Préparation cryogénique d'échantillons 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 reproducibly. Only then are meaningful results guaranteed. Most sample materials can be reduced to the required analytical fineness at room temperature by choosing a mill with a suitable size reduction principle (impact, pressure, friction, shearing, cutting).

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 size reduction principles only cause deformation. The perfect solution for these types of samples is cold or cryogenic grinding. This involves grinding aids such as liquid nitrogen (-196 °C) or 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.

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.

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 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 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.

Sticky or though 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 homogenized. The low temperature of the cryogenic process prevents the sample from clumping so that it is thoroughly homogenized and suitable for analysis.

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. The MM 500 control is able to maintain a selected temperature from -100°C to 0 °C. It offers the option to grind also larger sample volume pf up to 80 ml per batch.

The use of jars made of zirconium oxide or tungsten carbide is allowed for these temperatures. For very tough samples like the most plastics, pulverisation is only possible at the coldest option – the CryoMill. 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 Mills GRINDOMIX GM 200 or 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. Pre-frozen (Freezer, LN2 bath with no transfer of LN2 into the mill) samples are ok. Rotor and cutting mills, however, accept both dry ice and liquid nitrogen as grinding aids.

Due to the very low temperature liquid 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, MM 500 nano, MM 500 vario, MM 500 control and the CryoMill

The mixer mills e.g. the MM 400, MM 500 vario and CryoMill are perfectly suited for homogenizing small sample volumes with a maximum feed size of 8 mm. These mills feature two, respectively one or six, 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 or the MM 500 vario are made of steel or PTFE; single-use vials or steel tubes 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 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 or MM 500 vario.

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 two mentioned mixer mills, 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.

This mill is the most suitable machine to pulverize even the toughest plastic materials. The MM 500 nano and MM 500 control work with different grinding jars and offer also larger jars up to 125 ml volume – thus, up to 80 ml in one batch can be processed. The MM 500 nano offers also a very high energy input with up to 35 Hz, which is beneficial to crush even harder samples efficiently. For the MM 500 nano, the handling is the same as described for the MM 400 and MM 500 vario. A complete new way to do cryogenic grinding is realised in the MM 500 control. The jars are cooled via the thermal plates. Here, if also a CryoPad is attached, the temperature can be set at a value between -100°C and 0°C. The CryoPad controls the LN2 flow which is required to maintain the set temperature. This, this mill is optimal for samples like sweets and food, as here only “moderate” cryogenic temperatures of -40°C or even -20°C are required to pulverize those samples. As the temperature is lowered constantly, and the maximum negative temperature is -100°C, the use of tungsten carbide jars and jars made of zirconium is allowed for cryogenic grinding.

Cutting Mill SM 300

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 300. 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 200 and GM 300

Sticky and tough food samples such as cheese, raisins, wine gum or marzipan are perfectly homogenized with the Knife Mill GRINDOMIX GM 200 or 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 200 or 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

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.

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.