A Primer on Vacuum Concentration

Vacuum concentration (or centrifugal concentration) has been popular since the 1980s when it became a widespread application for drying plasmid DNA. The early vacuum concentrators were little more than closed centrifuges linked to vacuum pumps, and for the drying of samples in microcentrifuge tubes, such a evaporator design was satisfactory. Since its introduction the applications for vacuum concentrators have expanded dramatically and is now used for analyte concentration and solvent removal in addition to drying.

Evaporation vs. Concentration

Vacuum concentration removes one or more solvents from a solution by decreasing the atmospheric pressure over the liquid phase. By reducing the pressure exerted on the liquid phase, solvent molecules more easily enter the gas phase. The same effect is produced by applying heat. The resulting increase in solvent vapor pressure delivers more solvent molecules into the gas phase. Evaporation systems, such as the RapidVap, also work by exploiting the gas phase/liquid phase equilibrium at atmospheric pressure and room temperature. Evaporators pass inert gas over the solvent, thereby removing gas phase solvent molecules. The perturbation of the gas phase/liquid phase equilibrium concentrations results in more liquid phase solvent molecules escaping into the gas phase. In both cases, the result is a loss of solvent and a concentration of non-volatile molecules.

Bumping

Vacuum concentrators, such as the Labconco CentriVap Centrifugal Concentrators, include an enclosed centrifuge (air tight under vacuum), a solvent trap, and a pump, all of which are connected by vacuum tubing. By spinning the samples in a centrifuge and applying a vacuum, the violent boiling of solvents (bumping) is avoided. Boiling, also called "bumping," occurs when the external pressure over the surface of a liquid equals the vapor pressure of that liquid. Under these conditions, molecules easily break the intermolecular forces holding them in the liquid phase. Spinning the samples in a centrifuge prevents the sample from splattering while still allowing solvent molecules to escape into the gas phase. The solvent trap, which is often optional depending upon the type of pump used, catches and collects the evaporated solvent. Traps can be adsorbent cartridges that bind the vapor or cold traps that serve as a location for condensation. The vacuum pump removes both the initial atmosphere in the system and evaporated solvent. Pumps can be either low efficiency diaphragm pumps or high efficiency oil-based pumps. High efficiency pumps can reduce the atmosphere so efficiently that solvents can freeze.

Factors Affecting Solutes

A major factor affecting solutes during vacuum concentration is increasing salt concentration. As volumes decrease during vacuum concentration, both desired solutes along with other non-volatile solutes (buffer salts) are concentrated. Though many solutes such as plasmid DNA are not affected by salt, many proteins will denature as salt concentration increases.

Another factor dramatically affecting solutes, especially proteins, is temperature. As the gas above a solution is removed under vacuum, solvent molecules overcome intermolecular attractions and "jump" into the gaseous state. The evaporated molecules escape the liquid phase using their own internal energy, which lowers the liquid phase temperature. With aqueous solutions, the water can easily freeze when used in a centrifugal concentrator with a high efficiency vacuum pump. This freezing can be a problem as solutes and even biological components (e.g., organelles, membrane fractions, and protein complexes) can be irreversibly denatured or damaged by the freezing process. To prevent freezing, the chamber pressure must be increased. Unfortunately, this will slow the rate of evaporation.

Factors Affecting Evaporation Rates

Applying heat to the sample during vacuum concentration will increase the rate at which solvents evaporate, however, with high efficiency pumps, the atmosphere is essentially eliminated and heating the samples by convection is impractical. With no atmosphere, heat can’t be transferred from the chamber walls to the sample. Alternatively, when using a diaphragm vacuum pump sufficient atmosphere remains in the centrifuge chamber so that heat can be applied from the walls of the chamber to the samples on the rotor.

The chemical characteristics of the solvent itself are also a major factor in the rate of evaporation. Volatile liquids, such as small alcohols and many organics, readily evaporate without added vacuum. The time required to remove volatile solvents as compared to water is relatively short.

Many biological molecules are sensitive to changes in temperature. Proteins can be sensitive to extremes of both heat and cold. Prior to concentrating biological molecules, it is important to determine any stability issues.

For more information see Developing a Vacuum Concentration Protocol.

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