Evaporation-Controlled Automated Embedding and Polymerization Background


Vacuum conditions in a transmission electron microscope require that liquid water is removed from specimens before introducing them into the column. This is usually achieved by dehydration, i.e. replacing the water by a solvent such as acetone which in turn is replaced by a resin suited for ultrathin sectioning after polymerization. Resin embedding is traditionally performed using a graded series of dehydration solvent and resin mixtures. In the first step, the solvent / resin ratio is in favor of the solvent. This warrants low viscosity and thereby easy penetration of resin components into the specimen. Sequential steps aim at a stepwise increase in the resin concentration until finally the specimens are in pure resin, ready for polymerization.


A typical sequence can be as follows: 3:1 Solvent: Resin 1:1 Solvent: Resin 1:3 Solvent: Resin Pure Resin (2x)


A protocol based on sequential steps can take up to 48 or even 72 hrs for specimens which are dense and hard to infiltrate.


Resin embedding When during the embedding procedure a solvent-resin mixture is exchanged for a higher resin concentration mix, only the bulk of the volume is replaced; the specimen will slowly follow suit when the solvent diffuses from the specimen into the bulk mixture. This is a slow process as it is predominantly driven by diffusion and thus depends on the temperature and the viscosity of the resin. The exchange is furthermore driven by the difference in concentration of the solvent (gradient) and therefore depends on continuous refreshing of the bulk mix close to the specimen surface by liquid movement. Hence, the traditional way of embedding depends heavily on mechanical movement.


Removing solvent in an alternative way Instead of removing solvent using sequential steps with increasing resin concentration, solvent can also be removed by making use of the volatility of the solvent. Common solvents for dehydration and embedding are highly volatile whereas most resin components do not evaporate readily. Evaporation depends on the kinetic energy of solvent molecules. If the kinetic energy is high enough, a molecule can escape from a liquid into the gas phase. From the liquid point of view the evaporation rate is therefore related to the temperature of the liquid but also to the interaction the solvent molecules may have with other molecules such as resin components. From the gas phase point of view the evaporation rate depends on how easily molecules can stay in the gas phase. This is temperature


Table 1: Antoine Constants for common solvents


Solvent AB C Acetone


Acetonitrile Chloroform Ethanol


Methanol Water


T-range Vp 25°C


7.1327 1219.97 230.653 -64 – +70 31 kPa 7.33986 1482.29 250.523 -27 – +82 12 kPa 6.95465 1170.97 226.232 -10 – +60 8.20417 1642.89 230.3


-57 – +80 8 kPa


8.08097 1582.27 239.7 +15 – +100 17 kPa 8.07131 1730.63 233.426 +1 – +100


Data from DDSST GmbH, Germany.


An on-line calculator and source for the Antoine equation constants can be found at: http://ddbonline.ddbst.de/AntoineCalculation/AntoineCalculationCGl.exe


Data for propylene oxide were obtained from: http://www.lyondellbasell.com/techlit/techlit/2710.pdf


3 kPa


Figure 1: Saturated vapor pressure (kPa) vs temperature (˚C) of common solvents. At pressures slightly higher than the values on the vapor pressure line the solvent will not boil but still evaporate. At pressures below the vapor pressure line the solvent will quickly evaporate by forced boiling.


26 kPa


related as well, but also depending on the concentration of ‘solvent’ gas molecules and the presence of other molecules in the gas phase. A saturation condition is reached when as many molecules evaporate from the liquid surface as there are molecules from the gas phase returning to the liquid phase. This saturation condition is characteristic for each solvent and is reflected in the saturation vapor pressure. Saturation conditions and even near-saturation conditions are to be avoided for effective solvent removal. This is realized when the evaporated gas is continuously removed or diluted by forced movement of air over the liquid surface: an open container holding a volatile solvent such as acetone ‘dries’ more quickly at higher temperatures and under forced air flow. Air flow and on-going removal of the gas under vacuum prevent saturation.


Solvent vapor pressure Vapor pressure, or more accurately saturation vapor pressure, is the pressure resulting from evaporation when the number of molecules leaving a liquid equals the number that return to it from the vapor/gas phase. The vapor pressure increases with temperature. By definition, at the boiling point of a solvent the vapor pressure equals 1 atmosphere (101.3kPa). Under lower pressure conditions the boiling point is reached at lower temperatures. The vapor pressure graph presented in figure 1 shows the relation of vapor pressure and temperature. The data were calculated using the so-called Antoine equation, which in its turn derives from the Clausius-Clapeyron relation that describes the tangent at any point of the coexistence curve of a phase diagram.


Antoine equation: Log(p) =A-C/(B+T)


where p =pressure (mmHg) and T temperature in ˚C. A, B and C are substance specific constants which are valid for a limited temperature range. Table 1 shows a list of constants for common solvents. Please note that these values apply to pure substances only. Mixtures incorporating the solvent will show lower evaporation rates and vapor pressures will take longer to establish, especially with gradually decreasing solvent concentrations.


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