Critical Point Dryer
For preparing biological samples
Starting at 120 bars pressure, CO2 is a liquid.
If we decompress at 10°C (red line from 1→B), the P-T path crosses the liquid vapor curve, resulting in a (very tiny!) violent explosion of the cell sample as high density liquid boils into low density gas.
If we decompress at 50°C (green line from 2→3), the P-T path crosses from the supercritical fluid zone into the vapor (gas) state with a very gentle, gradational transition in density without rapid expansion, thereby preserving the cell's detailed features.
Keeping biological samples from exploding or shriveling in the SEM
The SEM chamber contains no air - it is kept in a vacuum state so the electron beam can get to the sample without being deflected by gas molecules. This is no problem for most geologists, chemists, physicists, archeologists, and artist users of the SEM. Biologists, however, study samples that are basically billions of tiny water balloons.
Water boils at 100°C (aka 212°F)...at sea level atmospheric pressures. Lower pressures at high elevations make it easier for water molecules to jump from a liquid to a gas state, so boiling temperature decreases with decreasing pressure. The vacuum in the SEM sample chamber is 1 × 10‒6 mbar (= 0.000001 mbar), which is 1 billionth of sea level atmospheric pressure! Water boils as soon as we get the sample into the chamber. When water boils, the gas expands and the biological specimens either pop! on a cellular level, or leak water more slowly and shrivel into raisins.
We could simply dry samples out by leaving them in the sun for a few hours, but that's how we make raisins. The reason slow drying of water makes raisins is because water has a high surface tension - it sticks to the sides of the cells. As the volume of water in the cell decreases, the walls of the cell get pulled together to form shriveled shapes.
Carbon dioxide, on the other hand, can exist as a liquid with nonpolar molecules that do not have so much surface tension. When CO2 evaporates, the sides of the cell do not get pulled together by cohesion, so the original shape of the cell can be preserved.
The critical point drying process first very gently replaces the water in the cells of biological specimens with liquid CO2 (usually by replacing the water with alcohol first, the replacing alcohol with liquid CO2 ). Getting directly from point A conditions where CO2 is liquid to point B where CO2 is gas without changing temperature will destroy the sample. The direct A→B path crosses the liquid-vapor boiling line, at which point the dense liquid will suddenly boil into an expanding gas, exploding the sample.
Critical Point Drying supercritical fluid path
The critical point dryer follows a circuitous path to get from point A conditions where CO2 is liquid to point B where CO2 is gas. It's still a path that gets us from A→B, but in a gentle fashion. The critical point dryer first raises the pressure of the sample from Ⓐ to point ①, then raises the temperature from a point in the liquid field into the supercritial fluid field ②. This transition happens with a gentle, gradational change in fluid density, so there is no catastrophic popping.
Maintaining constant temperature and decreasing pressure from point ② to point ③ is also a gentle, gradational change in properties from supercritical characteristics to vapor- (gas) like characteristics. Finally, the critical point dryer lets the sample cool to room temperature Ⓑ (a cold room as I've drawn this).
Voilà! A→B without distorting or damaging the sample by taking a roundabout way. The path is much gentler than the curve I've drawn here because I had to fit the graph on this page.
Using the Critical Point Dryer
We've been quite happy with the Tousimis Autosamdri-815. Several of our geologists use it for studying neotaphonomical processes (modern decomposition of things that become fossils).
The sample prep process is:
stabilize (fix) cell tissues by soaking in glutaraldehyde (this toughens the cell membranes so they don't distort during soaking and other handling), then soak sample in a series of increasingly pure ethyl alcohol solutions (some recipes also soak in acetone solution, depending on the type of sample)
load sample holder into chamber
pour a little 100% ethanol into chamber to keep things moist
secure the sample chamber lid with the knurled nuts
Adjust the lengths of time spent getting from point Ⓐ to ① to ② to ③ to Ⓑ by turning the vernier knobs.
Make sure the liquid CO2 is hooked up and valve opened to the regulator, press the start button, and the rest happens automatically.
The whole process takes overnight plus a morning, but almost all of that time is just samples soaking in different solutions, so it's zero trouble.