The sublimation step

The small, disoriented ice crystals formed in viscous samples by virtue of the restricted water motion, hinder mass transfer during the sublimation step, thereby resulting in less efficient drying and hence in samples more liable to structural changes. 

The freezing temperature also influences crystal size and hence the rate at which sublimation occurs. It is therefore essential to establish the best freezing conditions for each product. The determination of freezing points or eutectic points is probably the most complex task in the freezing protocol. The presence of any liquid constituent other than water in a liquid matrix lowers its freezing point. It is thus very important to pre-freeze the product to below the eutectic temperature prior to freeze-drying proper. In fact, any small pockets of unfrozen material remaining in the product may expand and compromise the structural stability of the freeze-dried product. 

The freezing points of biochemical products containing hygroscopic molecules (e.g. sugars, protein complexes) are well below zero by effect of some water molecules being tightly bound to molecules of the dissolved product. Eutectic points are usually determined from electrical resistivity measurements. 

As a rule, products should be frozen below — 20°C for no less than 3-4 h. 

In some cases, freezing or freeze-drying a highly concentrated solution may prove difficult and require diluting the sample prior to freezing so that an adequate amount of ice is formed in the freezing step. 

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The yield reported here is based on the total amount of ruthenium (both Rum and Ru°) available for formation of ruthenocene. An additional quantity of ruthenocene may be obtained by extraction of the pyrophoric residue from the sublimation step with benzene in a Soxhlet extractor under a nitrogen atmosphere. The benzene solution is filtered through activated alumina, the solvent evaporated, and the residue sublimed. 

The nickel complex, [Ni(NO)I], can also be prepared by the procedures outlined above. The compound is formed readily in reasonable yields, but it is significantly less stable than the iron and cobalt compounds. Moreover, although some of it can be sublimed, it decomposes slowly even below its sublimation temperature. The gram quantities used are the same as for cobalt, and no modification of the procedure is necessary until the sublimation step. No sublimation of the nickel complex takes place until the bath temperature reaches 155-165°C. At this temperature a small amount (2.5 g.) of product sublimes onto the cold probe. Anal. Calcd. for Ni(NO)I Ni, 27.22 I, 58.86. Found Ni, 26.8 I, 56.5. 

The sublimation step removes the water molecules that were separated from the solids as ice crystals in the freezing step. Sublimation is the process by which solid water is directly transformed into vapour water. When sublimation occurs during the freeze-drying step, it is commonly referred to as the primary drying process . The sublimation step does not affect unfrozen water associated to the solids (sorbed water), which is removed during the desorption step. 

The sublimation step requires very careful control of two of the key variables of the freeze-drying process temperature and pressure. 

The sublimation step separates the ice crystals formed during the freezing step. When an ice crystal forms, that which remains is the concentrated solute phase called the dry layer . This will become the freeze-dried material at the end of the process. Immediately following passage through the interface, however, the solids in the dry layer still contain a substantial amount of water (about 25-30 g per 100 g of solids), which continues to be strongly bound to the solids. Most sample materials will not be structurally or chemically stable unless most of this water (called sorbed water ) is removed. The process... 

Validation of the freeze-drying process has been found to rely upon the establishment of a range of product temperatures yielding a product with properties falling within a set of prescribed limits [3-5]. It is therefore essential to ensure that the product temperature is maintained within such limits during the sublimation step. 

The sublimation step should be started when the matrix is in a completely frozen state. In order to ensure that the matrix will preserve its frozen state throughout the sublimation step, the computer should be provided with the collapse or eutectic temperature of the sample. Since the vast majority (99%) of samples will have a collapse rather than a eutectic temperature, the software will need to base the product temperature on some knowledge of, or means of computing that for the sample concerned. Such a frequency distribution of collapse temperatures arises from the fact that the glassy state, formed in the interstitial region of the matrix, is non-stoichiometric in composition, so the collapse temperature can vary from container to container depending on the particular frequency distribution. 

The chamber pressure during the sublimation step (i.e. the primary drying process) has been found to be related to the product and shelf-surface temperatures [8] however, determining the shelf temperature required is more difficult as it depends on the nature of the heat transfer fluid used to control the shelf temperature and also on the particular design of the freeze-dryer. 

The sample container rests on a boundary layer at the top of the shelf surface. Such a layer is a region where the flow of heat transfer fluid is minimal or zero (i.e. the fluid is stationary). As a result, the sublimation step, which involves the transfer of heat from the fluid to the shelf surface, creates a temperature gradient across the boundary layer that depends on the thermal load exerted by the sublimation process and on the nature (viscosity, thermal conductivity and flow across the shelves) of the heat transfer fluid. The temperature gradient also depends on the number of shelves, their design and build, and on the pumping capacity of the circulation pump. These variables in turn depend on the size and particular manufacturer of the freeze-dryer, so the software used should include an input of data for the materials used, and for the dryer s design and build. 

The enthalpy change for the sublimation step is designated the lattice enthalpy, A//l(see Table 6.6 in the text). 

What would happen to the caffeine if the sublimation step were performed at atmospheric pressure ... 

Otherwise, as already emphasized in Section 3.3.3, for a given formulation, the water vapor mass transfer resistance values are directly dependent on the pore morphology of the freeze-dried zone, and, consequently, on the ice crystal morphology, that is to say on the freezing conditions and on the thickness of the freeze-dried layer which increases throughout the sublimation step. 

Consequently, it is confirmed that, for a given system, the mass transfer phenomena during the sublimation step of the freeze-drying process are significantly dependent on the morphology of the ice crystals and, in consequence, significantly dependent on the nucleation temperatures of the supercooled formulation. 

If the sublimation step is not yet finished, it could be interesting to estimate the time left to the endpoint. This can be easily done using a mathematical model that describes the dynamics of the process (e.g., the same as in the DPE algorithm) and uses the process parameters estimated by DPE. 

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