Lyophilization technique

Lyophilization, also known as freeze-drying, is a process commonly used in the food industry, biological samples preservation, and pharmaceutical applications, being a dehydrating technique suitable for heat-sensitive samples. This consist on the removal of water from a sample, either being from a solution or materials. During the process, the water is removed from previously frozen samples directly into vapor by sublimation, in decreased temperature and pressure conditions.

The process has 3 main stages, namely, freezing, primary drying, and secondary drying stages.

In the first stage, FREEZING, the water present in the sample is frozen, allowing for it to separate the other solute in the mixture. This can be done using a conventional freezer (-30°C), an ultralow temperature freezer (-80°C) or even with liquid nitrogen (-196°C), after which the sample(s) is placed in the freeze-drying equipment. This stage is completed in a few hours.

During the second stage, PRIMARY DRYING, sublimation takes over. Once the samples are placed in the lyophilizer, with adequate temperature and vacuum levels, the absorption of heat by the sample allows solid water to become vapor. Here, the vapor is direct to the collecting chamber, with reduced pressure below the equilibrium vapor pressure of ice with the aid of a vaccum pump (usually 0.03-0.2 mbar). Once in the chamber, the vapor condenses at the coil (usually below -40°C) and turns into ice once more. This stage takes the longest time (up to 3 days) and depends on many parameters, such as sample volume, solute concentration, thickness, surface area, collector temperature, vacuum level.

The third stage, SECONDARY DRYING, consists of removing non-freezing water to reduce the water content to acceptable values, and this step is considered the most critical since vestigial water can influence product quality and stability.
Samples containing volatile substances, such as other solvents rather than water, are problematic to freeze-drying. These substances affect the eutectic temperature, may enhance are not an option the vapor pressure at the sample surface, and require less heat for sublimation to occur. Thus, volatile containing samples will melt when placed in flasks exposed to room temperature. Also, equipment part and/or general equipment can suffer damage.

Lyophilization equipment

The existing equipment in the CQM/UMa facilities is a 4.5L Freeze Dry System by Labconco. This equipment was installed under Project reference: PTDC/CTM-NAN/1748/2012.

Supervisor: João Rodrigues (PI)

Unit Manager: Rita Castro (PhD)

Unit Manager Responsabilities

- Responsible for equipment management, approval of analysis/reports/quotations, and consumables acquisition.
- Responsible for equipment operation.

Regulation of operation of the Lyophilizer equipment

Lyophilizer Access and Functioning Rules

Only samples containing water as solvent are acceptable for lyophilization at CQM.

Each lyophilization session has a duration of 3 (three) days.

1. The lyophilizer equipment (Labconco® FreeZone 4.5L) is located in the LBCC - Laboratório de Bioquímica e Cultura Celular (Biochemistry and Cell Culture Lab), room 0.40.
2. The functioning, sample introduction, and removal, maintenance, and cleaning operations of the equipment is carried out only by the unit manager (UM) responsible for the equipment and/or a substitute designated by the first, in case of absence.
3. Applicants should contact the UM to be informed about the lyophilization schedule. Any alterations to this schedule will be informed in time by e-mail.
4.  Any researcher who requests lyophilization services will have to fill out obligatory, the corresponding service form, existing on the lyophilization service webpage on the CQM website.
5. Upon sample registration for the lyophilization service, the applicant will receive an automatic e-mail with all the information introduced in the form. There is no need to print the form.
6. The applicant must only bring the number of samples/flasks/tubes indicated in the filled registration form. Any extra vials will require a new registration form, and samples will be lyophilized in the next round. No exceptions will be allowed..
7. Frozen samples can be delivered in glass or plastic tubes or flasks, centrifuge microtubes, 15 mL or 30 mL centrifuges tubes (aka falcons), or others, considering that these should not be higher than a 50 mL falcon.
8. Samples volume should not exceed 30-50% of the total flask volume.
9. Samples submitted to lyophilization need to have only and only water as solvent.
10. Samples need to be fully frozen to be placed in the lyophilizer. If samples were exposed only to water as a solvent during the synthesis/preparation steps, samples may be frozen at -30ºC (-80ºC preferably, especially if microcentrifuge tubes are used). If, at some point, during the synthesis/preparation steps, any organic solvent was used, this must be removed, and samples in water must be frozen at -80ºC (or preferably with liquid nitrogen, as any residue of the organic solvent will prevent freezing). Failure to comply by these rules may result in no lyophilization of the sample, loss of sample during the lyophilization process, or equipment failure/damage.
11. Lyophilizer flasks, rubber lids, and metal/glass connectors must be handled with care and washed with soap, final wash with distilled water, and left to air dry.

Lyophilization services

The user fees are intended to cover CQM lyophilizer expenses, including the functioning, consumables, parts, and maintenance.

The price of services provided is divided into three classes, namely:

1. CQM Research groups
2. FCT National Centers and National Public Laboratories
3. Companies and private Research Centers

Choose the class that are best suited to your case or consult us using the address:

(prices: valid from January 2022; VAT not included, please add tax at rate of 22%)

Submit Sample

If you are not a CQM member and would like to use the services, please contact us via and check the pricing list. To access the Lyophilization Service, please submit here  and fill the sample registration form.

After the form submission, you will receive in your e-mail box an email confirming the successful sample registration and the filled form.

Sample Registration Form

References

Hidenori Kawasaki, Toshinori Shimanouchi, Yukitaka Kimura, "Recent Development of Optimization of Lyophilization Process", Journal of Chemistry, vol. 2019, 14 pages, 2019. https://doi.org/10.1155/2019/9502856
Labconco 4.5L Freeze Dry System Manual.

Funding

PTDC/CTM-NAN/1748/2012; Acquired: 2013