Sample Preparation & Processing Understanding Evaporation and Concentration Technologies Dr Induka Abeysena, Application Specialist & Rob Darrington, Product Manager, Genevac Ltd, Ipswich, UK.


This paper explains the basic principles of evaporation and concentration, and outlines some of the commonly used technologies. It also reviews the wide variety of systems used in evaporation, including pumps, cold traps and evaporators themselves.


Introduction


Solvent removal is an essential process across a broad range of applications in the pharmaceutical, chemical and biotechnology industries. A diversity of sample formats and solvents is used with no single technique for solvent removal providing a universal solution. Various commercial evaporation and concentration systems have been developed to accommodate the range of applications. These systems and associated hardware, vacuum pumps, cold traps and heating technologies, have recently benefited from exciting developments in freeze drying and centrifugal concentration technologies enabling enhanced evaporation performance and improved sample integrity. Further advances with new generation high-power cold traps are offering improved solvent recovery, thereby reducing the environmental impact of the evaporation / concentration process.


Having an up-to-date understanding of the processes of evaporation and concentration, their application at a practical level and using the latest equipment enables optimisation of protocols for improved and more rapid sample concentration.


Processes Involved With Solvent Removal


During solvent removal, energy is applied as heat such that the liquid is vaporised to gas, which is removed to leave a concentrated or solvent-free (dry) product. Many systems are referred to generically as ‘evaporators’. However, true evaporation is vaporisation to gas at the liquid surface; for many “evaporators”, boiling occurs rather than evaporation. The process of freeze drying involves neither evaporation nor boiling, but sublimation; that is, a shift from solid to vapour phase without a liquid phase.


The phase of a substance is determined by two major factors – heat and pressure – and the temperature at which boiling or vaporisation occurs is set by the pressure. Therefore, vacuum concentrators apply vacuum in the system to decrease a solvent’s boiling point, such that liquid vaporisation occurs at lower temperatures, e.g. water boils at 7.5°C at 10 mbar pressure. Similarly, in freeze dryers, heat energy supplied to a frozen sample at low pressure transfers sufficient energy for thawing but the pressure is insufficient for liquid formation, and hence the solvent sublimes to gas. Generated vapour is removed by a cold trap or condenser, where solvent is recovered.


Heat and Temperature


Solvent removal systems use an input of heat energy to induce solvent vaporisation, various heating mechanisms are employed, such as electrical heating blocks, lamps or low-temperature steam. Heat and temperature, although linked, are different and distinguishing between them is important. Heat refers to heat energy measured in Joules, whereas temperature measures the level of heat energy, i.e. the hotness or coldness of an object. Samples referred to as heat- sensitive are typically temperature-sensitive and a majority of samples can be heated without degradation provided temperature remains within defined boundaries. Applying vacuum in a system decreases a solvent’s boiling point, such that liquid vaporisation occurs at lower temperatures that are safe for the sample.


Heat and temperature are related by the equation Q = mcΔT, where Q is heat energy added, m is the mass of the object, c is the specific heat capacity of the heated object, and ΔT is the change in temperature. ΔT can be expressed in terms of heat added as ΔT = Q/mc. These equations hold true when all other parameters remain the same. However, at a change in


phase, added heat energy does not increase temperature since energy is required for the change in state, for example from liquid to gas. Therefore in true evaporative systems (no boiling), a sample is at the temperature of the system controlling it; whereas in a freeze dryer that actively freezes products, the sample is at the temperature at which it is frozen and then it’s temperature is governed by the ice sublimation temperature controlled by the vacuum level.


The opposite dynamic exists in vacuum concentrators that boil solvent. When a sample is wet and boiling, the sample is at the boiling temperature of the liquid. Figure 1 shows the relationship between boiling point and pressure for some common solvents. At this stage, it is possible to heat the system to high temperatures and a sample will not reach this temperature until the solvent is completely removed. Only when all solvent is removed will a sample warm to the temperature of the system. Accurate control and monitoring of sample temperature is


Figure 1. Relationship between pressure and boiling point for some common solvents


therefore essential. Typically control of the sample holder temperature offers effective protection, as the sample within cannot exceed this temperature unless the sample is heated directly and independently of the holder. To enable this necessitates using solid metal holders constructed from materials such as aluminium that can transmit the maximum amount of heat into the sample and are cooled by evaporating solvent. Real-time temperature monitoring is built into some systems, enabling accurate determination of when evaporation is complete thereby avoiding overheating of samples.


Different Drying Methodologies


Freeze Drying Freeze dryers are available as two basic types: one that actively freezes samples placed on chilled shelves similar to a laboratory freezer; the second type of (passive) systems do not actively freeze and instead utilise a manifold with attached flasks that contain sample either directly or within vials. A high vacuum is often employed such that samples remain frozen and hence well preserved as solvent sublimes and is collected in the cold trap. A typical freeze dried product is a diffuse ‘fluffy’ powder that has a very high level of dryness (due to the large surface area available for solvent removal) and is easy to weigh and redissolve. Some samples, such as DNA, may require careful handling during movement to avoid loss of the fine powder. Freeze drying is a comparatively slow batch process, although a range of configurations are available that can accommodate large batches of samples per cycle. Solvent bumping may occur, although this can be reduced by pre-freezing samples where feasible. The freezing process limits the technique to aqueous solutions or one of a few simple organic solvents that freeze easily e.g. tertiary butanol or 1,4-dioxane. Samples containing volatile solvents must be actively frozen at very low temperatures, which may demand vacuum control at very low pressure and be so cold that the condenser functions inefficiently.


Centrifugal Concentration


Centrifugal concentrators induce solvent boiling under vacuum and hence samples are cold but, in contrast to freeze dryers, not frozen, and so the process can be faster than freeze drying. Care has to be taken with centrifugal evaporation of aqueous samples that are prone to freezing. Centrifugal evaporators use cold traps to recover the vapourised solvent.


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