Critical Point Dryers technique - SPI Supplies
SPI-DRY™ Critical Point Dryers
An introduction for those not familiar with the technique
The Technique
Critical point drying is a method of drying tissue without collapsing or
deforming the structure of wet, fragile specimens, generally as part of
the sample preparation process for scanning electron microscopy (SEM).
Although it has some applications in transmission electron microscopy
(TEM), its major application, at least up until now, has been in tissue
preparation for SEM.
It is well known that allowing tissue to dry in air
or under vacuum (during the metallization process, for example) causes
damage to the surface which one wishes to examine in the SEM.
An example
is shown in the micrographs shown below:
SEM image (850X) of rose petal surface, CPD
SEM image (850X) of rose petal surface, fixed and air dried
In some cases, this deformation is acceptable during routine experiments,
but for the most part, it is not.
At one time, workers in the field tried
to "turn the other way" and pretend these drying artifacts did not exist.
But today's workers, pretty much worldwide, with their new high resolution
SEMs, will not tolerate drying artifacts.
And therefore, there is a
universal acceptance of the need for a CPD unit is virtually all life
science laboratories and even in some circumstances, depending on the
nature of the samples being examined, a growing number of materials
science laboratories.
Explanation of drying artifacts
The reason why tissue samples became damaged by normal air drying is that
very large surface tension forces are created in cavities of small
dimensions when there is a liquid/gas interface.
As tissue dries, the
liquid/gas interface travels through the surface of the material
collapsing the cavities between projecting structures.
In the case of
delicate liquid- containing samples, which become hollow when dried,
complete collapse often results.
The critical point drying method of
drying avoids these effects by never allowing a liquid/gas interface to
in this way the tissue is not exposed to surface tension forces.
The critical point:
The critical point of a liquid/gas system (e.g. water/steam, liquid
CO2/CO2gas) is its critical temperature and the
pressure associated with this temperature, that is, it is a point
Tc, Pc smaller on the T,P phase diagram.
the critical temperature, Tc the system is always gaseous and
cannot be liquefied by the application of pressure.
The transition from
liquid to gas at the critical point takes place without an interface
because the densities of liquid and gas are equal at this point.
If tissue is totally immersed in a liquid below its critical point
and the liquid is then taken to a temperature and pressure above
the critical point it is then immersed in gas (i. e. dried) without being
exposed to the damaging surface tension forces.
In order to carry out this procedure at a convenient temperature and
pressure, it is normal to replace the water (which has a very high critical
point) with some other liquid before carrying out the drying.
one substitution is usually necessary if the final liquid is not miscible
with water, e.g. if the final liquid is liquid carbon dioxide, the water
in the tissue is first replaced with acetone and then the acetone is
replaced with liquid CO2.
Another "route" is water-ethanol-Freon& 113-CO2.
because of environmental concerns, and the general unavailability any
longer of Freon 113, any route depending on Freon 113 is generally not any
longer practiced.
Liquid CO2 is the most usual drying medium
because it is inexpensive, convenient, and environmentally acceptable, at
least relative to Freon 113.
Nitrous oxide has also been used for this
purpose but it has never achieved much acceptance.
Fixation with
by the substitution of acetone or Freon 113 is carried out before
transferring the tissue to the
apparatus.
Final substitution with liquid CO2 and the drying
run are carried out inside the apparatus.
After the drying run, the
pressure is released and the dried tissue can then be metallized before
being inserted into the SEM for observation and imaging.
Usually, the
metal used is gold and this is done in a
The comparison micrographs shown above shows an example of tissue which has
been fixed in gluteraldehyde and osmium tetroxide, substituted with ethanol
and then Freon 113, and finally critical point dried from liquid CO2.
The difference between the two micrographs shows the obvious advantages of
using the technique of critical point drying on these kinds of samples.
The Apparatus Itself
The main body of the apparatus is a pressure vessel with integral water
jacket for heating and cooling.
The normal operating range of the pressure
chamber is 0-2000 psi and 10-50&C.
As can be seen in the
see various control valves, a thermometer, a pressure gauge and a support
stand are all attached to the vessel.
At one end of the cylindrical
chamber is a demountable window for viewing the process and in the
opposite end a removable access door for the specimen holder.
The viewing
window is an indispensable part of the design of the SPI CPD unit because
it is so important to make sure that there is no turbulence when the
CO2 is being run through the unit.
There are four pressure control valves. Built into the support column is an
over-pressure safety valve, sometimes called also a rupture disc, set at
Should this pressure be exceeded by overheating the chamber,
the value opens and reduces the pressure to ambient.
This rupture disc
must be replaced before the unit can be used further.
The manual value at the top rear of the body is used for admitting the
liquid gas to the chamber.
A transfer pipe with couplings is provided
for connecting the apparatus to a suitable siphon cylinder.
The manual
valve at the top front of the body is used for venting trapped air when
filling the chamber.
The valve at the bottom rear of the body is used
for draining transfer fluid after filling with liquid gas.
Both the vent and drain valves are used for causing a thorough mixing
action when substituting the transfer fluid with the liquid gas.
important that all transfer fluid be removed from the tissue and flushed
from the chamber if efficient drying is to be carried out.
Turbulence
and flushing are achieved by opening the inlet and drain (or vent) valves
simultaneously.
Care must be taken to ensure that the tissue remains below the level of the
liquid during this operation.
And one must avoid turbulence if their samples
are especially fragile in order to avoid artifacts and other damage.
The tissue holder consists of a boat shaped liquid holder in which are
placed tissue baskets with lids.
There is an automatic drain in the boat
which acts when the access door is closed with the holder inside the
This design fulfills two requirements:
that the tissue
remains wet during transfer to the apparatus and (b)
that the transfer
liquid can be totally removed before the drying run is started.
After replacing the transfer liquid (e.g. acetone, amyl acetate, Freon 113)
with the critical point drying liquid (e.g. liquid CO2) the
drying run can be started.
All the valves are closed and hot water is
circulated through the water jacket.
In the case of liquid CO2
raising the chamber temperature to 32&C causes a pressure rise from
about 800 psi to about 1150 psi.
At this point, the liquid/gas meniscus
becomes diffuse and then disappears.
The chamber now contains only gas.
The vent valve can be opened slightly and the gas bled off to leave dry
To ensure that recondensation of the liquid does not occur, we
recommend that the temperature should be taken at least 5 C& above the
critical point.
Safety precautions
Should any pressure leaks develop during the use of the apparatus, these
are easily fixed as nearly all the seals are standard nitrile rubber
O-rings or bonded seals.
For use with aggressive solvents and acetone,
we recommend the use of special EPDM bonded seals.
They cost a bit more
but they can be expected to last longer as well.
Return to:
2000 - . By Structure Probe, Inc.
All rights reserved.
All trademarks and trade names are the property of their respective owners.From Wikipedia, the free encyclopedia
Air-free techniques refer to a range of manipulations in the chemistry
for the handling of
that are . These techniques prevent the compounds from reacting with components of , less commonly
and . A common theme among these techniques is the use of a high
to remove air, and the use of an : preferably , but often .
The two most common types of air-free technique involve the use of a
and a . In both methods, glassware (often ) are pre-dried in ovens prior to use. They may be flame-dried to remove adsorbed water. Prior to coming into an inert atmosphere, vessels are further dried by purge-and-refill — the vessel is subjected to a vacuum to remove gases and water, and then refilled with inert gas. This cycle is usually repeated three times or the vacuum is applied for an extended period of time. One of the differences between the use of a glovebox and a Schlenk line is where the purge-and-refill cycle is applied. When using a glovebox the purge-and-refill is applied to an
attached to the glovebox, commonly called the "port" or "ante-chamber". In contrast when using a Schlenk line the purge-and-refill is applied directly to the reaction vessel through a hose or ground glass joint that is connected to the manifold.
An ordinary glovebox, showing the two gloves for manipulation, with airlock on the right.
The most straightforward type of air-free technique is the use of a . A
uses the same idea, but is usually a poorer substitute because it is more difficult to purge, and less well sealed. Inventive ways of accessing items beyond the reach of the gloves exist, such as the use of tongs and strings. The main drawbacks to using a glovebox are the cost of the glovebox itself, and limited dexterity wearing the gloves.
In the glovebox, conventional laboratory equipment can often be set up and manipulated, despite the need to handle the apparatus with the gloves. By providing a sealed but recirculating atmosphere of the inert gas, the glove box necessitates few other precautions. Cross contamination of samples due to poor technique is also problematic, especially where a glovebox is shared between workers using differing reagents,
ones in particular.
Two styles have evolved in the use of gloveboxes for . In a more conservative mode, they are used solely to store, weigh, and transfer air-sensitive . Reactions are thereafter carried out using Schlenk techniques. The gloveboxes are thus only used for the most air-sensitive stages in an experiment. In their more liberal use, gloveboxes are used for the entire synthetic operations including reactions in solvents, work-up, and preparation of samples for spectroscopy.
Not all reagents and solvents are acceptable for use in the glovebox, although again, different laboratories adopt different cultures. The "box atmosphere" is usually continuously deoxygenated over a copper catalyst. Certain volatile chemicals such as halogenated compounds and especially strongly coordinating species such as
can be problematic because they irreversibly poison the copper catalyst. Because of this problem, many experimentalists choose to handle such compounds using Schlenk techniques. In the more liberal use of gloveboxes, it is accepted that the copper catalyst will require more frequent replacement but this cost is considered to be an acceptable trade-off for the efficiency of conducting an entire synthesis within a protected environment
Main article:
A Schlenk line with four ports.
The other main technique for the preparation and handing of air-sensitive compounds are associated with the use of a Schlenk line. The main techniques include:
counterflow additions, where air-stable
are added to the reaction vessel against a flow of inert gas.
the use of
and rubber septa (stoppers that reseal after puncturing) to transfer liquids and solutions
, where liquids or solutions of air-sensitive reagents are transferred between different vessels stoppered with septa using a long thin tube known as a cannula. Liquid flow is achieved via vacuum or inert gas pressure.
A cannula is used to transfer
from the flask on the right to the flask on the left.
Glassware are usually connected via tightly-fitting and greased . Round bends of
with ground glass joints may be used to adjust the orientation of various vessels. Filtrations may be accomplished by dedicated equipment.
Commercially available purified inert gas (argon or nitrogen) is adequate for most purposes. However, for certain applications, it is necessary to further remove water and oxygen. This additional purification can be accomplished by piping the inert gas line through a heated column of copper , which converts the oxygen to copper oxide. Water is removed by piping the gas through a column of desiccant such as
or molecular sieves.
Air- and water-free solvents are also necessary. If high-purity solvents are available in nitrogen-purged , they can be brought directly into the glovebox. For use with Schlenk technique, they can be quickly poured into
charged with molecular sieves, and . More typically, solvent is dispensed directly from a still or solvent purification column.
Two procedures for degassing are common. The first is known as freeze-pump-thaw — the solvent is frozen under , and a vacuum is applied. Thereafter, the stopcock is closed and the solvent is thawed in warm water, allowing trapped bubbles of gas to escape.
The second procedure is to simply subject the solvent to a vacuum. Stirring or mechanical agitation using an
is useful. Dissolve once the solvent starts to evaporate, noted by condensation outside the flask walls, the flask is refilled with inert gas. Both procedures are repeated three times.
After being refluxed with sodium and benzophenone to remove oxygen and water, toluene is distilled under inert gas into a receiving flask.
Solvents are a major source of contamination in chemical reactions. Although traditional drying techniques involve
from an aggressive , molecular sieves are far superior.
Drying of toluene
Drying agent
Duration of drying
water content
Sodium/benzophenone
3 A molecular sieves
Aside from being inefficient, sodium as a desiccant (below its melting point) reacts slowly with trace amounts of water. When however, the desiccant is soluble, the speed of drying is accelerated, although still inferior to molecular sieves.
is often used to generate such a soluble drying agent. An advantage to this application is the intense blue color of the
. Thus, sodium/benzophenone can be used as an indicator of air-free and moisture-free conditions in the purification of solvents by distillation.
Distillation stills are fire hazards and are increasingly being replaced by alternative solvent-drying systems. Popular are systems for the filtration of deoxygenated solvents through columns filled with activated .
Drying of solids can be brought about by storing the solid over a drying agent such as
5) or , storing in a drying oven/vacuum-drying oven, heating under a high vacuum or in a , or to remove trace amounts of water, simply storing the solid in a glove box that has a dry atmosphere.
Both these techniques require rather expensive equipment and can be time consuming. Where air-free requirements are not stringent, other techniques can be used. For example, using a sacrificial excess of a reagent that reacts with water/oxygen can be used. The sacrificial excess in effect "dries" the reaction by reacting with the water (e.g. in the solvent). However, this method is only suitable where the impurities produced in this reaction are not in turn detrimental to the desired product of the reaction or can be easily removed. Typically, reactions using such a sacrificial excess are only effective when doing reactions on a reasonably large scale such that this by-reaction is negligible compared to the desired product reaction. For example, when preparing , magnesium (the cheapest reagent) is often used in excess, which reacts to remove trace water, either by reacting directly with water to give
or via the in situ formation of the
which in turn reacts with water (e.g. R-Mg-X + H2O → HO-Mg-X + R-H). To maintain the resultant "dry" environment it is usually sufficient to connect a
filled with
to slow moisture re-entering the reaction over time, or connect an .
Drying can also be achieved by the use of in situ
such as , or the use of
techniques e.g. with a .
Duward F. Shriver and M. A. Drezdzon "The Manipulation of Air-Sensitive Compounds" 1986, J. Wiley and Sons: New York. .
Brown, H. C. “Organic Syntheses via Boranes” John Wiley & Sons, Inc. New York: 1975. .
Williams, D. B. G., Lawton, M., "Drying of Organic Solvents: Quantitative Evaluation of the Efficiency of Several Desiccants", The Journal of Organic Chemistry 2010, vol. 75, 8351. :
Nathan L. Bauld (2001). . .
W. L. F. Armarego and C. Chai (2003). Purification of laboratory chemicals. Oxford: Butterworth-Heinemann.  .
Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K. and Timmers, F. J. (1996). "Safe and Convenient Procedure for Solvent Purification".
15 (5): 1518–20. :.
Rob Toreki (). . The Glassware Gallery. Interactive Learning Paradigms Incorporated.
Rob Toreki (). . The Glassware Gallery. Interactive Learning Paradigms Incorporated.
Jürgen Heck.
(reprint at Norwegian University of Science and Technology). .
. Technical Bulletin. .
R. John Errington. .
John Leonard, B. Lygo, Garry Procter. .
: Air-sensitive distillations
Air-free filtration
Air-free sublimation
Cannula: intra-bleed valve
Cannula: extra-bleed valve
Cannula: (Simple) no bleed valve
Cannula: two manifold system
Cannula: syringe valve
Teflon tap for air-sensitive NMR samples金沙江干热河谷区红地球葡萄单幅式小拱棚避雨栽培技术
cultivation technique of..
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金沙江干热河谷区红地球葡萄单幅式小拱棚避雨栽培技术
cultivation technique of single-row rain shelter of red globe in dry-hot valley region of jinshajiang
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