Critical Point Drying Preparation Techniques and Advantages
Here you can find information and downloads to explain the techniques and advantages of critical point drying. Details on this page cover:
(link to National Centre for Biotechnology Information website)
A summary of the critical point drying method
Critical point drying is an established method of dehydrating biological tissue prior to examination in the Scanning Electron Microscope. The technique was first introduced commercially for SEM specimen preparation by Polaron Ltd in 1971. The original design concepts, which included a horizontal chamber, are still embodied in the design of the E3000 and E3100 CPD models.
In recent years we have introduced two further models: the K850, which features built-in chamber cooling and heating, and the K850WM, which is designed for drying a 100mm/4” silicon wafer.
All three models have found general acceptance in many laboratories throughout the world. Together, these critical point dryers offer the user a choice most suited to the particular specimen preparation requirements.
The phase diagram shows the pressure to temperature ranges where solid, liquid and vapour exist. The boundaries between the phases meet at a point on the graph called the triple point. Along the boundary between the liquid and vapour phases it is possible to choose a particular temperature and corresponding pressure, where liquid and vapour can co-exist and hence have the same density. This is the critical temperature and pressure.
Critical point drying relies on this physical principle. The water in biological tissue is replaced with a suitable inert fluid whose critical temperature for a realisable pressure is just above ambient. The choice of fluids is severely limited and CO2 is universally used today, despite early work with Freon 13 and nitrous oxide.
With CO2 a critical point of approximately 35°C can be achieved at a pressure of around 1,200psi. Therefore if the water is replaced with liquid CO2 and the temperature then raised to above the critical temperature, the liquid CO2 changes to vapour without change of density and therefore without surface tension effects which distort morphology and ultra structure.
Since liquid CO2 is not sufficiently miscible with water, it is necessary to use an intermediate fluid which is miscible with both water and liquid CO2. In practice intermediate fluids commonly used are methanol, ethanol, amyl acetate and acetone.
CO2 grades required for critical point drying
Generally speaking, the grade we recommend is ‘normal’ grade - that is the one most commonly offered by industrial gas suppliers.
In most parts of the world, ‘normal’ grade of CO2, from the suppliers, is specified as ‘N4.5’ or 99.995% minimum purity with a maximum level of 50ppm of impurities.
There are, however, two other grades available by special order from most gas suppliers. One is ‘N4.0’ or 99.99% (less pure than ‘normal’ grade). The other is ‘N5.5’ or 99.9995% minimum purity. The N5.5 purity is easier to find in those parts of the world where there is high level of activity in electronics, since these customers often demand gases with higher purities.
We are not aware of anyone who has ever reported either superior results using N5.5 purity vs N4.5, or inferior results using N4.5 vs N5.5. However, we do want our customers to have the benefit of such detail about liquid carbon CO2 procurement in the event they should ever find reason to believe that their particular specimens might benefit from the higher purity product.
Remember, the requirement is for LIQUID carbon dioxide and NOT gaseous carbon dioxide. For this reason a cylinder with an internal ‘siphon’ must be specified. A siphon cylinder is normally denoted by a white stripe painted along its length. No pressure regular is needed.
CPD7501: pressure testing
Over 160 CPD7501 units are in use today around the world. We know of no reported cases of structural failure. However, our CPD, in common with all similar instruments, includes a small pressure chamber and high pressures are generated inside the chamber during the drying process. The instrument is protected by a safety valve containing a thin diaphragm guaranteed to rupture at 1,850psi at 20°C. All chambers are pressure tested to 2,500psi before shipment to the customer. Each chamber bears the serial number of that test and is supplied with the test certificate.
In some countries local pressure vessel regulations, such as the UK Pressure Systems Safety Regulations 2000, require routine pressure testing and certification of pressure vessels, such as the chamber in the CPD.
If this service is required then it is recommended that the unit is returned to Quorum Technologies, or other competent bodies, for routine pressure testing and re-certification. We offer a standard service that includes seal replacement, pressure testing, re-certification and carriage-paid return. For pricing and further information, please contact us or your local distributor.
NB: Always refer to the equipment operating manual for details of specific safety points for the product concerned.
CPD7501: replacing safety bursting disc
Warning - potentially lethal voltages are used in this equipment. Before making/breaking connections to the equipment, ensure power is OFF, the unit isolated from the mains supply and that it is safe to proceed.
The bursting disc assembly (pictured) is located inside the unit, to the rear of the chamber, and is accessed by removing the rear panel.
(a) Ensure all gas and water controls are fully closed.
(b) Ensure the unit is switched off and disconnected from the mains electrical supply.
(c) Release the securing screws and remove the rear panel.
(d) With a suitable spanner, unscrew and remove the hexagon-headed insert from the top of the assembly.
(e) Using tweezers, or other suitable implement, extract the ruptured bursting disc and copper washer.
(f) Insert a replacement copper washer (E3000-053) and safety bursting disc (E3000-052).
(g) Refit the hexagon-headed insert, taking care not to over-tighten, as this will damage the disc and/or washer.
(h) Refit and secure the rear panel.
E3000/E3100 and CPD7501: CO2 transfer hose fitting (cylinder end)
The fitting sizes are:
USA: ½" BSP female (230004310)
UK and rest of the world: 0.860 x 14 TPI female (230003410).
E3000/E3100: bonded seal durability
All models of E3000 and E3100 are fitted with nitrile bonded seals. Nitrile is a good general material in terms of its ability to withstand attack by solvents, such as ethanol. However, if acetone is used as the transition fluid then the EPDM seals have been found to be more resistant to chemical attack by that solvent.
In our experience, the main reason for short seal life is insufficient removal of the transition fluid during the critical point drying process (ie flushing with liquid carbon dioxide is not aggressive enough or the duration of flushing is too short). This can result in residual amounts of transition fluid being left in the chamber, which can attack and break down the plasticizer element of the nitrile compound, and in turn lead to hardening of the seal and subsequent leakage.
Generally it is the window seal, rather than the door seal, that is more likely to be affected. This is because the door seal usually has more direct exposure to atmosphere and therefore any residual transition fluid can more readily evaporate.
E3000/E3100: critical point dryer rupture discs
E3000, E3100 and CPD7501 models of critical point dryers are pressure tested to a pressure of 2,500psi. All models are fitted with an E3000-052 rupture disc with a bursting pressure rated at 1,850psi.
The E3000-052 rupture disc is held in place with a copper washer (E3000-053). During removal of a punctured bursting disc the copper washer may be damaged. For this reason, it is advisable to fit a new copper washer at the same time.
E3000: pressure testing
High pressures are generated inside the chamber during the drying process. The E3000/E3100 is protected by a safety valve containing a thin diaphragm guaranteed to rupture at 1,850psi at 20°C. All chambers are pressure tested to 2,500psi before shipment to the customer. Each chamber bears the serial number of that test and is supplied with the test certificate.
In some countries local pressure vessel regulations, such as the UK Pressure Systems Safety Regulations 2000, require routine pressure testing and certification of pressure vessels, such as the chamber in the CPD.
If this service is required then it is recommended that the unit is returned to Quorum Technologies, or other competent bodies, for routine pressure testing and re-certification. We offer a standard service that includes seal replacement, pressure testing, re-certification and carriage-paid return. For pricing and further information, please contact us or your local distributor.
NB: Always refer to the equipment operating manual for details of specific safety points for the product concerned.
E3000/E3100: replacing safety bursting disc
(a) To replace the bursting disc in the safety valve, unscrew and separate the pressure vessel from the stand pillar by rotating the vessel anti-clockwise relative to the base.
(b) Unscrew and remove the top of the safety valve using appropriate-sized spanners.
(c) Remove and discard the copper ring and ruptured bursting disc from the safety valve body.
(d) Fit a replacement bursting disc (E3000-052) and copper ring (E3000-053).
(e) Re-assemble in reverse order.
(f) Should safety valve bonded seal need replacing, use E3000-050 (pack of five).
E3000/E3100: securing the bonded door seal into door
Here is a simple way to prevent the bonded door seal from falling out of its recess while the E3000/E3100 Critical Point Dryer door is being screwed into place.
Bonded seals (pictured) consist of an outer metal ring with a nitrile or EPDM component bonded to its inner face. These seals (sometimes referred to as ‘Dowty seals’) normally push-fit into the recess in the E3000 and E3100 door. However, sometimes the fit is not exact and therefore when the door is positioned next to the CPD chamber the bonded seal can fall forward, making closure difficult. The solution is to very slightly deform the metal part of the seal so that it will then push-fit into the CPD door recess. The best way to do this is to grip the seal in a workshop clamping vice (across the circumference of the seal) and slightly deform the seal so that it push-fits into the recess in the CPD door.
NB: Bonded seals work by expanding sideways, and so a small deformation will not effect the sealing characteristics of the seal. All new E3000 and E3100 Critical Point Dryers are fitted with seals that have been carefully adjusted to be a push-fit.
E3000/E3100: viewing window maintenance
(a) To remove the viewing window, undo the four hexagon socket screws and remove the clear plastic safety guard.
(b) Unscrew and remove the viewing window slotted retaining ring, being careful not to disturb the end plate covering the water jacket.
(c) Clean and inspect the retaining O ring seal. Replace if hardened or damaged.
(d) Remove the viewing window and bonded seal.
(e) Clean and inspect the window and seal. Replace if damaged.
(f) Re-assemble in reverse order, taking care not to damage the window edges.