Cryogenic preparation of samples

Cryogenic size reduction and homogenization

A solid sample material should always be sufficiently prepared by size reduction and homogenization before it is subjected to chemical or physical analysis

Preparing samples for cryogenic grinding

Preparing samples

To obtain meaningful results, the analysis sample must fully represent the original material and the sample preparation process be carried out reproducibly. Most sample materials can be reduced to the required fineness at room temperature by choosing a mill with a suitable size reduction principle (impact, pressure, friction, shearing, cutting).

before and after cryogenic treatment

However, there are limits for size reduction at room temperature

For example when even a small temperature increase affects the sample in a negative way; or when the material is very elastic and the above-mentioned grinding principles only cause deformation. The perfect solution for these types of samples is cold or cryogenic grinding.

liquid nitrogen (-196 °C) for cryogenic grinding

This involves grinding aids such as liquid nitrogen (-196 °C)

or dry ice (-78 °C) which embrittle the sample by cooling and making it break more easily. Another advantage is the preservation of volatile components of the sample. In this article, we explain for which sample materials cryogenic grinding is applicable, which laboratory mills are suitable and which other aspects need to be taken into consideration.

materials for cryogenic grinding

For which materials is cryogenic grinding advisable?

  • Samples with elastic behavior
  • Samples with volatile components
  • Biological and medical samples
  • Sticky or tough food samples


environmental simulations

The general guidelines for categorizing environmental simulations

are regulated by the first part of the IEC 60068 standard. The following sections look at the specific requirements of the respective applications.

  • in transit
  • in storage
  • during installation
  • under actual operational conditions above the expected service life

Samples with elastic behavior

1. Samples with elastic behavior

Many polymers (plastics such as PP, PET, PA, etc.), as well as other materials, show visco-elastic behavior during grinding which results in plastic deformation. This means that a crack initiation - and thus a break-up - does not occur. Elastomers like silicone baking dishes or rubber tires which, due to their flexibility, are used at room temperature, have a so-called glass transition temperature far below room temperature.

Retsch cryogenic grinding mill

cryogenic procedure

The procedure

If elastic-plastic samples are immersed in liquid nitrogen, their temperature falls below the glass transition temperature; this reduces the ability of the material to resist high mechanical stress by elastic-plastic behavior or viscous flow. If this pre-cooled material is then ground in a mill, the sample shows brittle-breaking behavior. Cryogenic grinding is also suitable for hard plastics even though this material is brittle at room temperature. For a successful size reduction process, the temperature of the sample must not exceed the glass transition temperature.

Samples with volatile components

2. Samples with volatile components

Materials with volatile components like solvents (benzene, toluene, PCB, PCP, etc.) are difficult to prepare properly for analysis because a temperature rise during grinding may lead to a loss of the analytes. The increased particle surface resulting from the grinding process further promotes the emission of volatiles. The low temperature of liquid nitrogen or dry ice substantially reduces the high vapor pressure of the components and embrittles the sample matrix. Thus the volatile components are hardly affected by the relative temperature increase which occurs during the grinding process.

Biological and medical cryogenic samples

3. Biological and medical samples

If biological samples are prepared, for example for subsequent extraction of nucleic acids from yeast, bacteria, plants, or human/animal tissue, they may be highly temperature-sensitive during and after the process and may even be destroyed. In such cases, cryogenic grinding helps to improve the process by embrittling groups of cells and cell walls, making them disrupt more easily and slowing down the subsequent decomposition of the cell fragments. Undesired cell reactions are practically “frozen” by immersing the sample into LN2 so that cell activities can be observed at a later point in time.

grinding Sticky or tough food samples

Sticky or tough food samples

Sticky or tough sample materials like cheese, raisins, wine gum, or marzipan simply clump together when the ground is at room temperature and are not sufficiently homogenized. The low temperature of the cryogenic process prevents the sample from clumping so that it is thoroughly homogenized and suitable for analysis.

RETSCH grinders

RETSCH is the ultimate solution

For many materials pulverization with liquid nitrogen or dry ice is the only possible way of obtaining a sample suitable for subsequent analysis. RETSCH offers a range of laboratory mills that allow for gentle and efficient cryogenic sample preparation.

reduces both the cost and labor of cryogenic grinding


Their use considerably reduces both the cost and labor required for low-temperature grinding. A selection of suitable accessories ensures that the grinding process is carried out safely.

Samples, mills, and accessories


Gummy bears MM 400 • 2 grinding jars stainless steel 50 ml
• 2 grinding balls 25 mm stainless steel
• CryoKit, liquid nitrogen
10 pieces 1 min 30 Hz < 300 µm
Caoutchouc CryoMill • Grinding jar stainless steel 50 ml
• Grinding ball 25 mm stainless steel
• Liquid nitrogen
4 g 2 min 30 Hz < 500 µm
E. coli bacteria CryoMill • Grinding jar stainless steel 50 ml
• Grinding ball 25 mm stainless steel
• Liquid nitrogen
10 ml frozen cell pellets 2 min 30 Hz complete cell disruption
Plastic granulate ZM 200 • 12-tooth push fit rotor
• Distance sieve 0.5 mm
• Cyclone
• Liquid nitrogen
40 g 20 s 18,000 min-1 < 500 µm
Refuse-derived fuels ZM 200 • 12-tooth push fit rotor
• Distance sieve 0.75 mm
• Cyclone
• 300 g dry ice
150 g 30 s 18,000 min-1 < 0.75 mm
Wine gum GM 300 • Grinding container stainless steel
• Full metal knife
• Cryo lid with aperture
• Dry ice
500 g 40 s and 20 s 1000 min-1 und 4000 min-1 < 0.8 mm
Rubber duck SM 300 • Parallel section rotor
• 2 mm bottom sieve
• Liquid nitrogen
5 pieces 40 s 3000 min-1 < 2 mm
Pills with liquid filling RM 200 • Mortar and pestle stainless steel
• Liquid nitrogen
40 pieces 3 min 100 min-1 < 250 µm


Overview RETSCH mills for cryogenic grinding


Mill Feed size 1 2 Max. feed
quantity 1 2
Final fineness 1 2 Remark
Cryomill < 8 mm 1 x 20 ml 50 µm • Continuous grinding under cryogenic conditions
• User comes at no point into contact with LN2
• Zirconium oxide grinding jar available for cryogenic applications
MM 400 < 8 mm 2 x 20 ml 100 µm • Sample is placed in leak-free grinding jar of steel or PTFE and embrittled before grinding
• Intermediate cooling may be necessary
ZM 200 < 10 mm 4000 ml 300 µm • Embrittlement with LN2, e. g. of samples with glass transition temperature <-80°C
• Dry ice is best suited for materials with low thermal capacity
• Dry ice is preferably used for samples with a particle size <1 mm
• Use of cyclone is mandatory
GM 300 < 40 mm 2000 ml 500 µm • Dry ice is best suited for materials with low thermal capacity
• Dry ice cools the sample during grinding
• Full metal knife used together with stainless steel grinding container and a special cryo lid
SM 300 < 80 mm 4000 ml 2000 µm • Cryogenic grinding only with 6-disc rotor and cyclone
• Bottom sieves 2 – 20 mm suitable for cryogenic applications
RM 200 < 8 mm < 190 ml 10 µm • Dry ice or LN2 can be added during grinding
• Only use mortar and pestle made of stainless steel

If you have any questions, Geneq professionals are ready to assist:


    Source article from our partners





    Questions? Comments?