WHITE PAPER

AUTHOR

Dr. Tanja Hanke

Cryogenic preparation
of sample materials
Selection of suitable laboratory mills and
accessories for grinding processes with dry ice
or liquid nitrogen

A solid sample material should always be sufficiently prepared by size

reduction and homogenization before it is subjected to chemical or physical
analysis. Care should be taken that the analysis sample fully represents the
original material and that the sample preparation process is carried out
Product Manager reproducibly. Only then are meaningful results guaranteed. Most sample
materials can be reduced to the required analytical fineness at room tem-
perature by choosing a mill with a suitable size reduction principle (impact,
Retsch GmbH pressure, friction, shearing, cutting).
Retsch-Allee 1-5
42781 Haan, Germany 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
Phone: 02104/2333-100 the material is very elastic and the above mentioned size reduction principles only
E-Mail: t.hanke@retsch.com cause deformation. The perfect solution for these types of samples is cold or cryo-
genic grinding. This involves grinding aids such as liquid nitrogen (-196 °C) or
www.retsch.com dry ice (-78 °C) which embrittle the sample by cooling and make it break more
easily. Another advantage is the preservation of volatile components of the sample.
In this white paper 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.

www.retsch.com | White Paper | 1

WHITE PAPER

For which materials is cryogenic grinding advisable?

• 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 a plastic deformation. This
means that a crack initiation - and thus a break-up - does not occur. Elastomeres 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. 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 a 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.

before after before after

• 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 tem-
perature 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 samples

If biological samples are prepared, for example for subsequent extraction of nucleic
acids from yeast, bacteria, plants or human/animal tissue, they may react highly tem-
perature-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.

www.retsch.com | White Paper | 2

WHITE PAPER

• Sticky or tough food samples

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

before after before after

Overview Applications
Final fineness
Sample Mill Accessories Feed quantity Grinding time Speed
(d90)

Gummy bears MM 400 • 2 grinding jars stainless steel 50 ml 10 pieces 1 min 30 Hz < 300 µm
• 2 grinding balls 25 mm stainless steel
• CryoKit, liquid nitrogen

Caoutchouc CryoMill • grinding jar stainless steel 50 ml 4g 2 min 30 Hz < 500 µm

• grinding ball 25 mm stainless steel
• liquid nitrogen

E. coli bacteria CryoMill • grinding jar stainless steel 50 ml 10 ml 2 min 30 Hz complete cell
• grinding ball 25 mm stainless steel frozen cell disruption
• liquid nitrogen pellets

Plastic granulate ZM 200 • 12-tooth push fit rotor 40 g 20 s 18,000 min-1 < 500 µm
• distance sieve 0.5 mm
• cyclone
• liquid nitrogen

Refuse-derived ZM 200 • 12-tooth push fit rotor 150 g 30 s 18,000 min-1 < 0.75 mm
fuels • distance sieve 0.75 mm
• cyclone
• 300 g dry ice

Wine gum GM 300 • grinding container stainless steel 500 g 40 s and 20 s 1000 min-1 und < 0.8 mm
• full metal knife 4000 min-1
• cryo lid with aperture
• dry ice

Rubber duck SM 300 • parallel section rotor 5 pieces 40 s 3000 min-1 < 2 mm
• 2 mm bottom sieve
• liquid nitrogen

Pills with RM 200 • mortar and pestle stainless steel 40 pieces 3 min 100 min-1 < 250 µm
liquid filling • liquid nitrogen

www.retsch.com | White Paper | 3

WHITE PAPER

Application Videos Cryogenic Grinding

• CryoMill: Licorice and plastics with LN2

• Knife Mill GRINDOMIX GM 300: Raisins with dry ice
• Mixer Mill MM 400: Gummy bears with LN2
• Ultra Centrifugal Mill ZM 200: Plastic granulate with dry ice
• Ultra Centrifugal Mill ZM 200: Plastic pellets with LN2

Suitable Lab Mills for Cryogenic Grinding

When selecting a suitable laboratory mill for cryogenic grinding various aspects need to
be considered. On the one hand, the sample volume is crucial for the decision, on the
other hand the feed size and desired final fineness are important factors as well. The
mixer mills MM 400 and CryoMill are designed for processing small sample volumes.
These mills often achieve finer grind sizes, even with difficult plastic samples, than for
example rotor mills because the sample remains for a longer period inside the closed
grinding jar compared to the open grinding chamber of the rotor mills. The sample is
cooled continuously during the entire grinding process, in the CryoMill even with a
constant temperature of -196 °C.

Rotor mills, mortar grinders, knife mills or cutting mills process substantially larger
volumes and feed sizes than mixer mills. However, the size reduction principles of these
mills usually produce larger grind sizes, especially when grinding plastics. The Knife Mill
GRINDOMIX GM 300 is suited for cryogenic grinding mainly of food samples, with the
restriction that only dry ice and no liquid nitrogen can be used as the mill is not designed
for temperatures as low as -196 °C. Rotor and cutting mills, however, accept both
dry ice and liquid nitrogen as grinding aids. Due to the very low temperature liquid
Mixer Mill MM 400
nitrogen is particularly suitable for materials with a glass transition temperature below
-50 °C. Dry ice has the advantage of evaporating less quickly than liquid nitrogen;
moreover, it can be mixed with the sample for grinding thus extending the cooling effect.
This is particularly beneficial for materials of low thermal capacity which cannot hold the
low temperature, for example thin plastic foils. Sample feeding with dry ice is generally
easier, especially if the particles are smaller than 1 mm, than extracting the material
from liquid nitrogen. In addition, dry ice is safer to handle as the danger of asphyxiation,
for example, is much smaller. Also, dry ice doesn’t splash during grinding as it is mixed
completely with the sample material. Regardless of these aspects, appropriate safety
regulations should always be observed when dealing with cryogenic grinding aids.
The following section introduces a range of laboratory mills suitable for cryogenic
grinding.

• Mixer Mills MM 400 and CryoMill

The mixer mills MM 400 and CryoMill are perfectly suited for homogenizing small
sample volumes with a maximum feed size of 8 mm. These mills feature two,
respectively one, grinding stations into which the screw-top grinding jars - filled with
grinding balls and sample - are securely clamped for grinding. The closed grinding jars,
and thus the sample, are embrittled with liquid nitrogen.

Suitable grinding jars of the MM 400 are made of steel or PTFE; single-use vials of
1.5, 2 and 5 ml are also available. Care must be taken that no liquid nitrogen is enclosed
in the grinding jars. The frictional heat of the grinding process causes LN2 to enter the
CryoMill with 50 liter tank
gaseous phase, resulting in a considerable pressure increase inside the grinding jar.
With the help of tongs the closed grinding jar is placed for 2 to 3 minutes in an insulation
container filled with liquid nitrogen and is then clamped into the MM 400. Due to the
high energy input and the resulting frictional heat, the grinding process should not take
longer than 3 minutes to prevent the sample from warming up and to preserve its
breaking properties. If longer grinding times are required, these should be interrupted
by intermediate cooling of the closed grinding jars.
Unlike the MM 400 the CryoMill offers the advantage of continuous cooling of the
grinding jar with liquid nitrogen, reducing the temperature of jar and sample to -196 °C
within minutes. Thus a consistent temperature of -196 °C is guaranteed even for
long grinding times without the need for intermediate cooling cycles. Moreover, the
user comes at no point into contact with liquid nitrogen which makes operation of the
CryoMill safe and user-friendly. The automatic pre-cooling function ensures that the
grinding process does not start before a temperature of -196  °C is reached and
maintained. For heavy-metal-free grinding a zirconium oxide grinding jar is available.

www.retsch.com | White Paper | 4

 WHITE PAPER

• Ultra Centrifugal Mill ZM 200

Another mill which is perfectly suitable for cryogenic grinding is the Ultra Centrifugal Mill
ZM 200 which accepts larger sample volumes than the mixer mills. This high-speed
rotor mill even pulverizes some types of elastomeres, such as polystyrene,
without cooling. However, in most cases embrittlement of the material is required. The
sample, which can be granulate or pre-cut molded parts, is directly immersed into a
container filled with LN2 and is then slowly but continuously fed to the hopper of the
ZM 200 with a steel spoon. If the sample has a high fat content or is very heat-sensitive,
embrittlement usually improves the grinding process as the cooling counteracts the
effects caused by frictional heat. When using dry ice as grinding aid, this needs to be
mixed with the sample in a 2:1 ratio and the entire mixture is then pulverized in the
ZM  200. Dry ice is pure carbon dioxide and therefore evaporates from the sample without
residues. We recommend using a cassette in combination with a cyclone to ensure that
CO2 as well as residues of LN2 evaporate during the grinding process.

• Cutting Mill SM 300

Ultra Centrifugal Mill ZM 200
with cyclone The aforementioned aspects influencing the choice of a grinding aid are also relevant
when carrying out cryogenic grinding in the Cutting Mill SM 300. This mill is particularly
suitable for processing tough materials such as shoe soles or bitumen and accepts
larger feed sizes than the ZM 200. Even roughly cut and embrittled car tires can be
homogenized with the SM 300. The embrittled sample material is rather hard, therefore
we recommend to use the 6-disc rotor as it works more like a shredder. It is also suitable
to cut heterogeneous samples such as frozen chicken parts including bones. For
grinding thin plastic foil, which is often part of refuse-derived fuels, we recommend
using dry ice due to the continuous cooling effect during the grinding process.

• Knife Mill GRINDOMIX GM 300

Sticky and tough food samples such as cheese, raisins, wine gum or marzipan are
perfectly homogenized with the Knife Mill GRINDOMIX GM 300. Even chocolate, which
simply becomes paste-like when processed at room temperature, can be successfully
pulverized cryogenically. The sample is mixed with dry ice in a ratio of 1:2; after a
few minutes it is thoroughly cooled and the grinding process starts. The dry ice keeps
the sample cool all the time. Care should be taken not to use any plastic accessories
when carrying out cryogenic grinding in the GM 300 as these could be damaged during
the process. Suitable accessories include a grinding container of stainless steel, a
full metal knife and a lid with aperture to let the gaseous carbon dioxide evaporate.

• Mortar Grinder RM 200

Knife Mill GRINDOMIX GM 300 with
steel container In a mortar grinder samples are pulverized by pressure and friction and are submitted
to the grinding process for as long as it takes to achieve the desired final fineness. The
Mortar Grinder RM 200 is not a closed system; it is possible to add dry ice or liquid
nitrogen during the grinding process through a window in the cover. For cryogenic
applications mortar and pestle need to be made of stainless steel. Tablets with liquid
filling, for example, can be successfully pulverized in a mortar grinder.

www.retsch.com | White Paper | 5

 WHITE PAPER

Overview RETSCH Mills for Cryogenic Grinding

Max. feed Final
Mill Feed size 12 Remark
quantity 1 2 fineness 12

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

1
Depending on sample material and grinding parameters
2
Volume may differ for applications without cryogenic grinding aids

Conclusion
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 which allow for gentle and efficient cryogenic sample
preparation. 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.

www.retsch.com | White Paper | 6