Rotary evaporation vs freeze drying in protein concentration

Peptides and proteins have been hot stuff in the biotechnology field for awhile now. Not too long ago, I dedicated a post to protein purification, but I realized I’ve left the topic of acquiring proteins somewhat incomplete. In this post, I explore the next step after purification, protein concentration, by mainly concentrating on two commonly used techniques: rotary evaporation and freeze drying.

My sister has been busy with work lately, so I have been trying to support her by driving my nephew to school every morning. He needs to come back home by himself though and now that the weather is getting warmer, he prefers to take other means of transportation than the bus. A few mornings ago, we stood for several minutes in their garage considering if we should pack his bike, rollerblades, scooter, or longboard for his way home. Each offered distinct benefits in terms of speed, fun factor, cool factor and so on and all achieved the same goal, bringing him home from school.

Of course, life and lab life are pretty much the same. There are often multiple possibilities for reaching more-or-less the same result. Examples of this are endless, but I think since I just recently discussed protein purification with you, this topic is still stuck in my head. Let me show you, besides different means of transportation, different means for protein concentration.

As we’ve discussed before, peptides and proteins belong to one of the four fundamental classes of macromolecules found in nature. Proteins, made of amino acids, and peptides, made of a smaller number of amino acids, play an important role in nearly all fundamental processes in living cells.

In biomedical or biochemical research, peptides and proteins are frequently studied for their potential as therapeutic drugs. These biomolecules can be used as references in quantitative studies, growth factors and cytokines for cell culture, active enzymes for biochemical assays, carrier proteins for movement of molecules, inhibitors for blocking pathways or antigens for use with antibodies.

Proteins and peptides can be manufactured through chemical synthesis, using recombinant microorganisms or cell free expression systems, or via extraction from their natural environment. Isolating the proteins after they have been synthesized is a crucial step. And here is where the choice comes in. You can perform the protein concentration step by any one of several techniques, including spray drying , rotary evaporation , parallel evaporation or freeze drying .

In this post, I’d like to focus on two of the more frequently used techniques for protein concentration: rotary evaporation and freeze drying.

Rotary evaporation for protein concentration

If you have been reading the blog, you are aware, that a rotary evaporation system removes solvents from a sample via gentle evaporation and condensation of solvent. The solvents transform from liquid to vapor and back to liquid. The evaporation occurs under reduced pressure, allowing the boiling point of the solvent to remain lower than at atmospheric pressure.

There are several possible outcomes from a distillation process. Drying is the process where a liquid is being separated from a solid. Concentration is the process where you evaporate the solvent, while leaving some in the sample.

Freeze drying for protein concentration

Freeze drying or lyophilization is the gentlest process for drying various types of perishable materials. Freeze drying is based on sublimation, the direct transition of a substance from the solid to the gaseous state. Initially, the product is frozen (freezing step) and then dried by sublimation in an environment of reduced pressure (primary drying step). The low pressure enables the direct changeover of frozen solvent into vapor. Typically, the solvent to be removed from the product is water, but other solvents such as acetonitrile can also be efficiently separated.

Differences between rotary evaporation and freeze drying

There are key differences between using rotary evaporation and freeze drying for protein concentration. I’ve summarized parameter differences in the table below:

 Rotary evaporationFreeze drying
Minimal pressure appliedDown to 5 mbarDown to 0.03 mbar
Temperature rangeUsually from 40 to 60°CAmbient temperature (manifold or non-heated shelves)
End productDried or concentratedDried
Sample preparationNoYes, sample has to be frozen

Now let us consider a few other factors:

1. Sample preparation for protein concentration – Using a rotary evaporation system does not require sample preparation. If you opt to use a freeze dryer , you must freeze the sample prior to starting the process. Conveniently, you can use a dewar accessory filled with dry ice and a rotary evaporator to prepare the sample for freeze drying.

2. Concentration of water – For the protein purification step before the protein concentration step, reversed-phase chromatography is frequently used. For this method, mixtures of water and a second water-miscible solvent, such as methanol, acetonitrile or ethanol are used. Hence, water is the main solvent you need to isolate when performing protein concentration. The boiling point of the solvent is the main parameter you should consider if performing rotary evaporation. Below is a table with the boiling points of the most common solvents used in reversed-phase chromatography :

SolventBoiling point in atmospheric pressure (in °C)Distillation rate of solvent (in L/h)
Water1000.75
Methanol64.71.8
Ethanol78.371.8

Please note: the rotary evaporation was performed with a 60°C heating bath, 20°C cooling temperature, 1 L evaporating flask at 280 rpm.

As you can see, water has a much lower distillation rate compared to methanol and ethanol on a rotary evaporator. Water is the easiest solvent to isolate with a freeze dryer. Freeze drying is your best option, whenever you want to isolate water in large quantities.

3. Thermal stress during protein concentration – Both freeze drying and rotary evaporation use reduced pressures that enable us to dry at lower temperatures. Freeze dryers allow us to work at ambient temperature, whereas rotary evaporators often require to heat at a temperature above ambient temperature.

4. End product of protein concentration – In rotary evaporation, the end product depends on the characteristics of the compound. If you have a powdery compound, after rotary evaporation, you will obtain an uneven particle size powder. Freeze drying keeps products in their initial shape, meaning that a powder compound will have an airy, snow-like form.

Other criteria, such as time, sample input, budget and scalability also play a role in selecting the best method for your application. In summary, the figure below can help you select the most appropriate technique for your protein concentration:

rotary evaporation, lab evaporation, rotavapor, freeze drying, protein concentration

I hope this comparison of rotary evaporation vs freeze drying has helped you better understand the options you have available for efficient protein concentration. By the way, this is not the first time I compare different possibilities in the lab. Check out one of my first and most beloved posts, where I explore differences between silica and alumina use in flash chromatography and prep HPLC.

Who won the bike vs rollerblades vs scooter vs longboard battle? My nephew went for the long board, the cool factor won there. Luckily, the cool factor of rotary evaporation and freeze drying for your protein concentration is pretty much the same. Do you agree? Leave me a comment below! Ah, and just to keep you on your toes, I can already feel I have not exhausted all I need to say on the topic of protein concentration. Don’t miss future posts, where I will certainly dig into this application in even more detail.

Till next time,

The Signature of Bart Denoulet at Bart's Blog