On June 22, in Articles. Lyophilization, or freeze drying, is a technique often used in university and life science laboratories. During the freeze dry process, sublimation occurs as a result of a cycle of:. It's a technique well suited for sample preservation, restoration of water-damaged items and—of course—an ever-important vehicle for sample prep. Sounds simple enough, right? Of course there's much more to freeze drying and we've covered that in-depth on more than one occasion.
Say you know how to properly care for your freeze dryer and religiously stay away from common lyophilization mistakes like not knowing your sample's eutectic point, for example. Why are there still some samples that remain challenging time and time again?
What happens when you do everything right and your sample still makes you sweat? Don't kick your freeze dryer. Four key questions can help determine if and how a solvent can be freeze-dried:. How can the solvent be frozen? Find the most appropriate method that can reach a low enough temperature so that the sample is fully frozen. At which concentration can the solvent be frozen? Determine if the solvent must be diluted prior to freezing. Can the condenser collect the solvent?
Will the sample remain in a frozen phase during the process? Evaluate if you can maintain a pressure that is low enough to keep the sample frozen. In contrast to aqueous samples, organic solvents are tricky to freeze and require dilution prior to freezing.
Chemists might need to use liquid nitrogen for the solvents to reach a low enough temperature. Moreover, due to the low freezing point of organic solvents see table above , the condenser temperature might not be low enough to completely capture the solvents.
Instead, the organic solvents liquify in the condenser or leave the system through the pump as vapor. Because of this:. A scroll pump is recommended for all freeze-drying applications involving organic solvents. The low freezing temperature and triple point of the organic solvents makes it difficult to evacuate the system fast enough and to maintain a low enough pressure to avoid solvent melting, even at ultimate vacuum.
For diluted solutions, it is rather common to see solvents melting and evaporating, while water remains frozen. Whether this is problematic for you depends on your sample requirements. 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.
Example of compatibility of common organic solvents with Lyopvapor L It might be challenging to find a car charging station or bioethanol fuel station, but it might be worth the trouble in the long run. Same with using organic solvents in lyophilization. The ends might just justify the means. Excited to see if I will discuss chromatography or freeze drying with you next? Stick around to find out. Therefore, researchers with many samples to process, or mixtures of solvents, i.
In other laboratories—compound handling, for example—the aggressive nature of the organic solvents used makes a freeze-dryer unsuitable. Even in these environments, state-of-the-art centrifugal evaporators, such as the Genevac HT-4X, shown in Figure 1 , have some limitations. Purification laboratories receive samples dissolved in water and acetonitrile, with a low level of a modifier present, normally 0. Freeze-drying to remove these solvents is fraught with difficulties: 1 The acetonitrile requires either a very deep vacuum to freeze it, or a freeze-dryer that actively freezes the samples.
If it is not frozen, then bumping is inevitable, resulting in sample loss and cross-contamination. The centrifugal evaporator rapidly dries many different samples in parallel, without bumping. However, users report difficulties with a few samples dried in a batch. Not all of the TFA is removed and may damage the sample when in storage; in addition, the compound may interact with the water, boosting the boiling point. Residual solvent appears in nuclear magnetic resonance NMR analysis. While occurring occasionally, the implications of selecting a few samples by hand are prohibitive for automated laboratories; therefore the whole sample rack is reprocessed.
Samples prepared in water can be lyophilized in a centrifugal evaporator by pulling the best vacuum available. In the Genevac HT-4X with solvent-resistant scroll pump , the ultimate vacuum is below 0. This is like freeze-drying, and it is slow. Genevac developed a process whereby users evaporate some solvent using the speed of a centrifugal evaporator, then switch to lyophilization mode when only a few milliliters of solvent are left.
The effects of heating a sample during lyophilization were studied to determine if this gave a speed advantage. Initially, just water was used to develop the optimum conditions, and then water and acetonitrile were used to simulate samples taken from HPLC. A stock solution of 0. In fact, the freezing stage achieves cooling as well as freezing; therefore a separate stage 2 is not necessary.
However, the freezing stage with no heat appears to be essential. Without it, the samples dried normally and did not lyophilize; thus it was evident that the sample had not frozen at all. The time required to freeze-dry a sample is dependent on a number of factors, including sample volume, thickness, and surface area; the eutectic point and solute concentration of the sample; and the temperature of the collector and the maximum vacuum obtained.
In general, samples with a large surface area will freeze-dry faster than those that are thick or dense. While freeze-drying provides superior drying capacity, if time is of the essence, you may consider a vacuum concentrator or evaporator for your application.
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