SPI Supplies
25
SPI-DRY Critical Point Dryer
6.3.4
Substitution
After dehydration with ethanol, further substitution to amyl acetate or Freon 113 is necessary before
putting tissue in the pressure chamber. We suggest that the substitution should not be carried out
directly but through graded baths (25:75, 50:50 etc.) of the 2 liquids. 15 minutes per step should suffice.
6.4
Complete Operational Run
(a) Prepare the samples using either the method outlined in section 6.
(b)
Place the samples into either the mesh baskets or the microporous specimen capsules, making
sure not to allow them to air dry.
(c)
Place the mesh baskets or microporous specimen capsules into the liquid transfer boat, and fill it
with the substitution liquid.
(d)
Ensure that the CPD is properly installed, and that all connections, including water lines the CO
2
high pressure transfer hose, are tight.
(e)
Start the flow of cold water to cool the chamber to 20 C or below.
(f)
Load the liquid transfer boat into the chamber, and secure the rear door.
(g)
After ensuring that all of the control valves are closed, open the CO
2
tank valve.
(h)
Open the
VENT
valve slightly, to release trapped air in the chamber, and then fully open the
INLET
valve to rapidly fill the chamber with liquid CO
2
.
(i)
Leave the
INLET
valve fully open, and the
VENT
valve slightly open to maintain the liquid level.
Open the
DRAIN
valve to remove the substitution liquid. This flushing action should be continued
for 4-5 minutes.
(j)
Once all of the substitution liquid has been flushed from the chamber, fill the chamber with liquid
and close all of the gas control valves. Allow 1 hour for the liquid glass to impregnate the samples.
Please note, this time is only a guide for tissue samples less than 1mm thick. For larger or denser
samples, more time will need to be allotted for impregnation.
(k)
Repeat step (i)
(l)
Close the
INLET
valve and allow the liquid to fall to the level of the top of the liquid transfer boat.
(m)
Close all control valves, including the valve on the CO
2
cylinder.
(n)
Turn the cold water off, and turn the hot water (35 C
– 40 C) on. Slowly raise the temperature of
the chamber, and adjust the water temperature as needed.
(o)
While observing the CO
2
lever through the window, also monitor the
PRESSURE
and
TEMPERATURE
gauges.
(p)
Once the CO
2
has passed through the critical point (32 C, 1100 psi), turn off the hot water.
NOTE:
To avoid any uncertainty in reaching the critical point caused by thermal lag, it is
suggested to allow a suitable safety margin - ~36 C and 1200 psi.
(q)
Gradually open the
VENT
valve to release the chamber pressure. To avoid re-condensation of
the CO
2,
make sure to vent the chamber slowly (over a period of 4-5 minutes).
(r)
Once the chamber has de-pressurized, slowly open the
VENT
valve fully, to ensure the chamber
reaches atmospheric pressure before attempting to remove the rear door.
WARNING
The CPDA is a pressure vessel and operates at pressures in the range of 800 - 2000
psi (54 - 136 bar) which is potentially dangerous if handled carelessly.
WARNING
HAZARD TO HEALTH!
Risk of asphyxiation in poorly ventilated rooms.
As carbon dioxide is heavier than air, the concentration of exhaust gas will be
greater at ground level and the oxygen concentration correspondingly reduced.
