Concentrating heat sensitive samples without degradation

By Jenny Sprung, Senior Application Specialist

On July 02, 2013 in Case Studies

Typically, sample concentration or solvent evaporation requires heat to drive the evaporation process. For heat sensitive samples, adding too much heat can damage or modify the structure of the analyte. The most effective way to safely evaporate solvent from heat sensitive samples is to place the sample in a vacuum atmosphere. At reduced pressure, the evaporation phase change occurs at a lower temperature, allowing the sample to be concentrated without damage from excessive heat.

Common laboratory methods for solvent evaporation or sample concentration are lyophilization (also known as freeze drying), vacuum evaporation, nitrogen blow down and rotary evaporation. Several factors determine the optimal method for concentrating heat sensitive samples — sample volume, sample quantity, required temperature range, and solvent(s) being evaporated. These sample concentration methods and factors will be addressed in the following discussion.
 

Sample Size and Number

What is the sample volume and how many samples will you process per run? 

·       Lyophilizers handle a wide range of sample sizes and formats. Large volumes can be placed directly into flasks or on bulk trays. Micro plates, microtubes and test tubes can be placed inside a flask or used in bulk chambers with shelves. Serum vials that need to be stoppered can be put directly onto cooled shelves and sealed under nitrogen or vacuum after lyophilization is complete. The limitation of sample size is based on the freeze dryer’s capacity. 

·       A vacuum concentrator typically handles smaller samples, up to 25ml per tube. Rotors can run up to 250 micro tubes, as well as plates, auto sample vials, test tubes and other small containers. 

·       A nitrogen evaporator can process microtiter plates and flasks up to 400ml. Test tubes and vials can be used in a nitrogen evaporator, but for larger volumes, glassware specially made to fit the specific evaporator may be required.

·       Rotary evaporators run large samples up to 25L, but can only process one sample at a time. Specialty round bottom glassware with ground joints is used for a tight vacuum seal during the process. 

Temperature

It is important to know the temperature range that will maintain the integrity of your sample.  Some samples are not only heat sensitive, but can also be damaged by the freezing process. During the evaporation phase change, as solvent molecules enter the vapor phase they carry heat energy away from the liquid portion of the sample. This causes a phenomenon known as evaporative cooling, which results in reduced sample temperature and sometimes sample freezing. The evaporative cooling rate is highest during the early part of an evaporation process. During this time, it is possible to apply heat to the sample to prevent freezing without damaging the integrity of the sample. Heat is then reduced during the later stages of the process when the evaporative cooling is reduced.

In this example, the ability to control the heat set point is important to avoid sample damage during the later stages of process when the reduced solvent volume makes the sample more susceptible to heat damage.  Because there is a direct correlation between sample temperature and the solvent evaporation rate, the time required for the entire process is greatly affected by the amount of heat being added to sample. By following the temperature restrictions of your samples and monitoring the sample phase during the evaporation, protocols can be developed to produce the highest quality of samples in the shortest amount of time.

Solvents Used

A vacuum concentrator, nitrogen evaporator and rotary evaporator can typically handle most types of acids and solvents used, but the equipment must be designed to prevent damage from certain solvents or acids. For example, when HCl is used, it is best to use an acid resistant model to prevent degradation of the equipment’s components.

A lyophilizer's collector is typically stainless steel, but some can be coated with PTFE when used with solvents that may damage stainless steel. A lyophilizer also requires a temperature differential of 15˚ – 20˚ C between the eutectic temperature of the samples and the collector temperature — the collector being colder. Because of this requirement and because of their extremely low freezing point, alcohols can be difficult to freeze dry unless they are diluted to 10 – 15%. Evaporating the alcohols before lyophilization can be helpful to maintain the correct temperature differential.

Process

During the process of lyophilization, samples must first be pre-frozen. Pre-freezing can take up to 24 hours before the sample becomes solid. If the sample is temperature sensitive, pre-freezing could damage the sample prior to its concentration. If the sample is only heat sensitive, freeze drying is a good option. By using a tray dryer with a temperature control feature, shelves may be set and held at a temperature that will not harm the samples. If using a port-type freeze dryer, samples will eventually reach room temperature and is not recommended if samples are sensitive at ambient temperatures.   

If vacuum concentration is the method of choice, steps must be taken to prevent heating the chamber due to friction caused by the rotor hub. Some vacuum concentrators heat the chamber as high as 38˚ C, even if the temperature set point is ambient. By using a refrigerated vacuum concentrator, set point can be as low as -4˚ C to prevent the chamber from warming up. During concentration, evaporative cooling keeps the samples cold at the front end of the evaporation step. As the sample nears dryness, evaporative cooling stops and heat from the chamber can degrade sensitive samples. Using a refrigerated concentrator with controlled cooling keeps those samples from degrading after evaporative cooling ends.

Nitrogen blow down is another option for sample evaporation. Either using a dry block or water bath for evaporation, heat is typically used to quickly drive off liquid from a sample. If heat is not used, evaporation can take longer. As in the vacuum concentrator, evaporative cooling keeps the samples cool at the front end of evaporation, but the samples will warm to room temperature when evaporative cooling ends.

Because a rotary evaporator starts with large volumes, heat is recommended for fast evaporation and to help drive the molecules away from the collection flask into the recovery flask. A rotary evaporator spins the flask in a water or oil bath, which may cause friction from spinning, adding unwanted heat to the sample. 

Below is a chart showing the methods of evaporation above. 

Choosing the best method to evaporate samples doesn’t have to be guesswork. Another article that may help in your decision can be . By following these guidelines, you will be headed in the right direction to meeting your concentration needs.