Concentration of Solutes: Options for the Removal of Solvents from Samples

Solvent removal from solutions is commonly performed in laboratory and production processes. There are a number of different methods that can be used for solvent removal, including evaporation, vacuum concentration, lyophilization, reverse extraction, solute precipitation, and dialysis (solvent exchange). The objective of solvent removal may be to preserve solutes, as is routinely performed on protein solutions, to concentrate solutes for analysis, or as a step in the synthesis or modification of solutes.

Following is a brief discussion of the options for removing solvents. Not included in this overview are chromatographic methods for concentrating solutes.

Evaporation

Solutes that are not volatile can be concentrated by drawing the solvent into a gaseous head space. Two approaches can be used for solvent removal, one being by boiling (by applying heat or a vacuum - see below) and the other by directing a stream of (inert) gas over the solvent. In this latter approach, the gas essentially extracts solvent from the liquid phase by dissolving it into a gaseous stream (followed by dilution into the atmosphere). This is the basis of gas chromatography, i.e., a gaseous mobile phase dissolving solutes and solvents. As the gas flows, the decreased concentration of vapor-phase solvent molecules shifts the vapor/liquid phase equilibrium, drawing more liquid solvent into the vapor phase. By streaming the gas, the evaporation rate is increased. Evaporation systems, such as the RapidVap Evaporation Systems, direct a gas stream usually of inert nitrogen over the sample. The RapidVap evaporators have the option of applying heat and agitation to the sample to help quicken the evaporation process.

Vacuum Concentration

The removal of solvents can be effectively accomplished by boiling. Unfortunately, many solutes such as proteins are destroyed by the heat required to drive off solvents. However, solvents can boil by either applying heat or by lowering the atmospheric pressure. In both cases, the energy of molecular motion is greater than the intermolecular forces holding the molecules in solution. The result is that solvent molecules escape from the liquid phase to the gaseous phase. The difficulty with applying a vacuum (or applying heat) is that the force by which molecules move from liquid to gas causes the solution to splatter, which is also called "bumping." This causes sample loss and/or cross contamination between samples when multiple tubes are positioned together. In vacuum concentration devices, such as the CentriVap Centrifugal Concentrators, a vacuum pump is attached to an airtight, low speed centrifuge that, when running, prevents bumping by forcing the liquid down into the tube. The system can then run at high vacuum levels to speed solvent removal. The CentriVap Centrifugal Concentrators also have the option of regulating the centrifuge chamber temperature, which is useful for regulating sample temperatures at lower vacuum pressures.

Lyophilization (a.k.a., freeze drying)

Similar to vacuum concentration, the process of lyophilization goes one step further by lowering sample temperature to the point where the solution freezes and solvents are removed by sublimation. The freezing step can be done in the same preparation step or caused by the application of a vacuum which, in the process of removing the atmosphere, also removes heat. Normally the solution is always frozen before the vacuum is applied. Lyophilization can be a relatively complex process that is usually performed in multiple stages. The first stage is sample freezing, which is critically important to the overall process. Slow freezing of a sample causes large ice crystals to form which makes freeze drying easier, but may denature many temperature sensitive proteins. Freezing a sample rapidly results in small ice crystals which can impede freeze drying, but many proteins retain activity when flash frozen. The second stage, primary drying, occurs when the sample temperature is raised sufficiently to allow heat to flow into the frozen solution and drive the sublimation process. However during primary drying, if the temperature increases too much, the sample can thaw and "collapse." Primary drying removes 90% or more of the solvent, at which time secondary drying, the third stage, can commence by increasing sample temperature. Secondary drying is feasible as once the bulk of the solvent is removed during primary drying, the risk of melting is lessened. Secondary drying drives out residue solvent by applying greater amounts of heat. For instance, mannitol undergoes primary drying at temperatures below -23°C (depending on the formulation) while secondary drying is as high as 40°C.

Lyophilizers, such as the Labconco FreeZone Freeze Dry Systems, are an extremely effective tool for removing relatively large volumes of solvents while retaining activity of sensitive solutes. Freeze drying is very effective for concentrating and preserving biologically active proteins.

Reverse Extraction

Though extraction is traditionally viewed as a method to transfer a solute from one liquid phase (or solid) to another, it can also be used for solvent removal. The classic example of this approach is concentrating small volumes of DNA in aqueous buffer by adding dry n-butanol. Water is miscible with the alcohol while DNA is not. The net flow of water into the butanol phase results in a higher concentration of DNA in the remaining (original) aqueous buffer.

Dialysis

Semi-permeable membranes have been used for decades for removing small solutes (primarily salts) and solvents from solutions. Centrifugal concentration through a semi-permeable membrane and dialyzing solvents by mass action are typical methods for concentrating solutes by dialysis. In both cases, membranes with controlled pore size allow low molecular weight solutes and solvents to pass through the membrane while retaining the larger molecules. Centrifugal concentrators use centrifugal force to push the solution through the membrane while dialysis utilizes diffusion. Solvents can be removed by dialysis against concentrated solutions containing large molecular weight compounds or against a substance in the solid phase miscible in the dialyzed solvent. For example, water can be removed from a solution housed in dialysis tubing by placing the tubing in starch. As long as the pore size in the dialysis tubing is too small for a starch molecule to pass through, the water will move from the sample and solubilize the starch.

Precipitation

Altering a solution to render solutes insoluble is a long used method for concentration. This approach is routinely used to concentrate DNA by precipitation with salt and ethanol, and "salting out" proteins from solution by adding ammonium sulfate. Precipitation is practical if the solute is inert to the precipitation conditions. However, there is the possibility that biologically active molecules with labile structural motifs may become denatured and lose activity. Precipitation is especially practical for concentrating robust biomolecules such as carbohydrates, nucleic acids, and many structural proteins.

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