Critical Point Drying - a technical brief

Introduction

The advent of Scanning Electron Microscopy (S.E.M.) in the study of surface morphology in biological applications made it imperative that the surface detail of a specimen was preserved. The air (evaporative) drying of specimens causes deformation and collapse of most specimens, the primary cause of such damage being the effects of surface tension. The specimen is subjected to forces which are present at the phase boundary as the liquid evaporates. The most common specimen medium, water, has a high surface tension to air. By comparison that for acetone is several times lower. The surface tension could be reduced by substitution of a liquid with a lower surface tension with expectations of reduced damage during air drying.

However, the occurrence of what is known as 'continuity of state' suggests a drying technique for which the surface tension can be reduced to zero. If the temperature of liquefied gas is increased the meniscus becomes flatter indicating a reduction in the surface tension. If the surface tension becomes very small the liquid surface becomes very unsteady and ultimately disappears. When this 'critical point' is reached, it is possible to pass from liquid to gas without any abrupt change in state. If a specimen had been in the liquid it would have experienced a transition to a 'dry' gas environment without being in contact with a surface, avoiding the possibility of the damaging effects of surface tension. This is termed Critical Point Drying (C.P.D.) the basis of which are the classic experiments carried out over 100 years ago during investigations on the liquefaction of gasses.

The Critical Phenomena

The principle of the experiments which were initially carried out using Carbon Dioxide (CO₂) was to measure the change in volume with the application of pressure, of a fixed mass of gas, while maintaining the temperature constant and to repeat this for a range of different temperatures. The results are best understood by considering the graph obtained from plotting pressure (P) against Volume (V) for the series. This is indicated in Fig 1, the curves obtained are termed isothermals.

Fig 1.

Considering the 10⁰C Isothermal at low applied pressure, the C0₂ is gaseous (Vapour) and generally exhibits the characteristics of a gas (Boyles Law) over the range from 'r' to 's' . From the point 's' a very slight increase in pressure corresponds to a change from the vapour state to the liquid state, which is the phenomenon of saturation.From 's' to 't' the pressure is virtually constant while the volume is decreasing and at 't' the substance is all liquid.

From the point 't' the graph becomes almost vertical, indicating significant application of pressures for very little change in volume - liquids being virtually incompressible.

The 20⁰C Isothermal has similar general characteristics, however, there is less difference between points 'v' to 'w' compared to the difference in volume occupied, between the substances vapour and as a liquid.

This indicates that the densities of the saturated vapour and liquid are approaching each other, also the slight departure from the vertical 'w' shows the compressibility is greater than that at higher pressures. This evidence indicates that the properties of the liquid and gas states of the substance are becoming similar and will ultimately coincide. This in fact is realised at the 31.1⁰C Isothermal which does not show any horizontal discontinuity. The temperature at which this occurs is termed the Critical Temperature and has an associated Critical Pressure and Density and hence for a particular mass of gas, a Critical Volume. If a liquid was heated in a closed system so that the Critical Pressure could be attained, at the Critical Temperature, any visible meniscus would disappear, the surface tension would be zero and it would not be possible to distinguish between the properties of a liquid or gas. We therefore have continuity of state. Above this temperature the gas cannot be liquefied by the application of pressure and strictly speaking a substance should only be classified as a gas above its Critical Temperature, below this temperature where it could possibly be liquefied by the application of pressure, it is more precisely termed a vapour.

Critical Point Drying (C.P.D.)

It may now be apparent that we can utilise the Critical Phenomena as a drying technique, as it achieves a phase change from liquid to dry gas without the effects of surface tension and is therefore suitable for delicate biological specimens.

However, it is not surprising that the initial investigations were C0₂ as will be apparent from Fig. 2, showing a table of Critical Constants for some common substances. Even the practical achievement of the critical conditions would not assist the Biologist, as the specimens would suffer significant thermal damage if we attempted to apply the technique direct in the removal of water from the specimens.

Therefore, C0₂ remains the most common medium for which to apply the C.P.D. procedure, and is termed the 'Transitional Fluid', however, it is not miscible with water and we have to replace the water in the specimen with another fluid which is miscible with C0₂. This is termed the 'Intermediate Fluid'. Ideally and often it can also replace the water in the specimen, also serving as the 'Dehydration Fluid'. This is not exclusively the case, and additional steps may be used for particular circumstances. However, where it is being utilised for both processes, texts may refer to it under the different headings, Dehydration and Intermediate, depending at what stage it is being used in the specimen preparation schedule. Prior to any of these stages would be fixation of the specimen, this is typically a Glutaraldehyde-Osmium procedure.

Note :- The whole discipline of specimen preparation prior to the transitional stage is only mentioned in its most basic terms, procedures vary according to specimen and further references should be obtained.

1. Intermediate stage.

As mentioned previously, this involves dehydration and intermediate fluid, the following indicating a possible schedule.

(Wet Specimen) H₂0 > Acetone > C0₂ > CDP (Dry Specimen).

The specimen is usually processed through varying concentrations of dehydration fluid, culminating in complete replacement of the water with this intermediate fluid, because it has a low surface tension the specimen is less likely to experience damage due to evaporation while transferring to the chamber and being miscible with C0₂ (the transitional fluid) ensures satisfactory conditions after the flushing (purge) for the CPD process to commence.

(Wet Specimen) H₂0 > Acetone > 30%* -----100% > C0₂** > C.P.D. (Dry Specimen).

* 50/60/70/80/90 typically 10 mins. Each.

** Flush typically 3 times.

The table Fig. 3, gives an indication of some intermediate fluids. (Water is 73 Dynes/cm.)

Having transferred the specimen to the chamber in the Intermediate Fluid, the chamber is flushed several times to replace it with the transitional Fluid. The process, from which the complete technique derives its name C.P.D., can now be initiated.

1. Transitional Stage

It is apparent from the above(Fig. 1), the conditions for which the Critical Point Passage is obtained, (for C0₂, 31.1⁰C and 1072 p.s.i.) . However, it must be remembered that these Isothermals are obtained from a fixed mass of gas and an applied pressure for a series of constant temperatures. In the application to C>P>D. we have a fixed volume which is filled with the Transitional Fluid, some typical examples of which are given in Fig. 4. (Water is +374⁰C and 3212 p.s.i.).

Pressure is obtained by the effect of applying heat. While it can be readily appreciated that we can take liquid from below its critical temperature and obtain the transition to gas above its critical temperature, an understanding of the relevant 'start' and 'end' points and the cycle involved is required in evaluating the design and performance of C.P.D. equipment. It is still useful however, to utilise these CO₂ isothermals as indicated in Fig. 5, with the 'superimposed arrows' showing differing conditions for the C.P.D. device.

It is already acknowledged that this is not for the exact comparable circumstances, i.e. (for C.P.D. we would fill at CO₂ cylinder pressure and ambient temperature and not at the saturated vapour pressure at the lower temperature…the let down and decrease in pressure is as a result of venting and subsequent reduction in mass of gas: Not reduction in externally applied pressure…the relative volume is referring to the initial level of liquid in relationship to the total free volume available, this being the chamber plus ancillary fittings, associated with a practical system: not the variation in volume the substance experiences).

If we consider 'X' with the liquid CO₂ more than half filling the total available volume and we heat from 10⁰C to 35⁰C then we will make the transition from liquid to gas. The pressure rise will be rapid as the liquid expands and the level increased before the Critical Temperature is reached. This is termed 'going around' the Critical Point. Usually a pressure relief valve is employed to prevent excessive pressure increase. For condition 'Y', with approximately a half full condition, the liquid level will remain relatively constant, its density decreasing and that of the vapour increasing, becoming the same at the condition of critical temperature having been reached, together with the corresponding Critical Pressure.For the condition 'Z' with less half full condition, the level will fall, vaporisation occurring before the Critical Temperature is reached and the specimen may be uncovered and subjected to evaporation. Ideally, we wish to have a situation where the liquid fills the specimen chamber, while still only accounting for approximately 50% of the total volume available. This is to ensure that the specimens are not uncovered during initial flushing stages and in addition this should enable Critical Constants of Temperature, Pressure and Density to be achieved relatively simultaneously without the occurrence of either excessive pressure or evaporation conditions occurring.

Fig 6. The Chamber and Manifold arrangement of the K850 allows simultaneous achievement of Critical Temperature and Pressure.

It is also advisable to maintain a temperature somewhat above the Critical Temperature during the pressure let-down stage, to avoid the possibility of gas recondensing and also to control the letdown rate itself as there is evidence that time for pressure equalisation is advisable to avoid damage to the specimen.

*Emitech Ltd, Ashford, Kent.