A solvent solution

Removing high boiling point solvents when you can’t just turn up the heat can be tricky - we find out there is another way

The removal of solvents from thermolabile samples, typically biological, by the technique of centrifugal evaporation has been well established over the last 30 years. Samples are protected by evaporation of solvents at temperatures below or close to ambient in a partial vacuum. However, evaporator technology is put to the challenge when dealing with high boiling point solvents.

A High Boiling Point (HBP) solvent is usually taken to mean a liquid with a boiling point significantly higher than that of water. Figure 1 shows a range of solvents, with two well-known HBP solvents – DMF (Dimethylformamaide) (pink) and DMSO (Dimethylsulfoxide) (black) shown to the left of water (in yellow) on the graph. Other commonly used HBP solvents include NMP (n-Methyl-pyrrolidone) and DMAc (Di-Methyl-Acetamide).

Using a modern centrifugal evaporator and correct programming these apparently difficult, slow to remove solvents can be evaporated in a reasonable time. In principal removing HBP solvents should be straightforward providing that you heat the evaporator chamber to prevent condensation and also use as close to full vacuum as you can reasonably get.

Using a modern centrifugal evaporator – volatile solvents such as methanol will evaporate at a boiling point of -20°C or below. A very cold condenser is required to capture the vapour. But with HBP solvents, the boiling points of the solvents may be as high as +25°C or more. This presents an entirely different challenge, because the vapours condense too easily. They will condense on any object with which they come into contact that is below this temperature. If the right precautions are not taken, this could include the chamber walls, the rotor or the pipework to the condenser.

Preheating the evaporation chamber is possible on most centrifugal evaporators, switch on the heaters and wait until it achieves 40°C before you start the evaporation process. More advanced systems can do this for you, with a "Wait to Heat" function which means that the system will not start until the chamber has reached at least 40°C. A certain delay is associated with pre-heating the chamber. Some users short of time may wish to circumvent this delay. However, doing so is a false economy as the system cannot achieve the objectives without establishing the proper conditions. The correct approach is to plan ahead. Chamber temperature must not exceed that of the safe operating temperature for the samples, or it may cause them to be overheated and risk degradation.

Inside the evaporator chamber, stationary infrared lamps shine onto the metal swing or sample holder (depending on format used) as it spins past, and heat is then conducted from there into the samples. It is this heat flow that enables the removal of solvent. When the solvent is boiling, the sample temperature is governed by the boiling point of the solvent, but when the solvent is gone and the sample is dry, the sample temperature rises up to meet the temperature at which the sample holder (or swing) is controlled. Remember, at sea level boiling water cannot exceed 100°C until all of the liquid has been converted to vapour. At higher altitudes the boiling point (this fixed maximum temperature) is reduced and at even lower pressures this temperature is again reduced. So controlling the vacuum level controls the maximum temperature of the sample until it reaches dryness. Thus accurate vacuum level control is critical to sample protection.

To ensure that samples are not overheated the swing (or sample holder) must be kept at a temperature no greater than the maximum permissible temperature for the sample, throughout the evaporation run. In such a set-up the amount of heat flow into the sample is governed by the heat flow resistance (related to the design of the sample holder and swing) and the temperature difference between the swing (or sample holder) and the sample itself.

For example, if the sample temperature must not exceed 40°C, you must ensure that the swing will never go above this temperature. With a typical volatile solvent, the boiling point might be -20°C or less, so there would be 60°C temperature difference between the swing and the boiling sample. This temperature difference will allow substantial heat to flow into the sample, and boiling will be rapid.

To remove a HBP solvent such as DMSO – for evaporation at 2 mbar the solvent boils at around 38°C (see Figure 1). With only 2°C difference between swing and solution the heat flow is negligible and evaporation of the DMSO would be very slow. However, if the pressure is reduced to 1 mbar the boiling point is close to 27°C. The 13°C difference between the swing (at 40°C) and the boiling solvent will remove the solvent 6.5 times faster than at 2 mbar. If 0.5mbar can be achieved, the boiling point drops to 18°C, 22°C difference, resulting in 11 times faster evaporation/concentration than at 2 mbar.

In this case, the principle is clear for HBP solvents: the lower the pressure (high vacuum), the faster the removal of solvent. This cannot always be extrapolated to more volatile solvents for reasons of condenser performance.

Leaks anywhere in your evaporator system will have a significant impact on run times. All evaporator seals should be checked regularly and all pipe joints must be made air-tight after maintenance. Also the performance of your evaporator vacuum pump is crucial. Some pumps feature "gas ballast" to give the pump better solvent resistance but this reduces the ultimate vacuum level. Additionally it is important to know exactly the maximum permissible temperature to maintain the integrity of your sample in order to minimise evaporation time. Every extra degree that it is safe to use will help reduce evaporation time.

Evaporation of high boiling point solvents can be relatively straightforward given suitable equipment, namely a vacuum concentrator which can achieve a pressure below 0.5 mbar, and is able to heat the concentrator and sample holders in a controlled manner. Controlled use of very high heat during the early stages of evaporation can help gain a significant reduction in evaporation time, as can highly conductive sample holders which route heat energy directly to the boiling solvents.