If you have recently purchased any of our PCR, transformation, or enzyme kits you might have noticed that many of the tubes arrive seemingly empty. Over the past few years, Edvotek has supplied more and more of our kit components as freeze-dried, or lyophilized, materials. These reagents often appear as a dried out, flakey material that needs to be rehydrated before use, and are a key component to the success of your experiment. Lyophilization is a water removal process that we use to preserve many of our perishable reagents to extend the shelf life and make the materials more convenient for transport and storage.
What is Lyophilization?
At its core, lyophilization is a method that removes water from a sample by freezing the material and then subjecting it to a strong vacuum. It’s possible to manually control the process, but many modern lyophilizers are connected to computers and sophisticated sensors that can control the process from start to finish. The science of lyophilization takes advantage of “sublimation”, when a solid (in this case ice) changes directly to a vapor without going through the liquid phase. We often witness sublimation with dry ice, when the solid carbon dioxide sublimes at standard temperatures and pressures. Importantly, by adjusting the temperature and pressure of a a system we can control exactly which phase a substance will take at any given time.
Scientists use phase diagrams to examine the various phases of matter that a solution will take. As we all know, it is relatively easy to turn an aqueous solution into a solid – simply place it into the freezer. On the phase diagram we can observe this as the solid phase on the left (low temperatures). Importantly, if we maintain a low pressure but slowly increase the temperature of the sample it will begin to transition into a gas. Over time, this process will remove water from the sample, leaving behind the solid materials that had been dissolved. Importantly, this process is repeatable and is often gentler on the samples than desiccation or heating to remove water.
The lyophilization process occurs over three main phases – freezing, primary drying, and secondary drying – and uses a special piece of equipment called a lyophilizer.
- Freezing – The freezing step drops the temperature of the sample below the triple point, ensuring that it is completely solid and that it will not melt during the following steps. This temperature is vital – if the temperature is too high then the sample might melt during the following steps, rather than sublimating.
- Primary drying – Once the samples are frozen, the pressure inside the chamber is lowered until a strong vacuum has been achieved, and then the sample is heated slightly. From the phase diagram above, we can see that these conditions (low pressure and increasing heat) will cause our sample to sublimate. Primary drying can remove over 95% of the water in our sample.
- Secondary drying – After the majority of the water has been removed from the sample the temperature is again raised, often above the freezing point of water. This allows for the removal of remaining water and produces the final dried sample.
The advantages of lyophilizing samples
Many biological materials are sensitive to heat. Proteins can denature, DNA strands can break or be digested by enzymes, and bacteria will die if left for extended periods of time outside of a fridge or freezer. Fortunately, removing water from these components can prevent this and drastically enhance the shelf life and stability of the components. For many biologicals, this also allows for shipping at ambient temperatures, rather than more expensive wet or dry ice shipping. It also means that components are more stable and can often survive being forgotten in a warehouse over a long weekend!
Rehydration of lyophilized components is quick and easy. Each component will include unique instructions, but most of the time you will add between 1-10 mL of a buffer and gently vortex or flick the tube. Check the instructions provided for specific details on your components!
Edvotek has been providing lyophilized materials in our classroom experiments for over a decade. These include the antibodies used in ELISA experiments, the restriction enzymes included in many of our molecular biology labs, PCR beads, and all of our bacteria strains. If you haven’t already, give some of our lyophilized components a try in your next classroom lab!