconahol.

8. Follow the Labconco Steam Scrubber (steam scrubber) steps below to clean items using 

the steam scrubber. 
30

Labconco Steam Scrubber

Some cryomill pieces are best cleaned using the steam scrubber, Figure 34. These pieces include: 

titanium rods, Teflon bellows, stainless steel collection buckets, and stainless steel scoops. These 

items must be thoroughly rinsed with warm water before cleaning in the steam scrubber.

Do not clean any insulated parts from the cryomill using the steam scrubber: filling socket, 

inlet socket, end cap, outlet head, temperature pick-up, vibrating trough conveyor, securing 

clamps, outlet tube, and/or outlet socket. Instead, clean the above listed insulated items using 

warm water and Deconahol as previously described.

Figure 34. Labconco Steam Scrubber

1. Roll the steam scrubber to the sink located in the RM Production facility. 

2. Attach the distilled (DI) water line from the steam scrubber to the HML sink DI water 

faucet, and attach the tap water line from the steam scrubber to the HML sink tap water 

line, see Figure 35 and Figure 36. 
31

3. Place all drain lines inside the sink then plug the steam scrubber electrical cord into an 

electrical outlet

Figure 35. Labconco Steam Scrubber Distilled and Tap Water Lines

Figure 36. HML Distilled and Tap Water Lines

4. Load the cryomill parts inside the steam scrubber, add LabSolutions detergent to the 

detergent cup, and LabSolutions neutralizing acid rinse to the neutralizing solutions port, 

Figure 37. Close both the detergent cup and neutralizing acid rinse port. Safety Data Sheets 

(SDS) for the LabSolutions detergent and neutralizing acid rinse are available in Appendix 

A. 

Distilled 

Water 

Line

Tap

Water 

Line

Distilled 

Water 

Line

Tap

Water 

Line
32

5. Gently close and latch the steam scrubber door, but do not force the door closed. Items 

may need to be removed or rearranged in order for the steam scrubber door to close. Once 

the door is securely closed, the Science Plus program will appear on the steam scrubber 

screen (Figure 38). Turn on the DI and hot tap water from the sink knobs. Press 

“Run/Cancel” button to start the wash cycle. 

Figure 37. Labconco Steam Scrubber Detergent Cup and Neutralizing Solutions Port

Figure 38. Labconco Steam Scrubber Display Screen

Detergent 

Cup

Neutralizing 

Solutions 

Port
33

Cryomilling Overview

1. Frozen material must be crushed into <2.5 cm in diameter pieces in order to fit into the 

cryomill.

2. The vibrating trough conveyor (150) can be attached to the cryomill to ensure that the 

material uniformly enters the cryomill. Or, material can be distributed directly into the 

material grinding chamber (300). 

3. A vacuum jacketed, insulated LN2 hose (510), which is connected on one end to the LN2

piping system in the Marine ESB, is tightly attached to the cryomill. After reaching the 

desired cryomilling temperature, the LN2 is turned off.

4. The cryomill vibration motor (20) is turned on and the mill begins vibrating and rotating 

the titanium rods located within the material grinding chamber (300).

5. Material is distributed into the material grinding chamber (300) through the vibrating

trough conveyor (150), or the inlet socket (100) for cryohomogenization.

6. The material travels through the material grinding chamber (300) where the vibrating 

titanium rods pulverize the material into powder. 

7. The cryohomogenized material falls from the outlet tube (400) at the bottom of the machine 

and is collected in pre-frozen containers which in turn are quickly placed in an LN2 freezer

when full. 

8. The cryohomogenized material is passed through the cryomill as many times as necessary

to ensure homogeneity. A particle size analysis should be conducted to verify homogeneity.

9. The cryomill, cryomill parts, and RM production facility are cleaned. 

10. Upon completion, the cryohomogenized material can be bottled, or if necessary, packaged 

for shipment to the NIST Reference Material Facility in Gaithersburg, MD, for further 

processing. 
34

References

Pugh, R.S., Ellisor, M.B., Moors, A.J., Porter, B.J., and Becker, P.R. Marine Environmental 

Specimen Bank: Clean Room and Specimen Bank Protocols. NISTIR 7389, National Institute of 

Standards and Technology. 2006, 46 pp.

Palla VM-KT Cryomill Technical Documentation

Ness, Jennifer M., Standard Operating Procedure for Particle Size Characterization of Seabird 

Egg Homogenate NIST ROA 646.06-14-309

Prepared by:

Amanda J. Moors

Research Biologist

Rebecca S. Pugh

Research Biologist

Jody R. Evans

Research Biologist
35

Jennifer M. Ness 

Research Biologist

Debra L. Ellisor 

Research Biologist

Melannie J. Bachman 

Research Biologist

Reviewed by:

Paul R. Becker, Ph.D.

Project Leader - Research Biologist
36

Appendix A

Hollings Marine Laboratory Marine ESB Job Hazard Analysis
37

Hollings Marine Laboratory Marine ESB Job Hazard Analysis
38
39

Appendix B

Liquid Nitrogen (LN2) Safety Data Sheet

LabSolutions Detergent Safety Data Sheet

LabSolutions Neutralizing Acid Rinse Safety Data Sheet
40
41
42
43
44
45
46

Deconahol Safety Data Sheet
47
48
49
50
51
52
53

LabSolutions Powder Detergent Safety Data Sheet
54
55

LabSolutions Neutralizing Acid Rinse Safety Data Sheet
56