Freezedrying

This is one of the best methods for drying sensitive materials, but has relatively little mention in the literature. It is the only way water can be almost completely removed from tissue or organic material with minimal damage to the cell structure. The fresh herbage is first deep frozen as soon after harvesting as possible. It is transferred to the freeze-dryer chamber, and the methodology used is described below. A vacuum is applied, and a controlled supply of heat may be provided. This is to allow the ice to sublimate or evaporate, but never to melt. The extracted water vapour condenses on the surface of the refrigerated chamber at about -40°C. A small amount of water vapour escapes condensation and passes out to the vacuum pump and through the oil reservoir, thence through an oil mist filter to the atmosphere. If the oil is not hot, the water vapour will condense in the oil and sink to the bottom. The vacuum is measured using a Pirani gauge, with readout on a meter; this may have a separate electrical switch.

Notes:

A freeze-dryer must be purchased that either has a large built-in chamber, or to which a chamber can be attached. Some smaller ones are mainly for small-scale work when multiple samples are held in glass flasks or ampoules, which are attached to a manifold equipped with isolation valves.

It is vital to maintain clean contacts on the connection fitting of the Pirani gauge, because it is very sensitive to resistivity changes due to tarnishing or dust.

Freeze-drying methodology

The freeze-dryer is switched on and the pump started in order to warm up the oil within the pump housing. This is to prevent condensation of moisture in the oil. The gas ballast valve is also opened for the first half of the drying process, when most of the moisture is removed, to purge out any moisture from the oil. One manufacturer (ChemLab) recommends leaving the ballast valve open for the whole drying process. Although this will keep the accumulation of water in the oil to a minimum, it will mean the ultimate vacuum possible with the pump will not be reached, and samples will have slightly more residual moisture. When the condenser temperature indicator reads less than -30°C, the frozen sample may be loaded into the chamber. Samples may be placed in trays, paper bags or microporous bread bags, but preferably not in polythene bags, which could hinder the evaporation process. Samples of a lumpy consistency should be broken up while still frozen to speed the evaporation process. The chamber may have rubber seals which need greasing. The minimum amount of silicone grease should be applied, and the seals should be wiped scrupulously clean before the application, as any particles of sample material adhering to the seals will allow ingress of air owing to poor sealing at that point. With the chamber lid or door in place, the drain valve should be closed, and the vacuum valve opened. When the Pirani gauge reads 66.5 Pa (500 millitorr or 0.5 mmHg), the heater may be switched on. The drying time will depend on the nature and water content of the samples, but 2-4 days is normal. The condensation chamber will have a certain capacity, perhaps 3 l, so the total sample water content should not exceed this, and there is a loss in efficiency if about two-thirds of this value is exceeded.

At the end of the process, when the pressure has been about 13.3 Pa (100 millitorr or 0.1 mmHg) for several hours, the isolation valve is closed, the drain tap opened to allow ingress of air, and the defrost switch turned on. The lid may then be removed and the samples checked for dryness. Any larger samples should be inspected, to ensure there are no remaining areas of ice at the centre. The freeze-dried samples should be stored in a desiccator before milling, which should be done as soon as possible. Freeze-dried samples are somewhat more sticky than oven-dried ones, and the crisper they are, the better they mill. After milling, the samples should be stored in airtight sample tubes or grip-top polythene bags to prevent rehydration and fungal attack. Samples will not be as dry as oven-dry material, and will typically contain between 3% and 10% moisture depending on the sample. When results from the analysis of freeze-dried material have to be expressed in terms of oven-dry matter, a sub-sample must be taken at the time of weighing for a separate oven-dry matter determination. This will enable a correction for residual moisture to be made.

Microporous bags can be obtained from Cryovac at the website of Sealed Air Corporation:

http://www.sealedair.com/products/food/bakery_fs.htm Websites of freeze-dryer manufacturers/suppliers are: Virtis: http://www.virtis.com/ ChemLab: http://home-1.worldonline.nl/~chemlab/ CHRIST: http://www.phscientific.co.uk/html/ Heto: http://www.heto-holten.com/camel.htm

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