Pre freezing is the core pre process of experimental freeze-drying, which directly determines the morphology, sublimation efficiency, and final quality of the sample ice crystals. Its core goal is to completely crystallize the free water in the sample into ice crystals, avoiding problems such as foaming, shrinkage, and solute migration during the sublimation stage. The following are key precautions for the pre freezing process:

Accurately control the pre freezing temperature, which must be lower than the eutectic point of the sample

The eutectic point of a sample refers to the lowest temperature at which all components (water+solute) in the sample freeze together, and is the core reference index for pre freezing. The pre freezing temperature should be 5-10 ℃ lower than the eutectic point of the sample. If the temperature is higher than the eutectic point, the sample cannot be completely frozen, and local melting, foaming, and collapse may occur during the sublimation stage; If the temperature is too low (such as exceeding the eutectic point by more than 20 ℃), it will increase energy consumption and may also damage the activity of thermosensitive samples (such as proteins and bacterial strains).

In practice, if the eutectic point of the sample is unknown, it can be determined through pre experiments or by referring to empirical values of similar samples (such as pure water eutectic point of about -0.5 ℃, sugar containing solution eutectic point of about -10~-20 ℃, protein solution eutectic point of about -20~-30 ℃).

Strictly control the sample packaging form to ensure even freezing

Sample thickness: The flat thickness should be ≤ 10mm. If the thickness is too large, it will cause uneven heat transfer inside the sample, and even after the surface freezes, the inside will still be in liquid state, resulting in incomplete final freeze-drying. This can be achieved by increasing the surface area of the sample (such as using shallow trays or porous plates) or reducing the volume of packaging.

Sample form: Liquid samples need to be stirred evenly before being packaged to avoid excessive local concentration of solutes; Solid samples (such as plant tissue and meat) need to be cut into small pieces (particle size ≤ 1cm) to prevent internal freezing.

Container selection: Prioritize using containers with good thermal conductivity (such as glass freeze-drying bottles, penicillin bottles), and avoid using plastic containers with poor thermal conductivity; The container should be clean, dry, and free of residual impurities to prevent contamination of the sample.

Reasonably choose the pre freezing method and match the characteristics of the sample

Experimental freezing intervention freezing is divided into in-situ pre freezing and external pre freezing, and should be selected according to the sample type:

In situ pre freezing (direct cooling of freeze-drying machine shelf): suitable for sterile samples (such as vaccines, cell culture media), thermosensitive samples, and samples with high requirements for morphological stability. Note that the cooling rate of the shelf should be stable (recommended 1-3 ℃/min) to avoid rapid cooling that may cause excessive temperature difference between the inside and outside of the sample, resulting in the formation of large ice crystals (which can damage the sample structure and reduce rehydration); During pre freezing, the vacuum system should be turned off and only the cooling function should be turned on to prevent the sample from being pulled away.

External pre freezing (refrigerator, low-temperature freezer, liquid nitrogen pre freezing): suitable for pre freezing ordinary samples or bulk samples, with lower cost. Attention should be paid to avoiding direct contact between the sample and liquid nitrogen during pre freezing to prevent sample explosion; After the external pre freezing is completed, the sample needs to be quickly transferred to the freeze-drying machine drying chamber (transfer time ≤ 3 minutes) to prevent the sample from heating up and melting. At the same time, attention should be paid to the sterile operating environment to avoid contamination.

Ensure sufficient pre freezing time and ensure complete freezing of the sample

The pre freezing time should cover three stages: surface freezing of the sample → complete internal freezing → ice crystal stabilization. The specific duration is related to the volume, thickness, and thermal conductivity of the sample

Trace samples (≤ 1mL): External pre freezing for about 2-4 hours, in-situ pre freezing for about 3-5 hours;

Large volume/thick layer samples (≥ 5mL or thickness>8mm): need to be extended to 6-12 hours to ensure that the center temperature of the sample reaches the preset pre freezing temperature, and then insulated for 1-2 hours to fully stabilize the ice crystals.

The intuitive standard for determining whether a sample is completely frozen is that the sample is in a hard solid state, has no fluidity, and there is no liquid shaking sound when tapping the container.

Targeted pre freezing treatment of special samples

Samples containing organic solvents (such as ethanol and acetone extracts): The freezing point of organic solvents is lower than that of water, and the pre freezing temperature needs to be lowered (such as the pre freezing temperature of ethanol containing samples should be ≤ -40 ℃) to prevent the solvent from failing to crystallize; At the same time, after pre freezing, it is necessary to ensure that the solvent is completely solidified to avoid solvent evaporation during the sublimation stage and contamination of the vacuum system.

Thermally sensitive samples (such as proteins, antibodies, and bacterial strains): slow cooling and low-temperature insulation should be used to avoid rapid cooling damaging biological activity; Some bacterial samples can be pre frozen with protective agents (such as glycerol and sucrose) to improve their activity retention rate.

Easily oxidizable samples: During the pre freezing process, inert gases such as nitrogen can be introduced for protection to prevent sample oxidation and deterioration.

Avoiding operational errors during the pre freezing process

Misconception 1: The lower the pre freezing temperature, the better. Low temperature will increase equipment load and may also cause structural damage to the sample. It only needs to be controlled at 5-10 ℃ below the eutectic point.

Misconception 2: The longer the pre freezing time, the better. After the sample is completely frozen and kept stable, extending the time is meaningless and actually wastes energy.

Misconception 3: Seal the container during pre freezing. Containers should be kept breathable (such as penicillin bottles without stoppers or freeze-drying bottles without caps). If completely sealed, sample freezing and expansion can cause the container to explode.