Beyond Water: The Role of Solvents in Freeze Drying

Welcome back to the “Colorful Researchers” blog. My first two posts covered the basics of freeze-drying and spray-drying. In this blog, I would like to get more specific and focus on the role of solvents in freeze-drying.

In my first post on freeze-drying, I discussed the challenges I faced climbing to the summit of Aconcagua, Argentina's highest peak. Taking my body out of its comfort zone required meticulous preparation and specialist equipment to achieve the task. The same is true when it comes to pushing the boundaries of freeze-drying.

Freeze-drying instruments were initially intended to be used with water, and products not soluble in water were out of bounds. To fix this problem, chemists turned to solvents, and their use in freeze-drying applications has increased over the years. Organic-based solutions or inorganic acids and bases are now commonly used before the lyophilization of difficult products. When using solvents (or tackling great summits), many considerations must be made before the process begins. Read on to discover how to incorporate organic solvents in your laboratory freeze-drying workflow without damaging your equipment or sample.

Why use solvents?

Solvents are helpful for preparing pharmaceuticals or other products where the product’s solubility and stability in water might be limited. Some samples, such as proteins or cells, may undergo structural changes or degradation in the presence of water. Solvents such as DMSO can act as cryoprotectants during freezing steps, which maintains the overall structure from crystallizing ice. Ethanol with microbicidal properties ensures the sterility of the solution, leading to a longer shelf life of the freeze-dried product. In other cases, solvents are required to separate those biomolecules or phages from unwanted by-products appearing during synthesis. Acetonitrile, trifluoroacetic acid, and methanol are some frequently used solvents. The other aspects to be considered are the accelerating effects of solvent on the drying time. This means it may help to dry the final product in a shorter time. 

How can I determine when to use solvents?

Organic solvents like acetonitrile, acetone, methanol, and ethanol have lower eutectic or glass transition temperatures than water
To determine whether a solvent is necessary and can be freeze-dried, you need to understand the vapor-pressure curve of the solvent (see table below):

                               
Unlike aqueous samples, organic solvents are notoriously hard to freeze; in fact, they are often used in anti-freeze! Due to the low temperatures indicated in the table, these solvents may require dilution before freezing. You may need to use liquid nitrogen to reach a low enough temperature. 

What are the challenges of using solvents?

Solvents can present several challenges due to their toxicity and the extreme conditions required to dry them.

• Toxicity & Safety: Solvents like acetonitrile and methanol are toxic and require careful handling, appropriate safety measures, and compliance with environmental regulations. If the solvent amount is too high, the system will not be able to maintain the required pressure, and everything will melt and evaporate. The process must be terminated at this point.
• Removal of Solvents: Due to the low freezing points, the condenser temperature might not be low enough to capture the solvents completely. Organic solvents can liquify in the sample and leave the system as vapor through the pump. Therefore, a scroll pump is recommended for all freeze-drying applications involving organic solvents.
• Instrument/Material Compatibility: Not all materials are compatible with organic solvents; some may react or degrade. Freeze-dryers contain plastics (acrylic drying chamber), elastomer (seals and gaskets), which can react adversely with inorganic and organic solvents. Cleanliness is very important, as well as checking the instrument and replacing worn parts before starting a cycle. 

Tips on using solvents:

• Eliminate as much solvent as possible before freeze-drying, using a rotary evaporator or other instrument if necessary.
• If the first one is not feasible, dilute solvents with water whenever possible to influence solvent concentration in your favor.
• Be prepared for periodic replacement of parts if etching becomes problematic.
• Carefully cleanse the freeze-dryer after each cycle
• Do not allow condensate to sit in the condenser. Immediately conduct the defrosting step of the instrument with the drain valve open. Wash out the condenser with water and ensure it is clean and dry.
• Use a dry pump when handling solvents other than water. Make sure the pump's exhaust port is in a fume hood to avoid solvent exposure.
• The use of an ultimate vacuum pump is recommended due to the low pressures (lower than 0.05 mbar) required to keep the solvents in solid form.

Pushing the limits of any process, be it mountain climbing or freeze drying, involves a little more planning and often the use of specialized equipment; however, following the advice provided here, you should have no problem performing the most advanced freeze-drying techniques to date. 

Bueno Chao,
Bruno