Evaporation-Controlled Automated Embedding and Polymerization (cont.)


An example applying to acetone dehydration


and epoxy embedding Many ultra structural studies involve the use of epoxy resins and acetone. Acetone is highly volatile. The vapor pressure curve for acetone is singled out and presented in figure 2. From the graph it is clear that at 25°C the vapor pressure is ~30 kPa, in other words acetone boils at 25°C if the pressure is lowered to 30 kPa or below. In the case of boiling evaporation occurs not only at the surface of a solution, but throughout the whole volume. This is unfavorable for embedding mixtures as it leads to splashing and potential loss of specimens. At pressures slightly above the saturated vapor pressure, evaporation is still proceeding progressively but only from the surface of the mixture. This is a safe condition for a mixture of resin and acetone to gradually evaporate acetone from the surface. When a preset raise in pressure is exceeded, the pressure is released by air admission into the recipient up to a preset level. The evaporated acetone is diluted with air and removed by the vacuum pump. The dilution prevents that saturation will ever occur. The remaining mixture will become more concentrated in resin. Thus by carefully controlling temperature and vacuum pressure, air admission and flushing the acetone can be removed quantitatively and within hours; upon completion the specimens are left in for all practical purposes pure resin ready for polymerization after topping up with pure resin to compensate for the removed solvent.


Figure 2: Saturated Vapor Pressure of Acetone


Introducing the EMS


The EMS POLY III is an instrument for the embedding of specimens by the proper combination of pressure and temperature. Central to the instrument is a specimen chamber that is temperature controlled and which can be heated up from room temperature to 70°C. The pressure in the chamber can be reduced from ambient pressure to a controlled level with an inbuilt vacuum-pump. The instrument chamber accepts up to 52 BEEM specimen vials. The vacuum pressure the specimens are exposed to is controlled within narrow limits and warrants solvents will evaporate in a controlled way with the risk of explosive boiling. The exhaust of the vacuum pump is connected to a chemical vapor resistant hose, which can vent into a fume hood in compliance with laboratory practices and general health and safety regulations when working with volatile substances. The instrument features preset programs which can be modified according to the user’s preference. In the presets pressure and temperature settings have been coordinated and optimized for an efficient removal of solvent from the specimens eliminating the risk of boiling. Bulk removal of solvent is followed by steps for the thorough removal of trace amounts. As a practical approach the instrument can be loaded by the end of a workday and (when using acetone or propylene oxide) by the next morning the vials are ready for polymerization after the vials have been topped up with pure resin. A lengthy and sometimes tedious manual procedure now reduced to a few simple steps.


Alternatively, to free up the instrument for a next run, polymerization can take place in a stove. The instrument is (after cooling) immediately available for a next run.


Instrumentation


From the graphs it is obvious that fluctuations in temperature as well as in pressure may seriously influence the evaporation process. Without proper control of the vacuum pressure and the temperature the solvent may boil off explosively when the saturated vapor pressure is undercut. Furthermore, extraction of solvent forces the release of the latent heat of evaporation which leads to a significant drop in temperature in the mixture and concomitantly a drop in the evaporation rate. It is imperative that the conditions of pressure and temperature are thoroughly controlled.


Which solvents can be evacuated with the EMS POLY III? The instrument handles any of the solvents currently used in embedding protocols. In principle any solvent can be safely and gradually evaporated as long as the proper settings for pressure vs temperature are observed. The one important rule is that the pressure should initially NEVER be lower than the saturated vapor pressure at that temperature. Values can be calculated with the Antoine Equation or derived from the graph (Figure 1). For the bulk removal of solvent it is advised to work at a pressure that is approximately 10% higher than the saturated vapor pressure. Failing to do so may lead to sudden boiling of the solvent and spilling of mixture and specimens from the vials. The risk of sudden spilling is higher with more volatile solvents such as propylene oxide and instrument settings need to be accurate.


Important: at no stage should solutions be allowed to bubble. Gas bubble formation needs to be avoided to prevent splashing and damage to the specimens when bubbles should form inside the specimen.


Although extensive testing has been carried out, specific combinations of solvent and resin need to be tested for their suitability for embedding and polymerization in the Poly III on an individual basis.


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6