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An introduction to Freeze Drying
An introduction
to Freeze Drying
State of the Performance
State of the
Performance

An Introduction to Freeze Drying

© Jouan Nordic A/S Text by James M. Flink and Henning Knudsen Printed in Denmark for Center-Tryk ApS, Holbæk 2002

An Introduction to Freeze Drying

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Introduction Goal of booklet, whom it is for Organisation, 2 parts- introduction and supplements

Describing some terminology Definitions of synonyms

• drying-dehydration

• product-sample-material-solids

• water-moisture-solvent

• freeze drying-lyophilisation Definition of solution

• solution-solids/solute-solvent Note on glossary

What is drying?

Why do we want to dry our sample?

How can we dry our product?

What is freeze drying and why choose to freeze dry? An initial discussion

What is a typical freeze dryer?

A closer look at material properties

Introduction to heat and mass transport

A closer look at the freeze drying process freezing, freezing rate and mass transport sublimation, the interface and retention of structure desorption, moisture gradients, end-point

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11.

Questions to ask when choosing a freeze dryer, or when operating a freeze dryer Material properties

freezing requirements, conc., comp., temp.

stability behaviour, temp., limits, final m.c. Capacities required

• batch capacity, loading in chamber

• rate of production

Freeze drying parameters

• heater temperatures

• chamber types, sample surface, areas in containers

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Supplements

A) Water properties

B) Material properties

C) Mass transport

D) Heat transport

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1. Introduction

This booklet is intended as an introduction to the freeze drying process for persons who will be using freeze drying in the future, or are cur- rently using a freeze dryer and wish to know more about how it works.

To accommodate readers having different goals, this booklet is divid- ed into 2 parts. The first part, which comprises the main text, gives a short, concise discussion of various aspects of freeze drying, regarding both the process and the materials to be freeze dried. In the second part are found supplements to some of the main text topics in which fuller explanations of the phenomena involved in freeze drying are giv- en. We suggest the main text to be read first in its entirety to obtain an overall view, and thereafter the supplements of interest to be examined.

2. Describing some terminology

Before beginning the main text, it would be an advantage to define a few terms.

The terms drying and dehydration are used interchangeably to describe the process of complete removal of water from a material.

The terms product, sample and material are used interchangeably to describe the »stuff« that we are going to freeze dry, or the »stuff« that we obtain after freeze drying.

The terms water and moisture are used interchangeably to describe the »stuff« to be removed from our sample by freeze drying.

The terms freeze drying and lyophilisation can be used interchangeably to describe the process which is commonly called freeze drying.

Lastly, we should define 3 terms which relate to our sample, solids or solute and solvent. Solvent is the liquid material in which the solids or solute are found. In this booklet, water is the solvent. Solids can be subdivided into 2 groups; those which are soluble (present as individual molecules in the solvent) and those which are insoluble (present as groups of

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molecules in the solvent). Soluble solids and solvent make a solution, insoluble solids and solvent make a suspension. A sample can contain both soluble and insoluble solids in the solvent.

3. What is drying?

Drying or dehydration is a process where the solvent in the sample, in our case water, is physically removed from the sample such that the solids are obtained in an essentially dry (eg. water-free) condition.

4. Why do we want to dry our sample?

There are many reasons for wanting to dry a material as listed below. It is necessary to remember that the major goal of a drying process is to obtain a dry material which meets one or more of the goals listed in Table1, the end-product thus being the most important considera- tion in freeze drying.

Table 1:

Product-based goals for freeze drying

Preparation of tissue specimens suitable for structure (histological) studies using various microscopic techniques, including electron microscopy.

Sample preparation as a step in chemical/ biochemical analysis procedures.

Long-term storage stability of materials, such as foods, microbial cells, enzymes for assay kits, blood, etc.

Recovery of products from a reaction mixture in an easily handled (dry) form.

To discuss these goals of freeze drying we can, as examples, use some of the materials listed in Table 2 where freeze drying has been found desirable. It clearly appears from the wide variety of products in this

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list that the differences in specific goals to be achieved by freeze dry- ing a product and the differences in its properties will result in widely varying requirements on the freeze drying process.

Freeze drying is used to attain long-term storage stability of food prod- ucts and, for that matter, other biological materials (microbial cul- tures, enzymes, vaccines, blood fractions and pharmaceuticals). Here the goal involves the arresting of biological and chemical reactions which are responsible for degradation in wet material by means of reduction of water activity in the product.

In

some cases, especially for the biological materials mentioned above,

it

is also essential that the inherent biological activity is preserved fol-

lowing the drying process and, further, that it is recoverable when rehydrated. Retention of biological activity is thus an additional requirement beyond that of long-term storage stability.

Table 2:

Some typical materials which are dried by freeze drying

Micro-organisms (bacteria, yeasts) including starter cultures in the meat and dairy areas)

Viruses

Vaccines and antitoxins blood fractions

Enzymes (for analytical systems and/ or reactions in general)

Tissues and bone materials for transplants

Biological reagents and standards

Food materials (beverages, fruit, vegetables, meat, fish)

Whole animals for museum display

Archeological items (wood, leather, paper)

Pharmaceutical preparations

Vitamins

Most types of sensitive agents in chemical/ biochemical laboratories (including free radicals and physiological media)

Tissues and cells for electron microscopy

A somewhat different goal with freeze drying is the preparation of tis-

sue samples for electron microscopy. Here it is most essential that the structure of the tissue remains unchanged in the drying process.

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Freeze drying can also be utilised in some types of chemical analyses when it is desirable to have the sample present as a dry powder, either to meet »water-free« requirements of the analysis system, or to con- centrate the product prior to analysis, thus increasing analysis sensi- tivity. In this case it is an essential goal that in the freeze drying process the sample does not change chemical composition due to instability of sample components. This goal can place strict requirements on the drying conditions utilised.

Another similar use for freeze drying is the recovery of intermediate reaction products from dilute reaction systems, especially where further reaction steps are best conducted at higher concentrations. Here the retention of chemical and/ or biological activity will be an essential requirement for accepting the drying process.

It can be seen from above examples that the various end-uses for the product will place different requirements on the process such as retention of biological activity, retention of physical structure, preven- tion of chemical changes in storage, etc. It is, thus, important in the following discussion to keep in mind that it is the product which defines which of the possible drying methods can be used and what process conditions will be possible. It will be noted later how the prop- erties of the product and the above mentioned usage-based require- ments together will act to define the range of conditions which can be used in preparing the freeze dried product.

5. How can we dry our product?

There are many different drying methods which can be used to pre- pare dry materials. Some are most suited to large-scale industrial pro- duction, some to laboratory preparation while some are, in principle, suitable to either scale of operation. A number of methods are listed below together with their primary use area. We have already mentioned freeze drying as a method for preparing dry materials. Some of the dry- ing methods listed are most suitable for liquids (for example: spray drying, drum or roller drying) while others are best suited for solids (pneumatic drying, fluidised bed drying, tunnel or cabinet drying). Freeze drying is somewhat special as it is suitable for both solids and liquids.

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Freeze drying is also particularly special with respect to its physical ba- sis (eg. how it works). This topic, the physical basis of freeze drying, will be a significant part of the remainder of the main text. These spe- cial attributes are important to explain the wide range of applications of freeze drying in the laboratory environment; however, it is also nec- essary to note that the wide-spread acceptance of the freeze drying process is also due to the ease with which freeze drying can be con- ducted in the laboratory, even by persons not having any special knowl- edge of the process.

Table 3:

Examples of drying processes (Code letters: (I) = primarily industrial scale, (L) = primarily laborato- ry scale, (I,L) = both industrial and laboratory scale)

Freeze drying

(I, L)

Drum drying

(I)

Cabinet drying

(I, L)

Belt drying

(I)

Tunnel drying

(I)

Critical point drying

(L)

Spray drying

(I, L)

Vacuum drying

(I, L)

Flash drying

(I)

Microwave drying

(I)

Fluidised bed drying

(I)

Macrowave drying

(I)

While one can freeze dry samples without knowing much about freeze drying, it is the aim of this booklet to give various staff levels, from the freeze dryer operator to the project manager, a better understanding of the freeze drying process, and especially the relationship of process conditions to product properties so that they can obtain a fuller understanding of both the opportunities and the limitations associated with freeze drying.

6. What is freeze drying and why choose to freeze dry? An initial discussion

Freeze drying is a 3-step process. In the first step, the product is frozen solid so that the water present in the material is converted to ice. (In fact, a small portion of the water is strongly bound by the solids and does not freeze - more information on that is found in the supple- ments). In the second step of the freeze drying process, the ice formed

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in freezing is removed from the product by direct conversion from sol- id to vapour by a process called sublimation. In the third step of the process, the water which was strongly bound to the solids of the sam- ple (called sorbed water) is converted to vapour and removed from the product, this process being referred to as desorption.

There are 2 main differences between freeze drying and all other drying processes; (1) the material is solidified prior to drying, and (2) the removal of the major amount of water from the sample occurs through direct conversion of water from the solid state to the vapour state. In all other drying processes, water is present in the material as a liquid, and water removal occurs by vaporisation, (eg. water is converted from the liquid state to the vapour state). The mobility of liquid water in most drying processes vs. the immobility of solid water (ice) during freeze dry- ing has major consequences on the properties of the dry product ob- tained. Furthermore, the sizable differences in temperature levels at which water is converted to vapour is also important. The sublimation process (in freeze drying) occurs at low temperature (generally about -20°C to -30°C) whereas for vaporisation of liquid water, the temper- ature for the liquid-to-vapour transition will generally occur at higher temperatures in the range of +50°C to +100°C

In situations where the immobility of the components of the dry materi- al is important, like the preparation of dry tissue sections for microscop- ic analysis, freeze drying would be the logical choice. In cases where chem- ical stability or retention of biological activity is important, choice of low temperature conditions for removal of the major amount of water from the product would dictate to choose freeze drying.

That freeze drying may not be optimal for all drying situations relates primarily to cost considerations (both capital costs and operating costs) since freeze drying is generally more expensive than most other drying processes. Thus, for cases where specific product-quality fac- tors are of lesser importance, it is possible that the higher cost of freeze drying will not be justified. (For laboratory situations, operating cost considerations are generally not significant). As we have noted earlier (Table 1), there are many areas where particular product-related requirements will necessitate the use of freeze drying if a product of satisfactory quality is to be obtained.

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In the chapters below, the freeze drying process will be examined in further detail and related to the properties of the materials to freeze dry. But before doing that, we will examine the physical layout of a typ- ical freeze dryer, as the reader may already have one in the laboratory.

7. What is a typical freeze dryer?

When describing a typical freeze dryer, it must first be noted that although all freeze dryers are built up of the same basic system com- ponents they may vary greatly in size and appearance. Even if one were to investigate the differences between laboratory and industrial freeze dryers, it would quickly become clear that a freeze dryer for food pro- duction of tons per day capacity, such as for freeze dried coffee, basi- cally operates on the same principles as a laboratory freeze dryer preparing grams per day of tissue samples for microscopic analysis.

As this booklet is primarily intended for persons utilising laboratory freeze dryers, the examples discussed here will be based on a labora- tory system. Figure 1 - on the nest page - shows a number of labora- tory freeze dryers which vary in size and appearance. For this discus- sion, we will concentrate on a single model, the small freeze dryer in the foreground, as it clearly shows all the basic elements of a typical freeze dryer.

On the left can be seen the vacuum pump (1), which is connected by means of a tube and valve to a refrigerated condenser system (2) which removes the water. On the front of the freeze dryer are a number of controls and measurement devices (3), and lastly, there are a number of sample chambers shown (4), some of which are connected to the condenser via tubing and valve systems (5).

All the shown freeze dryers are built up of the same basic components, though in most cases the vacuum pump and refrigerated condenser are hidden in the cabinet under the chamber for aesthetic reasons. Sev- eral types of tube and valve systems (5) can be used to connect vari- ous chambers to the refrigerated condenser. Some of the connections are rather long and may have a complicated geometry.

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Figure 1. Typical configurations of laboratory freeze dryers.

➃ ➃ ➁ ➇ ➃ ➆
of laboratory freeze dryers. ➃ ➃ ➁ ➇ ➃ ➆ ➀ ➂ ➅ ➄ ➄ ➂
of laboratory freeze dryers. ➃ ➃ ➁ ➇ ➃ ➆ ➀ ➂ ➅ ➄ ➄ ➂

freeze dryers. ➃ ➃ ➁ ➇ ➃ ➆ ➀ ➂ ➅ ➄ ➄ ➂ 1. Vacuum

➄
dryers. ➃ ➃ ➁ ➇ ➃ ➆ ➀ ➂ ➅ ➄ ➄ ➂ 1. Vacuum pump

1. Vacuum pump

2. Refrigerated condenser system

3. Controls and measurement devices

4. Sample chambers

5. Tubing and valve systems

6. Tubing and valve systems

7. Shelf controller for temp. reg.

8. Serial printer (via RS 232C)

The typical freeze dryer can be visualised in a simple diagram (Figure 2) where the numbers given correspond to the components noted above. In Supplement C, similar diagrams will be used to describe mass transfer as related to freeze drying.

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Figure 2.

Schematic diagram of a typical freeze dryer.
Schematic diagram of a typical freeze dryer.

8. A closer look at material properties

We have earlier noted that the sample is composed of 2 major com- ponents, water and solids. Each of these components is important to determine how the sample behaves during freeze drying, and thus both general and product-specific knowledge regarding the properties of water and solids is important for conducting successful freeze drying. In this chapter, we will consider some basic aspects of the general prop- erties of water and solids.

Figure 3. A p-T diagram showing phase equilibrium lines, the triple point, and the critical point.

water and solids. Figure 3. A p-T diagram showing phase equilibrium lines, the triple point, and

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Water: - Water can exist in 3 forms (called states), referred to as solid (as ice), liquid (as the solvent in our sample), and vapour (as steam). The state of pure water depends at any time on its temperature and the pressure in the system. If solids are present in the water, then the composition of the sample will also have an effect on the state of water. The various relationships between the states of water are given in various state diagrams. Two are of particular interest: the tempera- ture-pressure diagram for pure water (Figure 3) and the temperature- composition diagram (Figures 4a and 4b). These are discussed in detail in Supplement A. It can also be mentioned now that to change the state of water, it is necessary to either supply or remove energy to the water molecules.

We have already noted that several changes in the state of water are important in freeze drying. One is the freezing of the water (a change from liquid state to solid state) while another is sublimation (a change from solid state to vapour state). The energy transfer required for these changes in the state of water is quite different. For example: to freeze a gram of water requires removing 80 calories from the water, while sub- limating a gram of ice requires supplying 680 calories to the ice.

Figure 4. Typical temperature-composition phase diagrams for aqueous solutions

supplying 680 calories to the ice. Figure 4. Typical temperature-composition phase diagrams for aqueous solutions 14

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In the freeze drying process, freezing water occurs at atmospheric pres- sure (perhaps in a chest freezer, etc.), and thus the temperature of the change in state (liquid state to solid state) is the well-known freezing point of water (0°C).

As we do not generally freeze dry water, we are quite interested in the presence of the solids in our sample, as this will have an effect on the temperature at which liquid water will freeze to solid water. The effect of added solids on the freezing point is given by a freezing point dia- gram (Figure 5), which comes from Figure 4. Freezing point diagrams are described in detail in Supplement A. At this point, we can simply note that cooling a solution below its freezing point results in the for- mation of solid water (ice). As the remaining unfrozen liquid water still contains all the initial solids, we can note that the freezing of this water to ice gives, at the same time, an increase of the concentration of solids in the remaining, unfrozen liquid. As the temperature gets lower and lower, more and more ice forms, and conversely, less and less unfrozen liquid is left. We also observe that the more solids we had initially (i.e. the higher the solids concentration in the original sam- ple), the less ice and the more liquid we will have at any given sample temperature.

When freezing to very low temperatures, we find that material displays one of two different types of freezing behaviour. In some solutions (called eutectic formers), the liquid suddenly solidifies at a tempera- ture which depends on the type of solids in the sample. For NaCl solutions, this temperature is -21.6°C, while it is much lower for CaCl 2, about -55°C. In the other type of solutions (called glass formers), the liquid does not solidify, but rather just becomes more and more vis- cous, until it finally ends as a very stiff, viscous liquid. These differences in freezing behaviour will be important in our later discussions regarding the possibilities for successfully freeze drying different materials.

With the sublimation change of state, there is no liquid state present. Just as most people can remember that the boiling point of liquid water at atmospheric pressure is +100°C, people in freeze drying know that typical important sublimation values for ice are 1 mbar at -20°C and 0.128 mbar at -40°C. This means, for example, that subliming ice having a temperature of -20°C will give a vapour having a pressure of

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Figure 5. Freezing point curve for sucrose solutions.

Figure 5. Freezing point curve for sucrose solutions. 1 mbar. A more complete listing of temperatures

1 mbar. A more complete listing of temperatures and pressures is given in Table 4. These relationships of temperature and pressure are particularly important to understand mass transport (the movement of water from the sample to the condenser) in a freeze dryer.

Solids: - First we can note that, when discussing the solids in a sample and their influence on freeze drying, it is necessary to include as a part of the »solids« - those water molecules directly associated with (eg. »bound to«) the solids. In freeze drying, there are a number of mate- rial-related factors which are important to determine whether a high- quality product will be obtained. These factors are called stability fac- tors and can be divided into 2 major groups, structural stability and chem- ical stability. The properties of the material, relative to these stability fac- tors, will determine how well a product can be freeze dried.

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Table 4:

Temperature-water vapour pressure relationship

Temperature (°C)

Water vapour pressure (mbar)

-30

42.421

-20

23.374

-10

12.275

-

0

6.103

-10

2.599

-20

1.034

-30

0.381

-40

0.128

-50

0.039

-60

0.010

-70

0.025

-80

0.0005

The structural stability of a material relates to its ability to undergo freeze drying without physical changes, particularly as regards size and shape. The material property most closely related to structural stabil- ity can be called stiffness. This property particularly describes the abil- ity of the material to bear its own weight and resist the influence of external forces on its geometry. For a given material, stiffness will depend on its temperature and moisture content (Figure 6). Here we see that for a given material there is a wide variety of temperature-mois- ture content combinations which give regions of structure stability or structure instability. A different material will have other combinations of temperature and moisture content defining the regions of stability or instability. The relative stiffness of different materials will depend on their molecular weight and molecular structure (in particular their bonding patterns).

Solids of lower molecular weight or with lower levels of bonding, higher sample temperatures and higher moisture contents all tend to give a material less stiffness. Sample behaviour during freeze drying can depend on whether the solids were eutectic formers or glass formers during free- zing as these 2 forms can differ greatly as to stiffness.

These points are discussed further in chapter 10 and in the Supple- ments.

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Materials having poor structural stability generally end up shrunken or puffed, or may be glassy-looking and sticky. Such samples are said to have collapsed during freeze drying. Poor structural stability (collapse) in freeze drying, besides requiring a longer time to dry, also gives a poor quality product.

Figure 6. Structural stability diagram

quality product. Figure 6. Structural stability diagram Table 5: Temperatures for structure transitions, °C

Table 5:

Temperatures for structure transitions, °C

Collapse during freeze drying

Apple juice

Citrate buff

-42

-40

Coffee extr.

-20

Dextran

-9

Fructose

-48

Gelatin

-8

Glucose

-40

Inisitol

-27

Lactose

-32

Lemon juice

-36

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Maltose

Methocel

MSG

Orange juice

Ovalbumin

Phosphate buff

PVP

Sucrose

Sorbitol

-32

-9

-50

-24

-10

-80

-23

-32

-45

Collapse of dry material

Fructose

37

Maltose

96

Glucose

35

Maltotriose

88

Lactose

101

Orange juice

Sucrose

52

Maltodextrin (DE=10) 226

56

Maltodextrin (DE=20) 174 Maltodextrin (DE=25) 150

Xylose

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While some materials are in practice classified as very difficult to freeze dry, essentially all materials can be freeze dried in theory. It is »only« necessary to choose the proper freeze drying conditions for the par- ticular properties of the material to be dried. Critical temperature lev- els in freeze drying for a variety of materials (called collapse temperatu- res) are given in Table 5. To freeze dry without structural loss requires maintaining temperatures in the frozen areas of the sample below this collapse temperature.

In some few cases, commercially available standard freeze dryers can- not attain the process conditions required by the properties of the material (such as the -48°C collapse temperature for fructose), and it must be accepted that either (1) the material cannot be freeze dried with structural stability (though it can be dried without structural sta- bility), or (2) that it will be necessary to construct special, perhaps very costly, equipment to accomplish the freeze drying.

Table 6:

Chemical reactions which affect the chemical stability of freeze dried materials:

Non-enzymatic browning (including maillard reaction and acid- catalysed dehydration)

Protein denaturation

Nutrient destruction

Lipid oxidation

Nucleic acid breaks

As related to freeze drying, the area of chemical stability is very broad, due both to the many materials which are freeze dried and the num-

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ber of chemical reactions which can occur. A list of some important reactions is given in Table 6. For a number of reactions (such as microbial survival), the reaction mechanisms are poorly understood. Some of the reactions do not occur much during freeze drying, but rather during the subsequent storage of the dry product.

Beyond the chemical composition of the sample, the other factors hav- ing a significant influence on the chemical stability of the material are its temperature and moisture content. It is generally known that chem- ical reaction rates increase with increasing temperature and fall with decreasing moisture (though some exceptions relative to the influence of moisture are known).

During freeze drying, the sample will have widely varying temperatures and moisture contents the magnitudes of which will depend on the process conditions chosen. To achieve our goal of a successful freeze drying, the process conditions chosen must take into consideration the properties of the materials relative to sensitivity to chemical reac- tions.

The stability factors (both structural and chemical) of the material will be important when placing constraints on the operating conditions in our freeze dryer. These considerations will be taken up again in chap- ters 10 and 11. The Supplements also give further information on these relationships.

9. Introduction to heat and mass transport

Simply stated, transport is the movement of something from one place to another. With respect to freeze drying, we are interested in 2 types of transport: mass transport (the movement of »stuff«) and heat trans- port (the movement of energy). We will shortly discuss each of these individually.

When analysing transport behaviour in freeze drying, we are especial- ly interested in how fast the movement of heat or mass is occurring. We can define this rate of transport to be controlled by two factors. One factor, which tends to cause the transport to occur, is called the

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driving forces, while the other factor, which tends to prevent the trans- port from occurring, is called the resistances.

The rate of movement of our »something« depends on the ratio

driving forces

resistances

We can see that a high transport rate can result from either a high driv- ing force, a small resistance or a combination of intermediate values of each. However, we should note that without a driving force, there will be no transport even if there were zero resistance.

Mass transport (See also Supplement C) Mass transport is the movement of matter from one location to another. In freeze drying we are interested in the movement of water, in particular from our product to the freeze dryer condenser. In freeze drying, the driving force for mass transport is pressure difference while the resistances arise from flow friction between the moving water molecules on the one hand and (1) other moving water molecules, (2) air mole- cules in the freeze dryer and (3) the walls of tubes and valves of the freeze dryer on the other hand.

We have noted earlier that water at different temperatures has differ-

ent pressures, and particularly, it was mentioned that ice at -40°C has

a lower pressure than ice at -20°C (See Figure 3 or Table 4). Thus, if

we connect one space which is cooled to -40°C by a refrigeration machine with another space where we keep our product at -20°C, we will have a difference in water pressure between the spaces. Such a sit- uation is shown in Figure 7. This pressure difference (driving force) causes a movement of water molecules from the sample at the higher pressure to the refrigerated space at a lower pressure (The refrigerated space in a freeze dryer is called a condenser because it condenses the water vapour to ice again).

A number of resistance factors are hindering the flow of water mole-

cules. Water molecules tend to move in straight lines until they hit some- thing, at which time they bounce off in another direction. The more times they hit something (be it another water molecule, air molecules, walls of tubing or bends, or small openings in valves), the more diffi- cult it is for them to move in the desired direction of flow. In such a

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Figure 7. Mass transport parameters presented on the typical freeze dryer schematic.

parameters presented on the typical freeze dryer schematic. case, we would say that our system has

case, we would say that our system has high resistances to water flow. In Figure 7 are shown 2 tube configurations for connecting the sample chamber with the refrigerated space. The upper tube being wide- diametered and straight will give fewer collisions between water mole- cules and the tube walls and will thus be a low resistance pathway rel- ative to the lower tube with its small diameter, many bends and partly closed valve.

In freeze drying, there are limits to the pressure differences that we can achieve. In accordance with theory, the condenser cannot have a lower pressure than absolute zero (perfect vacuum) while the ice pressure of the product is limited in practice by the melting properties, this defining a maximum temperature and pressure for the sample region. Thus, it is quite important to keep the mass transport resistances as low as possi- ble. Removal of air and non-condensable gases is most important, hence we have a vacuum pump attached to the system. Beyond that, the

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dimensions of the freeze dryer should reflect the magnitude of the water transport loads to be placed on the system. In general, connecting tubes should be short and have relatively large diameters. The number of valves should be kept to a minimum and those used should be wide-throat types. In addition, the design should have a minimum of sharp bends in the con- necting tubing. We will return to these very important considerations in chapter 11.

Heat transport (See also Supplement D) Heat transport is the movement of energy. Energy movement is very important in freeze drying, as each time there is a change in the state of water (as in freezing, sublimation, desorption and condensation), it is necessary to remove or supply energy to the water molecules. Thus, without heat (energy) transport, no freeze drying can occur.

The driving force for heat transport is the difference in temperature from one location to another. Energy moves from locations of higher tem- perature towards locations of lower temperature.

Resistances of heat transport depend on the physical situation, which - for simplicity in our discussion of freeze drying - can be divided into 3 groups: conduction, convection and radiation. Conduction is the mode of en- ergy transport in solids where no movement of mass is possible. Con- vection is the mode of energy transport in liquids, vapours and gases where energy is transported by the movement of molecules in space. Radiation is the mode of energy transport by which energy is transported across »empty« space (such as from the sun to earth).

The magnitude of the resistances for these 3 transport modes depends on many factors. For conduction, heat transport resistance is very dependent on the thermal properties of the material. Copper, for exam- ple, has much less resistance to heat transport than polystyrene foam. Thus, we use copper in heat exchangers where we want high heat trans- port and plastic foam for insulation where we want low heat trans- port. In freeze drying, we will find that conduction is an important mode of energy transport, both to the sample from the apparatus as well as inside the sample. For convection, heat transport is very dependent on the number of molecules available to carry the energy. In the freeze dryer where we have removed all the air, the number of molecules in the space is generally small, and convection is generally

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Figure 8. Heat transfer modes in freeze drying.

Figure 8. Heat transfer modes in freeze drying. not too important. As the space has so

not too important. As the space has so few molecules, we can con- sider it to be almost empty, and thus radiation can be an important mode of heat transport. In actuality, combinations of conduction and radiation heat transport are quite common, either by design or where good thermal contact required for conduction is not achieved. Typi- cal configurations are shown in Figure 8.

For heat transport, the driving force can theoretically have any mag- nitude wished, but in practice, there are temperature limits above which the product should not be subjected. These temperature limits give a maximum level for the heat transport driving force. Some of the heat transport resistances are under our control (such as how good the sample contacts its holder, this determining the magnitude of one of the conduction resistance) while other heat transfer resistances are less controllable (eg. the resistance regarding radiation).

To conclude this chapter, we can note that a number of the practical problems encountered in freeze drying are related to inadequate con- siderations regarding heat and mass transport, and especially a fail- ure to appreciate the relationship between these transport phenome- na and the material properties of the sample. Some of the considera- tions which should be made when establishing a freeze drying process and some of the problems which can arise at that time will be discussed further in chapter 11.

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10. A closer look at the freeze drying process

We have already noted in chapter 6 that freeze drying is a 3-step process consisting of freezing, sublimation and desorption Before examining these 3 steps in more detail, we will consider the following 2 items.

First, even though the freeze drying process has 3 steps, it is generally very common to conduct the freezing step in a chest freezer, in a refrigerated liquid bath or with liquid nitrogen, using equipment not directly associated with the freeze drying apparatus. In these cases, only the sublimation and desorption steps take place in the freeze dryer itself.

Secondly, while we can say that all locations in the sample have com- pleted the freezing step before we begin the sublimation step, the same situation is not precisely true when describing the relationship between the sublimation and desorption steps. While at each location in the sample sublimation proceeds desorption, if we consider the whole sample at one time, some regions are in the sublimation step while other regions, which have already been through sublimation, are in the desorption step. Thus, even though we discuss sublimation and des- orption as separate steps, it is important to realize that they are occurring simultaneously in the freeze dryer at different locations in the sample.

The freezing step As noted above, freezing is often not conducted in the freeze dryer but in a separate apparatus. The choice is generally based on factors of cost and convenience, though technical considerations regarding freez- ing rates and freezing temperatures should be included in the choice of freezing methods.

The freezing step must be completed before starting the sublimation and desorption steps. Freezing has 3 main effects on the sample which are of major importance with respect to freeze drying. These are (1) partial dehydration of the sample, (2) stiffening of structure and (3) deter- mination of sample morphology (eg. 3-dimensional structure).

In the freezing process, liquid water in the sample is converted to ice. As converting some of the liquid water to ice leaves a liquid phase con-

25

taining an unchanged amount of solids dissolved in less water, we would say the solids in the remaining liquid have become more con- centrated, or conversely, we could consider that the solids have become partially dehydrated. As the ice formed is still present in the sample, freezing only causes a physical separation of the water (as ice) from the solids. Removal of this water (ice) from the sample first oc- curs in the sublimation step. Also, the fact that the ice is still located in the sample means that the temperature must be maintained low enough so that the sample remains frozen when it is transferred from the freezing device to the freeze dryer. Otherwise some ice will melt and the degree of partial dehydration will be reduced. The degree of par- tial dehydration achieved during freezing will depend on the type of material and the sample temperature history during freezing; for »glass« formers, there will always remain some unfrozen water associ- ated with the solids. (See also Supplement A).

The structural stiffness of the frozen sample depends on the combi- nation of the stiffness of the ice regions and the stiffness of the unfrozen (concentrated solution) regions. Ice itself is quite resistant to deformation, and thus acts as a support for the unfrozen regions the stiffness of which can be much more variable. The relative amounts of ice formed and material remaining unfrozen will depend on the tem- perature of the sample and the initial concentration of solids in the sample, so the efficiency of ice as an overall structure support will depend on these factors. The stiffness of the unfrozen material (solids plus the tightly bound, unfrozen water) will depend on the molecular weight of the material as well as the temperature and water content of the sample. This is similar to the relationship shown in Figure 6 but at the higher moisture content region. (See Supplement B for more details).

Sample morphology refers to the physical distribution of the compo- nents of the sample in space after it is frozen; that is the size, location and orientation of the ice crystals formed by the freezing process. The morphology obtained in freezing will influence the freeze drying process as well as some properties of the dried product. Sample morphology depends on several physical properties of the sample (such as con- centration, viscosity, presence of cell walls or membranes, etc) as well as on the heat transport conditions in the freezing step.

26

In general, we can note that while slower freezing gives smaller num- bers of larger ice crystals which tend to be more orientated in one direction, rapid freezing gives large numbers of small ice crystals which are not oriented in one direction (Figure 9).

Figure 9. Influence of freezing rate on ice crystals morphology in the frozen sample.

rate on ice crystals morphology in the frozen sample. Viscous samples, which have poorer water transport

Viscous samples, which have poorer water transport possibilities, will give smaller ice crystals. Samples with smaller crystals and disorien- tated crystals will tend to have a higher resistance to mass transport in the sample during sublimation. This will tend to result in slower dry- ing and in the sample being more sensitive to structural alterations in the sublimation step.

In spite of above comments, the freezing step must be chosen so as to obtain the required product properties. For example, to preserve the fine structure of the sample for microscopic analysis, it is necessary to use very rapid freezing so as to minimise the size of ice crystals. This product-based requirement on the freezing step means that the remaining freeze drying conditions must be chosen with thought to the higher mass transport resistances to be found in such a sample.

In another case, trying to freeze or freeze dry a very concentrated solution may prove difficult, or impossible. In this case it may be nec- essary to dilute the sample prior to freezing so that an adequate amount of ice will be formed in the freezing step.

The sublimation step The sublimation step removes the water molecules which were sepa-

27

rated from the solids as ice crystals in the freezing step. The sublima- tion step does not involve the unfrozen water which is associated with the solids. This associated (or sorbed) water is first removed in the des- orption step.

As noted earlier, sublimation is the direct transformation of the solid water to vapour water. To accomplish this transformation requires energy, which means that we must have heat transport to the ice crystals if we are to get vapour formed. Thereafter, the removal of this vapour will be governed by the mass transport conditions present in the system.

In order to remove the water vapour formed, a pathway from the ice crystal to the sample surface will be required. This means that subli- mation always starts at an open surface and thereafter moves inwards into the sample. After sublimation of some of the ice, we will find that the sample can be divided into 2 regions, the dry layer (from which the ice crystals have sublimated) and the frozen layer (where the ice crystals are still present). The meeting place of these 2 regions is called the ice interface, sublimation interface, freeze drying interface or simply the interface. At the interface, the water content makes a sharp change from that nominally in the frozen region (that of the original material) to that in the dry layer (starting at about 25-30% water and falling to 0-3% water).

Figure 10 shows a schematic diagram for the movement of the ice interface and the resultant moisture distributions between the frozen and dry layers. In actuality, the moisture distribution is more compli- cated. It is also here at the interface that the structure supporting effect of the ice ceases, due to its removal from the structure by sublima- tion. This leaves a porous, open structure composed of the solids and associated water. Retention of this porous, open structure now de- pends solely on the stiffness of the unfrozen solids.

To have sublimation occurring at the interface, the energy required for the solid-vapour transformation must be transported through the sam- ple to the interface. This will require temperature differences, referred to as temperature gradients, between some heating source and the interface. The magnitude of the gradient (ie. temperature difference) required to give a given rate of water loss will depend on the resistances to heat transport present in the system.

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Figure 10. Moisture content distribution and corresponding schematic diagrams showing frozen and dry layers at various stages of freeze drying.

frozen and dry layers at various stages of freeze drying. The frozen layer has a much

The frozen layer has a much lower heat transport resistance than the porous dry layer, so much lower temperature gradients are required for heat transport through the frozen layer than through the dry layer. Since the dry layer surface must be open so water vapour can be removed from the interface, it follows that heat transport to this sur- face from the heat source must occur by radiation.

Heat transport from the surface to the interface through the dry layer will occur primarily by conduction along the solids.

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Figure 11. Heat and massflows together with corresponding temperature and moisture gra- dients for conduction and radiation heating.

moisture gra- dients for conduction and radiation heating. Heat transport through the frozen layer will also

Heat transport through the frozen layer will also be by conduction, though heat transport from the heat source to the frozen layer surface can be by conduction, convection or radiation, depending on the con-

30

figuration of the freeze dryer. Figure 11 shows schematic examples for temperature and pressure gradients in sample freeze dried with ideal conduction or radiation heat transfers. Also note that the sample is divided into dry and frozen layers.

After the vapour has been formed at the interface, it must be trans- ported out of the sample to the refrigerated condenser. This requires mass transport, and thus there must be a pressure difference, called a pressure gradient, from the interface to the refrigerated condenser sur- face. As noted above under sample morphology, the mass transport resistances in the sample are established in the freezing process. As all the vapour which is formed (this being determined by the magnitude of the heat transport) must be removed, the water pressure at the interface will establish itself at a level relative to the refrigerated con- denser pressure so that the resultant mass transport of vapour away from the interface will balance the vapour formation rate at the inter- face as determined by the heat transport. Herein lies a potential prob- lem since, in accordance with the equilibrium conditions given in Fig- ure 3, an increase in the water pressure at the interface will also result in an increase in the ice temperature, and the structural stability of the material depends very much on this interface temperature. It is in this way that an increase in heat transport to the sample can eventually result in a loss of sample structure.

It has been noted earlier that the stability factors of a material place limitations on the freeze drying conditions which may be used. In the above, it is noted that to conduct the sublimation step, it is necessary to transport energy to the ice crystals and to remove the vapour formed. To accomplish this requires establishing temperature and water pres- sure gradients, which if not properly chosen with consideration for the stability properties of the material to be dried, will result in an alter- ation of product properties. In particular, if one wishes to retain prod- uct properties, it is usually necessary to place limitations on the rate of heat transport to the interface.

In some cases, however, improper mass transport conditions (such as trying to put too much water vapour through a long, small-diameter hose) will give poor freeze drying behaviour, even when heat transport has not been intentionally increased. Lastly, there are some materials which are so sensitive with respect to their stiffness that it is almost

31

impossible to freeze dry them without a loss of structure. Some of these points are examined more in depth in chapter 11.

The desorption step In the sublimation step described above, we removed the ice crystals that were formed in the freezing step, from the sample. When an ice crystal is removed, what remains is the concentrated solute phase in what we call the dry layer. This will become our freeze dried material at the end of the process. Immediately following the passage of the interface, however, these solids in the »dry« layer still contain a signif- icant amount of water (generally about 25-30 g water/ 100 g solids), this being the strongly bound, unfrozen water mentioned earlier under the freezing process. Most sample materials will not be structurally or chemically stable unless the major amount of this water (called sorbed water) is removed. The process by which this more strongly associated water is removed is called desorption.

When we talk of sorbed, associated or bound water, we refer to water which exerts a lower pressure than pure liquid water exerts at the same temperature. This pressure reduction results from the binding of the water to the solids, with the strength of the binding determining how much the pressure is reduced. Since the strength of the water-solids bond increases as the amount of associated water per unit of solids decreases, the pressure exerted by the water decreases as the product gets drier.

There are diagrams (Figures 12a and 12b), called sorption isotherms, which give the relationship between the water pressure and water con- tent of a material at a given temperature (Figure 12a). The water pres- sure is often expressed in a normalised form that is relative to the water (vapour) pressure over pure water at the same temperature. The nor- malised water pressure is termed the water activity (Aw= p/po) and sorp- tion isotherms generally use this representation (Figure 12b). A materi- al will have different sorption isotherm values as temperature changes such that at each sample moisture level, the water pressure increases as the temperature of the sample increases.

The water pressure exerted by the sorbed water molecules is one of the two pressures determining the mass transport driving force required for removing the sorbed water. The other pressure is the water pres-

32

sure at the refrigerated condenser. Theoretically, our product will cease to lose water (ie. mass transport stops) when the water pressure at the sample is equal to the water pressure at the condenser since the driv- ing force will then be equal to zero.

Figure 12. Typical water sorption isotherms, Ts > T2 > T1.

12. Typical water sorption isotherms, Ts > T2 > T1. Using a series of sorption isotherms

Using a series of sorption isotherms for our material, we can for each sample temperature find the moisture content of the material for which the corresponding water pressure is equal to the condenser water pres- sure. This will then be the theoretical lowest final moisture content possible for the sample at that temperature. In actuality, the final mois- ture content will be higher, as the rate of water loss decreases sub- stantially as the pressure differences between sample and condenser get smaller and smaller.

If the final moisture content is not low enough to ensure product sta- bility, it will be necessary to alter the freeze drying conditions so that additional water transport from the sample occurs. This additional water loss can be achieved in 2 ways (See Figure 13).

(1) One can lower the condenser temperature (from say -50°C to -80°C). This will result in a lower water pressure at the condenser, and thus the water pressure gradient will become zero (ie. mass transport ceases) at a lower water pressure in the sample, which will correspond to a lower sample moisture content. Since, in reality, it is the reduc-

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Figure 13. Effect of changing sample temperature and/or condenser temperature on the min- imum possible sample moisture content.

on the min- imum possible sample moisture content. tion in driving force (pressure gradient) as the

tion in driving force (pressure gradient) as the sample dries which lim- its the final moisture level, this approach has a limited effectiveness for most normal materials.

A modern laboratory freeze dryer has a refrigerated condenser oper-

ating at about -50°C (p = 0.04 mbar). The further pressure decrease

achieved by operation at very low condenser temperatures (say -80°C;

p = 0.0005 mbar) is relatively small when compared to the saturation

water pressure which exists at the sample temperature (0.023 mbar for +20°C). While the extra pressure decrease at the condenser would appear to permit a lower final pressure at the sample, it has almost no effect toward increasing the pressure gradient and thus the actual water loss.

We must conclude that extremely low condenser temperatures are only necessary in special cases where the material being freeze dried has a very low structural stability and thus can only tolerate low tempera- tures. Here the sample water pressure itself would be low and thus the change in condenser pressure will have a significant effect on the over- all pressure gradient.

(2) The second approach, raising the sample temperature, offers more promise as the increase in water pressure in the sample is quite sizable.

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For a given water pressure at the condenser (ie. fixed condenser tem- perature), increasing the sample temperature will result in a sizable increase in the pressure gradient, and at the same time, sample and condenser water pressures will equalise at a much lower sample mois- ture content.

A sample temperature increase from +20°C to +30°C will result in a

saturation pressure rise from 23.3 mbar to 42.3 mbar, which is almost

a doubling of the gradient. Structural and chemical stability consider-

ations will place limits on the degree to which the sample temperature can be raised. It is interesting to note, however, that since stability gen- erally depends on both temperature and moisture content, the potentially decreased stability due to a temperature rise may be some- what balanced by increased stability associated with reduced moisture content; in the end, though, there will exist limits to the magnitude of the temperature increase possible.

We can further note that as in the other 2 freeze drying steps, it is also necessary in the desorption step to input energy to form water vapour from the bound water molecules. As heat transport resistances of the dried material are high, to have the required energy input, there will exist temperature gradients in the sample. The fact that temperature varies in the sample will mean that the sample will have a distribution of moisture contents in the dry layer. These gradients are shown in Figure 11. It is essential that at the end of the freeze drying process, all locations of the sample have moisture contents which are below the level required for product stability. It is not adequate that the average moisture content for the sample is below the level required for stability. This means that after the last ice crystals disappear and the sublima- tion step is completed everywhere in the sample, sufficient time must be allowed for all locations to desorb their remaining bound water. The last region to desorb to the desired level will be the region adja- cent to the last disappearing ice crystals.

As heat transport is slow (due to the high heat transport resistances), and the water pressure difference is low (due to the decrease in sam- ple water pressure as drying proceeds), the desorption step is gener- ally slow, especially considering the relatively small amount of water to be removed (generally 1-10 % of the total amount of water origi- nally present). Thus, all factors which can speed up the desorption

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step, such as raising the sample temperature, will have a significant influence on reducing the overall process time.

Summary of chapter 10 Since chapter 10 covers an extensive amount of material, it may be worthwhile making a short summary here. In chapter 10, we have seen that the freeze drying process can be divided into 3 steps, the first (freez- ing) in which the liquid water is divided into 2 groups, ice (solid water) and solute-associated water. The extent of this division depends on the sample temperature and the initial composition.

The second step (sublimation) involved removal of the ice crystals by input of energy. The energy input must be controlled so that the vapour produced can be removed without causing conditions for structural breakdown, especially at the sublimation interface. In practice, this generally means control of heating temperature though some sub- stances are inherently difficult to freeze dry.

The third step (desorption) also requires the input of energy. Following the sublimation of the ice crystals at a given location, the solid- associated water diffuses from the concentrated solution region to the vapour pathways.

The ultimate final moisture content of the sample is attained when the water pressure of the solid-associated water is equal to the water pres- sure of the condenser.

11. Questions to ask when choosing or operating a freeze dryer

When choosing a new freeze dryer, or when operating an existing freeze dryer, there are a number of factors to consider which will have an important effect on the success of a freeze drying process. While many of these factors have been mentioned at various points in the text, we will now directly present some of these practical considerations.

The most basic considerations: What do we wish to obtain as a dry prod- uct and why are we freeze drying it?

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The following questions should be the first one to be asked. Which of the various reasons for freeze drying are important for the product under consideration? How important is structural stability, or how important is the retention of the biological activity, etc? We could decide, for example, that low temperature drying with no chemical change is the goal, and that the total retention of structure is of lesser importance. In this case, making special efforts to design our freeze drying process so as to also retain structure may cause us unnecessary complications.

The envisaged end-use of the product can also have a practical signif- icance with respect to the best configuration of the freeze drying apparatus, especially regarding the choice of chambers and sample holders. This is discussed further below.

Material-related considerations: What are the characteristics of the mate- rial in general, and of our sample in particular?

After one knows why the sample is being considered for freeze drying, the next most important questions are related to the properties of the material to be freeze dried. It is important to have information about the chemical composition of the sample and the concentration of the solids. This information will be important in determining the freezing conditions that are required to adequately freeze the product.

Factors affected by the chemical composition of the sample From the composition, one will get an idea if the sample will be a eutectic former, or a glass former. Eutectic formers have definite struc- ture-loss temperatures, while glass formers have a more diffuse behaviour. Composition information is also important to evaluate the stability factors (both structural and chemical) of the product as these will directly depend on the chemical makeup of the sample. This information will be useful to determine which process parameters are permissible during the drying (primarily temperature levels) and dur- ing the subsequent storage (both temperature and moisture levels).

The influence of the sample composition on the chemical instability can be shown by considering the effect of the presence of amino groups (as amino acids or in proteins) in a carbohydrate-based sample. Mail- lard degradation of the carbohydrate will be possible (as well as

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changes in protein properties) when the sample contains the amino groups.

In a similar way, the presence of reducing carbohydrates can poten- tially reduce the stability of proteins (and thus equally well, enzymes) during both freeze drying and subsequent storage. It will be necessary to place limitations on the permissible freeze drying processing condi- tions if we want to avoid this type of chemical change during the pro- cess.

The chemical composition will also influence the structure stability. The presence of small amounts of high molecular weight materials will greatly improve the probabilities for structure retention during freeze drying. This higher molecular weight material can either be naturally present in the sample, or could be a component that one might con- sider adding to a sample primarily composed of structure-sensitive, low molecular weight species. Additionally, the moisture content at the end of freeze drying which is required for structure stability during storage is also dependent on the composition of the sample.

Factors affected by the concentration of the solids in the sample Initial sample concentration is important as it determines the amount of ice which forms at each freezing temperature. This influences the structural strength of the frozen material, and especially the stability at the interface. Since the pathways for vapour flow through the dry layer are the places from which ice crystals have sublimated (see Fig- ure 10), it is obvious that vapour removal conditions during sub- limation and desorption will depend on the amount of ice crystals formed, and thus on the initial concentration of the sample.

Samples with very high solid concentrations, in which little ice is formed, will be difficult to freeze dry due to the reduced stability at the interface and the limited number of vapour pathways in the dry layer. It can also be mentioned that samples having very low solid concen- tration, while technically not difficult to freeze dry, can be difficult to recover at the end of the drying process. In this case, there can be so few solids present that the original structure formed during the sepa- ration of the ice crystals cannot support itself once the ice is subli- mated. Thus, it appears that, in practice, there exist minimum and maximum sample concentrations if freeze drying is to be successful.

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An additional sample property of importance is the physical status of the sample (ie. is it liquid or solid?). This will primarily be important on the basis of sample handling considerations, such as determining what type of containers can be used, or how the sample can be frozen.

It is important to understand the properties of the original sample, and to determine if these should be altered so as to obtain a material that fulfils the goal of the product. In many cases, it will not be nec- essary to alter the material to achieve a successful freeze drying, but in some cases, alteration by changing the composition and/or concentration of the sample will be necessary if the material is to be successfully freeze dried under the desired process conditions, or successfully freeze dried at all.

DryPro 3000 freeze dryer with manifold for flask bulk drying

DryPro 3000 freeze dryer with manifold for flask bulk drying

Condenser-related considerations

Capacity-related questions: How big a freeze dryer do I need? The refrigerated condenser as a capacity measure. When buying a freeze dryer, there is always the question of how big a dryer is required. While a number of different size criteria could be used, laboratory freeze dryers are usually rated by the capacity of the condenser to hold ice (ie. water from the sample). This capacity says nothing about how fast the vapour can be transported to the con- denser, only how much ice it can hold before defrosting is necessary. Once we know the sample concentration, this capacity information

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will allow us to calculate how much product can be processed before

it is necessary to defrost the condenser.

As the dryer cannot be used during the defrost period (generally 2-4 hours, longer if the condenser does not have built-in defrost heating), frequent defrosting is not desirable. On the other hand, the condens- ing capacity costs money, so an overly large condenser is not neces- sarily the answer either.

As a minimum, the condensing capacity must be adequate to handle the amount of product to be processed in a single batch, with some excess capacity for special situations. This means that the condensing capacity is related to the product holding capacity of the drying cham- ber.

It is possible to add more product to the dryer than one has condens-

ing capacity for - for example if one keeps adding manifold elements to

a freeze drying system with external manifolds. A similar situation can

arise by inserting an extra tray or two in a bulk sample chamber. Such overloading of the system will result in incomplete drying of the sam- ple, no matter how long the freeze drying continues.

The condenser size can be larger than that required for a single batch. In this case, several sequential freeze dryings can be conducted before the condenser need be defrosted. In a laboratory setting, this can have some value as much of the working day can be used to observe the sample in the initial stages of the drying process instead of this time being used for defrost.

Another important measure of freeze dryer capacity to be considered

is the rate at which the condenser can remove heat and thus condense

vapour. If 2 freeze dryers can condense 10 kg of ice between defrost cycles, our freeze drying possibilities will be different, depending on their being able to condense at 0.5 or 2.0 kg/h at the desired process condenser temperature. The system with the higher condensing rate will have more flexibility, but this is achieved at the cost of a more expensive apparatus. For a condenser of a fixed heat removal capaci- ty, the more water vapour the condenser is required to freeze in a giv- en time, the higher the temperature (and thus the pressure) will be in the condenser.

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Increases in the rate of water vapour transport to the condenser can arise from several sources (to be discussed below), all of which are related to increasing the rate of production of product (either by dry- ing faster, or by handling more product in a batch). To have an ade- quate pressure gradient from the sample to the condenser, it is neces- sary to ensure that the condenser has an adequate heat removal ca- pacity at the anticipated rates of water vapour transport so that it can maintain the desired operating temperature and pressure.

DryPro 3000 freeze dryer with stoppering arrangement, HSC 500 controller and printer

DryPro 3000 freeze dryer with stoppering arrangement, HSC 500 controller and printer

Freeze drying chamber considerations

The choice of freeze drying system: chamber or manifold? While undergoing freeze drying, the sample is located in or on a sample holder. There are many types of sample holders available (such as ampoules, bottles, flasks of all sorts, trays, etc., see Figure 1) and choosing which one to use will depend very much on the ultimate usage of the product. The sample holders are generally placed in a vac- uum-tight enclosure called the freeze drying chamber. When a manifold freeze drying system is used, the sample holder actually acts as the freeze drying chamber as well.

When deciding which type of sample holder is most suitable, we must first ask the question »How am I going to utilise my product?«. Sam- ples that are to be utilised in ampoules, such as pharmaceuticals or chemical reagents, are best dosed into the ampoules in liquid form and then freeze dried in the ampoule. This method gives fewer hand- ling steps, which reduces risks of contamination and further ensures more reproducible dosages.

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The size of ampoule required will partly depend on the amount of ma- terial in a dose. It is a general rule of thumb that the thickness of the sample should be less than 15-20 mm. Due to the shape of an ampoule, the ampoule headspace, which is empty volume over the sample, will be an important consideration limiting the sample volume suitable for an ampoule. For larger sample volumes, it is necessary to either spread the sample on the walls of the ampoule (by altering the freezing procedure), or utilise bulk processing.

Ampoules can be freeze dried on either a manifold system, or by being placed on trays, which are then loaded »en masse« in a chamber.

A manifold system is simpler and cheaper to use when one is process-

ing few units at a time. In particular, one saves the cost of a chamber and can sometimes utilise a smaller capacity vacuum pump. A mani- fold also allows the ampoules to be flame-sealed under vacuum. How- ever, once the number of sample units (be it ampoules or flasks) becomes significant, the amount of manual handling with a manifold system will increase sizably, and it will be advantageous to use a cham- ber system.

A

manifold system can also be used together with larger jars or flasks.

In

this case, the jar or flask acts as a small freeze drying chamber. Mod-

ern freeze drying flasks have greaseless connections (based on rubber seals) and are available in either plastic (polycarbonate) or glass. For particulate sample, the wide-mouth type freeze drying flask will be pre- ferable, as it is easier to insert and remove the sample from this type of flask than from the standard conical-neck, round-bottom labo- ratory flask. For liquid samples, the preferences between wide-mouth and conical-neck flasks are less clear, as wide-mouth flasks are easier to empty while conical-neck flasks are easier to use with shell freezing.

With a manifold system, it is possible, with care, to place samples independently on the manifold or remove samples from the manifold as they are completed, which means that samples of various sizes can be freeze dried simultaneously.

In some cases, the manifold system can also be utilised for freeze dry- ing different materials at the same time. These possibilities for greater handling flexibility could be useful in a chemical laboratory where freeze

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drying involves a variety of small sample lots as part of an analysis pro- cedure.

With a chamber system, the samples are placed directly on shelves, or on removable trays. Having decided to use trays, it is no longer neces- sary to place the samples in ampoules or bottles, as it is possible to place the sample directly in the tray (called bulk loading). Bulk load- ing in a tray has advantages and disadvantages. The sample holding capacity of a bulk-loaded tray will be about 40 % higher than an equal size ampoule or bottle-loaded tray, due to the tray area lost around the round ampoules or bottles, and the tray area lost to the walls of the ampoules or bottles. Bulk loading also results in a better thermal contact between the sample and the tray, this giving less resistance to heat transport.

Bulk-processed liquids will generally form a low density, porous cake following freeze drying. Low bulk density of the dry material will give poor economy with respect to packaging, handling and storage (due to the large volume per unit weight), and thus bulk freeze dried liquid samples are usually crushed or ground to a powder to achieve a high- er bulk density and thus a better economy. With bulk loading, sealing the sample under special gas atmospheres or vacuum is usually not possible. Thus, while some protection can be achieved by first expos- ing the sample to an inert gas before opening the chamber, it will even- tually be exposed to air upon removal from the chamber. In some cas- es this can be important with respect to the later chemical stability in storage.

By using special freeze drying stoppers, it is possible to seal tray-loaded vials or bottles under vacuum or inert gas in special chambers at the completion of freeze drying.

There is a wide variety of chambers available which are designed to achieve one or more of the various freeze drying goals. For example, special chambers have been designed specifically for freeze drying small tissue sections at low temperature, through the use of external cool- ing systems. In such a chamber, there are built-in facilities to further treat the sample (crosslinking, embedding, etc) at the completion of the freeze drying steps to prepare it for microscopic analysis. Most

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chambers, however, are simpler, as the usual freeze drying goals do not make such high requirements.

After having presented the pros and cons of manifold and chamber systems, it is perhaps best to conclude that it would be most ideal if the freeze dryer is designed so it can utilise either system. This is espe- cially true when the freeze dryer is used in the laboratory environment, as this will give the highest degree of flexibility.

DryPro 6000 with horizontal manifold

DryPro 6000 with horizontal manifold

Freeze drying parameters

Freeze drying capacity as related to chamber and sample: How much product do we need freeze dried per day? There are many chamber and sample factors (such as sample thick- ness, sample surface area, chamber heating, etc), which will influence on how much product can be freeze dried in a particular period of time. It is by varying these factors that we have the most control over the freeze drying process, and it is here that we can get into most trou- ble! We will discuss how each of these factors influences the freeze dry- ing process.

Sample Thickness - Sample thickness affects both the freeze drying time and the amount of material that may be present in the sample hold- er. It is obvious that the thicker the layer of material in an ampoule or on a tray, the more sample we can load into the dryer. This would give

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us a higher loading capacity. However, thicker samples also take longer to dry.

Figure 14. Freezing configurations which give increased sample surface area.

configurations which give increased sample surface area. During drying, a very thick sample will eventually get

During drying, a very thick sample will eventually get a large dry layer thickness, which has high resistances to heat transfer and mass trans- port. To transport energy to the interface across such a thick, high- resistance dry layer requires a sizable temperature difference (driving force) and since most products will have a quality-related upper tem- perature limit, it will generally be difficult to maintain the same drying rate with a thick sample that was possible for a thinner sample of the same material. Thus, for thick samples, it is also necessary to accept a further slowing of the drying process.

45

Thus, if drying is to be completed in a reasonable time, one should try to observe the practical rule of thumb for maximum sample thickness (generally given as 15-20 mm). In some cases, this is impossible (for example when freeze drying whole animals), and in this case it is nec- essary to expect that freeze drying may take days or weeks.

When increasing sample thickness, it is necessary to consider that this will also increase the total water loading to the condenser. It is neces- sary to be certain that using extra thick samples does not place more water in the chamber than the condenser can remove. If the condens- er becomes overloaded, the drying will never be completed.

Sample surface - Another method for increasing the capacity of the freeze dryer is by increasing the drying rate (ie. less time required to dry a giv- en amount of material) so more material can be processed each day. There are several ways in which this can be accomplished. One method is to increase the surface of the sample. Transfer of heat to the sam- ple and transfer of water away from the sample occurs through the surfaces of the sample, and the larger surface the sample has, the high- er the rate of water removal possible.

The surface can be increased in various ways such as by wall coating dur- ing freezing (called spin or shell freezing), by using larger freeze drying flasks, by granulation, or by using more flasks or trays (See Figure 14).

If freeze drying flasks are filled with a given amount of liquid sample, the liquid sample will sit in the bottom of the flask in a pool which, when frozen, will have a given thickness. The larger the radius of the flask, the more area the liquid will cover, and the thinner the sample layer will be. The combination of increased transport area and reduced thickness will give a sample which dries more rapidly.

If the flask is tilted at an angle and rotated during the freezing step, the liquid can be distributed on the wall surface of the flask, which will result in a sample having a much higher transport surface and a much lower thickness.

The above approaches only apply to liquid materials. Granulation (grinding) of the frozen material to small chunks (called granules) is applicable to both liquids or solids. Here one increases the specific sur-

46

face of the sample by decreasing the particle size. Granulation can be simulated by freezing the liquid as droplets, either by spraying into an immiscible chilled liquid, into liquidified gas or by impingement on a very cold metal plate.

A last method for increasing sample surface in the freeze dryer is by

attaching more flasks of sample to a freeze dryer manifold, or by plac- ing more trays into the drying chamber. Here the surface increase is achieved without decreasing sample thickness. There is again a chance that the amount of water load placed on the dryer can become larger than the capacity of the condenser, which (as noted above) can result

in a failure to complete the drying process.

When attempting to increase the drying rate as noted above, a major

risk of product quality loss arises if the rate at which water is being transported from the sample to the condenser becomes so large that

a high pressure drop is required to overcome the mass transport

resistances in the manifold or chamber piping and valves. For example,

the more sample surface is placed on a manifold, the more water per unit time will have to be moved from the sample to the condenser.

When the condenser has an adequate heat removal capacity to con- dense all the arriving water, the required pressure difference (driving force) for mass transport will be obtained by an increase of the water pressure at the sample. This pressure increase will give rise to a tem- perature increase at the sample as well, which eventually can result in structural changes, depending on the properties of the material. As the water transport rate to the condenser increases, the condenser tem- perature will start to rise as well, since the heat removal capacity of the condenser becomes inadequate relative to the vapour delivery rate.

To maintain the necessary water transport flow rate, it is necessary that the sample water vapour pressure increases even more, so that the water pressure difference between sample and condenser is main- tained. On the basis of the same reasoning as given above, increases

in sample water pressure (and thus sample temperature) will result in

further potential for structure deterioration.

To summarise this situation, it is essential that the amount of water that is to be transported in the freeze dryer piping and valves is planned

47

in accordance with the carrying capacity of these units, the condensa- tion rate capacity of the condenser, and the properties of the materi- al being freeze dried.

One can say that there exist maxima for the amount of sample and sample surface that can be placed on trays in a chamber or in flasks on a manifold. These maxima are flexible as materials that are inher- ently more structurally stable can tolerate higher loadings than the more sensitive materials. A further factor entering into this considera- tion will be sample heating, which is discussed immediately below.

Sample heating - We have earlier discussed that to sublimate ice and des- orb bound water (i.e. to conduct the last 2 steps of the freeze drying process), it is necessary to supply energy to the sample. Even when we make no conscious effort to supply energy during freeze drying, nature is in actuality assuring that the required energy is being transferred.

In Figure 15 we can see that if we have our sample is in a flask mount- ed on a manifold, there will be heat supplied from the room air on the outside of the flask to the cooler wall of the flask, through the process of convection.

The heat will be transferred by conduction through the wall of the flask towards the sample, and after crossing the flask wall/ frozen layer interface, is conducted further to the interface in the sample where the frozen layer and dry layer meet.

At the interface, the energy is used to sublimate some ice crystals so that the product becomes a little dryer. For samples on a tray in a drying chamber, the course of the heat transfer will be a little different, but in effect we would note that even if we make no effort to supply heat, nature will do it.

We have now noted that heat is both required for freeze drying and will be supplied by nature (if we don’t do it), and we can immediately remark that the rate at which heat is supplied to our sample by nature is quite arbitrary and will most likely be either too fast or too slow. As we have noted earlier, it will be the properties of the sample which will determine which rate of heat transfer can be tolerated. Thus, if we have a sample which is sensitive to structural change, the natural heat trans-

48

fer rate may be too fast, while if we have a relatively insensitive sam- ple, the natural rate will be too slow. In most cases, the natural rate is slow enough so that freeze drying can occur with what is called »no external heating«.

When we come back to the problem of freeze dryer capacity, we will recognise that the faster we can remove the water from the sample, the shorter the time required in the drying chamber per batch, and thus the more batches can be processed in a given period. In most cases, the heat transfer rate from nature to the sample is lower than the product can tolerate, and thus the drying will go slower than the possible optimum.

The easiest way to increase the capacity of the freeze dryer is to increase the rate of heat transfer to the sample by means of artificial, control- lable heating. With the presence of artificial, controllable heaters, we can regulate the rate of heat transfer to our sample in accordance with the sensitivity of the sample to physical or chemical changes. Increa- sing the rate of heat transfer to the sample by using heating at tem- peratures above ambient is the most usual method to increase the rate of freezing drying and thus, the capacity of the freeze dryer.

As has been noted, with each method to increase freeze dryer capacity, there are limits to the possible increase, and this observation is especially true for the heating of the sample. We have noted earlier that the sam- ple will have temperature limits which should not be exceeded in regard of either loss of structural stability or chemical changes.

As has already been noted in the two sections on the relationship of capacity to sample thickness and sample surface, increasing the rate of water transport by increasing the rate of heat transferred to the sam- ple can eventually result in an overloading of the mass transport path- ways (eg. the piping and valve system of a manifold).

This will result in a water pressure rise at the sample, which is neces- sary so that the pressure difference for mass transport is large enough to give the required rate of water transport away from the sample. As noted earlier, the pressure rise at the sample gives a concurrent tem- perature rise at the sample, and if this is allowed to become large enough (for example, by a steady increase of the heat transfer rate),

49

Figure 15. Sample heating configurations in freeze drying.

Figure 15. Sample heating configurations in freeze drying. the stability temperature of the sample will be

the stability temperature of the sample will be eventually exceeded and the sample will undergo structural changes the consequences of which have been discussed earlier.

50

If we care to look at the sample on a more microscopic scale, we will

note that if we consider the situation at the interface (the rate of heat

transfer to the interface and the rate of water transfer away from the interface), we will find that the mass transport resistances of the dry layer will be important in determining the structural stability of the interface region, and thus the allowable (or optimum) rate of heat transfer. Thus, it is essential to consider both the »microscopic« and »macroscopic« heat and mass transfer situations.

The methods and problems associated with an artificial, controllable heating of the sample will somewhat depend on the relationship of the sample to the heating medium. In general we can identify 3 types of relationships: one for heating samples on manifolds and two for heat- ing samples on trays in the freeze drying chamber. The situation for heating samples on a manifold has been described above as being a combination of convection and conduction (Figure 15). We can increase the rate of heat transfer to the flask on the manifold by heat-

ing the air, by blowing the air faster over the outer surface of the flask, by removing any condensed ice layer on the flask (this ice layer acts as

a resistance to heat transfer), or by submitting the flask to a radiant heat source.

We can note that if contact between the sample and the inside wall of the flask is not very good (for example, due to breakage of the sam-

ple), the rate of heat transfer will become quite low and the drying time will become long. In this case the use of a radiant heat source, such as

a heat lamp, can improve the drying rate as it can transfer heat directly to the sample surface. But, such a direct transfer can be dangerous and quickly lead to overheating if not carefully controlled.

The situation for samples on trays in the drying chamber depends on whether the trays are in direct (and good) contact with the heating source, or whether the trays are suspended between the heating source without direct contact (Figure 15). In the first case, the sample is heat- ed primarily by conduction from the heat source, through the various layers of tray, sample holder and sample frozen layer.

In fact, it is similar to the case of the flasks on the manifold except that due to the vacuum in the freeze drying chamber, convection is not too significant as a heat transfer mechanism. This means that if the con-

51

tact between heating plate and sample holder surfaces is not good, the conduction heat transfer will not be good, and the drying rate will

fall significantly. This is especially important for the cases of contact between trays and heaters (the trays often become warped due to rough handling) or ampoules/ flasks and trays (most ampoules have

a slighty concave bottom). Eventual heat transport control must account for these non-ideal situations.

The second heat transport mode for drying samples on trays in the freeze drying chamber is through the use of radiant heating to the sam- ple and the tray surface. To have a reasonable heat transfer rate, it is necessary to use much higher heater temperatures than with the conduction-based heating systems.

Radiant heating has its advantages (it tends to be more even across a tray, as well as less sensitive to tray mishandling), and disadvantages (it is more energy-demanding due to heat losses from the dryer cham- ber associated with the higher temperatures). In addition, since it is usual to provide radiant heat from 2 sides, part of the heat transport will go via the dry layer.

As the dry layer has a high resistance to heat transport, it is necessary

to use quite high temperatures at the sample surface to give a reason-

able heat transport to the interface. This gives the risk of heating dam- age to the dry sample through overheating. Thus, with radiant hea- ting, we have another control condition to consider: the overheating

of the dry layer with subsequent chemical or structural damage.

To heat or not to heat, and how to heat, that is the question The question whether to heat or not depends very much on the re- quired throughput of the freeze dryer and the sensitivity of the sample. The quality of the product obtained will quite surely not be better when one uses heating, but at the same time, there will be a range of hea- ting rates at which quality will not be changed by the heating. At heat- ing rates higher than the limit, the quality will fall, though how »rap- idly« will depend on which quality factors are most important for determining the value of the product (eg. retention of enzyme activity vs. rate of solubility).

If the throughput rate is adequate, one can then be satisfied with the

52

natural heat input. On the other hand, if experience shows that the capacity of the dryer when using natural heating is becoming inade- quate relative to the amount of material to be dried, it could be suggested to first examine the possibility of obtaining improved drying rates by increasing the sample surface or decreasing the sample thickness, though taking care not to exceed the overloading limitations noted above. If further capacity increases are required, there is little alternative to utilising artificial heating to accelerate the drying process. Again, the limitations noted above must be respected so that the required product quality will be maintained.

The how of heating partly depends on the type of system involved. For a manifold system, heating can occur by means of heater-blowers, elec- tric resistance heaters or heat lamps, or by special arrangements which allow the use of a circulating liquid from a heating bath. In this latter case, the freeze drying flask could have a built-in jacket, or the manifold and flasks could be arranged so that the flasks could be dipped into an open trough through which the liquid was circulated. In general, a circulating liquid heating method for a manifold freeze drying unit will be awkward to use if there are more than a few flasks on the manifold.

In a chamber-based freeze dryer, heating is generally conducted either by circulating a pre-heated liquid through hollow heating plates from a reservoir of a closed-loop system, or by means of electric heating units (such as silicone rubber heaters) bonded to the heating plates. Electric heating units will be cheaper for small laboratory drying sys- tems while the circulating liquid system will be more suitable for larger installations. With the circulating liquid, it is possible to both heat and cool so temperature control can go in either direction. On the other hand, the time constant for a circulation liquid system will generally be larger than the electric heating system, making control less responsive. When all factors are considered, it will seem that the ulti- mate solution would be a combination of both systems.

Yet, a liquid circulation system with both heating and cooling will, in general, be preferred when drying heat-sensitive materials as temper- ature control can be more precise.

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Supplement A:

Water properties

Equilibrium phase diagrams for water and aqueous solutions show the various states in which water can exist under defined conditions of tem- perature (T), pressure (P) and composition (X). By states, we mean the physical condition of water, e.g. solid water (ice), liquid water and vaporous water (water vapour). The basis for representing states in terms of temperature, pressure and composition stems from these being somewhat controllable parameters. Lastly, by equilibrium, we mean that if we do not change the environment conditions (T, P or X), the state(s) present will then remain unchanged for all time.

Since there are 3 controllable variables (T, P, X), the phase diagram for water exists as a 3-dimensional diagram, with axes for T, P and X. As the 3-dimensional diagram is quite complicated, it is usual to discuss water properties in a series of 2-dimensional diagrams. The two of most interest to freeze drying are the pressure-temperature relation- ship for pure water (called the P-T diagram at X=0) and the tem- perature-composition diagram at atmospheric pressure (T-X diagram at P = I atm).

The pressure-temperature diagram

A word first about units of pressure. Most freeze drying literature up

to the most recent times has used pressure units of mm Hg or torr

(which are equal), though recently there has been a trend to increased usage of the SI units Pascal (Pa) and millibar (mbar). Most phase diagrams use atmospheres (atm) as the units of pressure. The relation- ship between these units is I atm = 760 torr = 760 mm Hg = 1013 mbar

= 101300 Pa.

A lot of information is given by a P-T diagram. The axes denote the

pressure exerted by water molecules and the temperature of these water molecules. There are 3 state regions (denoted solid, vapour and liquid) which are bounded by the lines AB, BC and BD, which give the combinations of temperature and pressure at which water in one state will change over into another state, that is the conditions for coexis- tence of two states.

54

Figure A 1. Pressure-temperature diagram for pure water.

Figure A 1. Pressure-temperature diagram for pure water. Some of these points are well known to

Some of these points are well known to us; for example point E is located at +100°C and 1013 mbar (atmospheric pressure). We know that at +100°C we can convert liquid water to vapour water, a process we call boiling at 1 atmosphere pressure. The »boiling« we see is the water molecules exerting a pressure of 1 atm, this equaling the total air pressure.

If we lived at the top of Mt. Everest where the local atmospheric pres- sure is only 0.31 atm (pressure falls with increasing altitude), our water would boil at +71°C (shown at point G). We should note that even though it is not +100°C, we still have a transition of liquid to vapour water.

We make use of this same information (lowering the pressure so water boils at a low temperature, as at point H) when we use a vacuum evap- orator to change liquid water to vapour water under gentle temperature conditions. The line BD denotes the interconversion of liquid water and vapour water. When we form vapour water from liquid water, we call the process evaporation while the reverse process is called condensation.

55

With the exception of special circumstances, this line ends at 0°C, a temperature with which we are familar. We can see that point B sits at the intersection of the 3 regions, and as such has a special significance and name, the triple-point of water (it is actually +0.01°C, but we will assume it to be 0°C here). If we look at the line C, we see that it is almost vertical, which means that the transitions between solid water and liquid water (crystallisation also called freezing and melting) occur at 0°C, essentially independent of the vapour pressure of the water.

The last line, AB, which separates the solid water and vapour water regions, is probably less well-known than the first two lines, which is unfortunate because it is here that freeze drying occurs. The line AB gives the corresponding pressures and temperatures at which solid wa- ter is converted directly to vapour water (called sublimation), or vapour water converted to solid water (called condensation). In these regions, there is no liquid involved in the transition.

Just as we can remember the standard value for boiling temperature in our head (+100°C = 1 atm), so can people working with freeze drying remember specific values of interest to them. Thus, if we have ice at 0°C, it will exert a pressure of 4.6 torr (point B); conversely if we can keep the water vapour pressure over ice at about 1.33 mbar, then the ice will have a temperature of about -20°C (point J). If we go still lower, to -40°C, then the ice will exert a still lower pressure, about 0.133 mbar (point K). The »trick« of freeze drying is to use these pressure dif- ferences to remove water from our product. This is discussed in chap- ter 9 of the main text and Supplement C.

To summarise, we have seen that water can be changed between its 3 states and that the changes which occur depend on either the water pressure or temperature, which are controllable variables. It should be noted that these conversions (called phase transitions) only occur with the addition or removal of energy to the water molecules.

The composition-temperature diagram The X-T diagram is used to show the influence of added solutes on the phase transition behaviour of water. For simplicity, only the solid-liquid phase changes (for an ideal solution) are shown. At a higher temper- ature, we would find the corresponding liquid vapour line. (At the pres- sure chosen, there is no solid-vapour line.)

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Figure A 2. Temperature-composition diagram for a eutectic-forming solute.

diagram for a eutectic-forming solute. When discussing freeze drying, it is easier to present

When discussing freeze drying, it is easier to present separate X-T dia- grams for the 2 solid-liquid transition behaviours encountered. One type of X-T behaviour, characteristic for salts such as buffers is denoted eutectic forming (Figure A 2), while the other, characteristic for sugars and a number of other biological materials, is denoted glass-forming (Figure A 3).

There are many similarities between the diagrams, which simplifies their discussion. There are 2 major phase transition lines (AB or ADB and BC or CD) for the two components of the system, water and solute. In addition, the eutectic-forming diagram has a line DBE, which defines the lowest temperature, the eutectic temperature, at which liquid can exist in such a system.

The absence of this line on the glass-forming diagram indicates that there is theoretically no temperature at which liquid does not exist. It is important to note the word »theoretically«, however, since at a low enough temperature (below the glass transition temperature) the product acts like a solid. However, the product is in fact a highly vis-

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Figure A 3. Temperature-composition diagram for a glass-forming solute.

Temperature-composition diagram for a glass-forming solute. cous liquid even though it resembles a solid, and thus

cous liquid even though it resembles a solid, and thus cooling below the glass transition temperature does not give a real liquid-solid tran- sition.

We can see from these 2-component diagrams (water and solute) that the composition axis is a »mirror image« for each component (ie. 100% water means 0% solute, etc). Thus the phase transition temperature at 0% solute is the crystallisation (freezing)/ melting temperature of pure water (0°C) as discussed under the P-T diagram. This point is shown in Figures A 2 and A 3 as T m . Similarly, if we have pure solute (100% solute), the crystallisation/ melting temperature is shown as Tc . We can see that if we add solute to water, or water to solute, the freez- ing or crystallisation temperature (ie. the temperature at which the sol- id and liquid interconvert) is decreased.

Using the water side (line AB - of most interest relative to freeze dry- ing) we can describe what happens as we cool a solution which is ini- tially at temperature T, and composition X s. To avoid complicating the discussion, the system is assumed to always be at equilibrium; in real

58

life we can have non-equilibrium conditions such as subcooling or supercooling arising, which will give a more complicated behaviour. Initially, cooling the solution lowers its temperature with no change in composition. We remain in the liquid region until the temperature falls

to T fs, which is the freezing temperature corresponding to Xs. This is shown in Figures A 2 and A 3 by the line SR. When the solution reach- es temperature T fs , continued cooling will cause the system to cross the phase boundary and the first ice crystals are forming. Ice crystals are the solid-structure form of water molecules and are pure (ie. 100% water). With this crystallisation of some of the water molecules, there are now fewer liquid water molecules remaining in the solution, and thus the composition changes in the direction of increased % solute. If we continue down the SR line to point U, we find that we are in the phase region, liquid plus solid water. At point U, we have a mix of ice crys- tals (X=0) and a more concentrated solution (X=X p) at temperature

T fp.

If we now consider a second solution at temperature T of a higher ini- tial concentration of solute X p, we can see that it is necessary to cool this solution to T fp before the first ice crystals form. At this point, the unfrozen solution from S and P act identically as the temperature is further lowered. The major difference between these two solutions will be the percentage of the initial solution which is present as ice. The more concentrated the initial solution, the less ice is present at any temperature.

Returning now to solution S, further cooling leads to the point at which we differentiate between eutectic-forming and glass-forming solutes. For eutectic-formers, there exists a unique temperature and composition com- bination (called the eutectic temperature and eutectic composition) at which the water and solute together form an intimately mixed solid phase called the eutectic. Cooling the sample at the eutectic tempera- ture gives this phase transition and further cooling below the eutectic temperature does not result in further concentration of the sample as it has totally solidified.

In freezing glass-forming solutions, the liquid phase continuously gets more and more concentrated. Eventually, more water cannot be removed from the liquid phase to the growing ice crystals because the material has become stiff due to the low temperature and high solute

59

content. We say that the sample is »solid«, but in fact it is a very stiff, viscous liquid. While this »solid« appearance is not a stable condition, the rate at which this »liquid« flows is generally so small that we can- not notice its flow over reasonable lengths of time. The rate of flow is, however, very sensitive to temperature change and the »solid« reverts to an obvious liquid condition over a narrow temperature range.

In brief, we have noted how cooling a solution results in the formation of solid water (ice) and the concentration of the remaining liquid phase. The more concentrated the initial solution, the less ice will be formed at a given freezing temperature. Lastly, we have two basic types of solutions, eutectic or glass formers, the first resulting in a completely solid phase at low enough temperatures, while the second results in highly concentrated, viscous liquids, known as glasses. These differ- ences are of some importance with respect to the freeze drying be- haviour.

Supplement B:

The material

We can note some general factors which we have called stability factors are important for obtaining high-quality freeze dried products. The properties of a material relative to these stability factors influence the »freeze dryability of the material«, in particular the constraints under which the freeze drying process must be conducted.

These stability factors are divided into two major groupings: (1) structural stability and (2) chemical stability.

Structural Stability of Materials

The structural stability of a material relates to its ability to go through the freeze drying process without changes in size and shape. Poor struc- tural stability generally gives products which are shrunken, puffed, glassy-looking, and which are often sticky.

With respect to structural stability, eutectic-forming and glass-form- ing solutes show differing behaviours. The alteration of structure for

60

eutectic-forming solutes involves melting and dissolution, while glass- forming solutes involve changes in the »stiffness« of the material.

Melting/ dissolution and structure (eutectic-forming solutes) It was noted earlier (Supplement A) that at the eutectic temperature, the unfrozen eutectic solution solidifies as a mixed system of ice crys- tals and solute crystals. Thus at sample temperatures below the eutectic temperature, the sample is totally solid, being solely composed of crystalline species. As a solidified eutectic rises in temperature, a major change will occur in structure when the eutectic temperature is reached as the entire solidified eutectic will melt at a single tempera- ture. A further increase in temperature will result in a melting of some of the remaining ice so that the solution concentration will decrease in accordance with the freezing point curve. Since most eutectic- forming solutes are low-molecular-weight materials, the melted eu- tectic has a very little structure-stabilising ability.

Ice crystals themselves serve as a supporting structure for the frozen material, and thus the overall stability of a eutectic-forming sample in which the eutectic has melted will partly depend on the initial con- centration of the solution and the eutectic concentration. These determine the proportion of the initial sample which is present as ice crystals at the eutectic concentration and temperature. A material hav- ing a high eutectic concentration and low initial solid concentration will convert much of the water to ice before achieving the eutectic con- centration. Such a sample could macroscopically appear to be struc- turally stable, even though the eutectic might be melted.

Structural breakdown of the dry eutectic-based material occurs through a melting of the crystalline structure (if temperature is increased at a constant moisture) or in a dissolution process (if mois- ture is increased at a constant temperature). Moisture must be added at a level corresponding to a saturated solution before the structure changes.

»Stiffness« and structure (glass-forming solutes) The concept of stiffness is used to describe the physical rheological prop- erties which characterise the structural stability behaviour of a glass- forming material. Stiffness is a measure of resistance of a material to the action of outside forces which are acting to change its shape. (Stiff-

61

ness is related to viscosity in liquids, or to the viscoelastic elements in

a solid.)

To have a stable structure when freeze drying glass-forming solutes, the sample must have adequate stiffness throughout the freeze drying process. There are three major factors which influence stiffness:

molecular composition, sample temperature and sample moisture content. Since structural instability involves a deformation or flow process, it is a time-dependent property. It means that we define a material to be structurally stable if the deformation over a chosen period of time is less than we can measure. Perhaps, if we looked longer, we would note some flow and thus call the material unstable. Thus, stability also depends on the time chosen for observation. For freeze drying we usually define the observation time to be the duration of the freeze drying process.

Temperature and moisture influence on stiffness. The influence of temperature and moisture on the sample stability for

a

single material (ie. fixed molecular composition) is shown in Figure

B

1. There is an interaction of moisture content and temperature which

define the sample stability boundary (ie. boundary between stable and unstable samples). If we lower the moisture content of a sample, it will be stable at a higher temperature (for the same observation time) or

vice versa.

Also noted in Figure B 1 is the effect of changing the observation time on the temperature-moisture content relationship for the stability boundary (here t 1 is less than t2). To retain structure during the freeze drying process, it is necessary that the material always remains in the region of stability. This means that for each moisture content existing in the sample during drying, there is a critical temperature (often called the »collapse temperature« in the scientific literature) above which that region of the sample cannot be allowed if structural stability is to be secured.

From the general shape of the stability boundary in Figure B 1, we can see that collapse temperature is highest for zero moisture content, and decreases with increasing moisture content until we reach a moisture content range above which the collapse temperature does not increase further. The region of 0- 5% moisture is of most interest when con-

62

Figure B 1. Influence of temperature and moisture content on the structural stability bound- ary of a material.

on the structural stability bound- ary of a material. sidering the stability of the final freeze

sidering the stability of the final freeze dried product, while the other end of the moisture content scale (up to 25- 30% moisture and above) is of most interest in the freeze drying process itself.

The fact that at higher moisture contents, collapse temperature does not decrease further with increasing moisture content results from the special behaviour of water in the freeze drying system. As we lower the temperature below 0°C, a portion of the liquid water wil be convert- ed to solid water (ice). Thus, while the overall average sample mois- ture content remains unchanged (since both liquid and solid water are counted in the overall average), the liquid moisture associated with the solids is somewhat lower, and it is only this liquid moisture asso- ciated with the solids which is of importance in determining the flow or deformation behaviour of the material. As we have noted earlier when discussing the X-T diagram (Supplement A), in the freezing pro- cess, we eventually attain a solid-associated moisture content where further removal of liquid water from the solids is hindered by the increasing sample stiffness. Hence, at average moisture levels higher

63

than this solid-associated moisture concentration, the collapse tem- perature will remain constant.

For frozen systems of glass-forming solutes, the presence of ice has a significant influence on the sample structural stability. We have noted earlier for glass-formers (Supplement A) that (1) as we freeze a solution we obtain a mixture of ice and a more concentrated solution and (2) the amount of ice formed at a given temperature decreases as the initial solution concentration increases.

Figure B 2. Influence of material molecular weight on the structural stability boundary.

molecular weight on the structural stability boundary. The overall structural stability of the sample depends on

The overall structural stability of the sample depends on its overall stiff- ness, which comprises the stiffness of the ice phase and the stiffness of the concentrated solute phase. Pure ice crystals are quite stiff even at a temperature close to 0°C, so the ice phase present in a sample will make an important contribution to the overall sample stiffness.

In discussing the influence of temperature and moisture on the stiff- ness of a sample containing both ice and material phases, we can note from Figure A 3 that if we slightly raise the temperature of the frozen sample, we cause two actions to occur; one is the conversion of some

64

solid water (ice) to solute-associated liquid water associated with the solute (ie. an increase in the actual moisture content of the solid material even though the overall sample moisture content is unchanged) and the other is an increase in the sample temperature.

Figure B 3. Relationship of material molecular weight and maximum temperature giving structural stability for a given sample moisture content.

structural stability for a given sample moisture content. As each of these two actions results in

As each of these two actions results in a decrease in sample stiffness, frozen samples with ice are much more sensitive to a temperature rise than a sample in which there is no ice present. This means that the frozen material can be especially sensitive to a loss of structure during freeze drying.

Molecular composition and stiffness.

Each material will have its own temperature-moisture content curve giving the structural stability boundary. Figure B2 gives a series of curves for 3 members of a family of compounds. Examples of such families could be the glucose-based saccharides (glucose, maltose, maltotriose

starch) or amino acid poly-

mers (amino acid, dipeptides,

proteins). The

lower the molecular weight of the material, the lower the stability boundary curve. This means that at any moisture content, the lower

maltodextrins (starch hydrolysates)

polypeptides,

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the molecular weight, the lower the temperature at which structural collapse will occur. Increasing the molecular weight of the sample

results in stability being retained at higher temperatures. Figure B 3 shows that the data from Figure B 2 can be presented as a straight- line relationship (here for moisture content Ma). That 1/MW is linear with collapse temperature means that the effect of molecular weight

is most noted at low molecular weight (ie. 1/MW is high), and that

when we get two compounds of much higher molecular weight, fur- ther increases in molecular weight have little additional effect on col- lapse temperature.

Chemical Stability of Materials

The chemical stability of materials as related to freeze drying is an extensive subject due to the large number of materials that can be freeze dried and to the many chemical reactions that can occur. These reac- tions may occur in the freeze drying process or may occur in the stor- age of freeze dried material.

A number of these chemical reactions are noted below. It can be men-

tioned that our understanding about how these reactions work is quite variable. For some, such as the survival of microorganisms, the over- all reaction schemes are not fully understood, while we have a good picture of the reaction mechanism for others.

The freeze drying or storage parameters which most influence the chemical stability are the moisture content and the temperature of the material. It is well known that chemical reaction rates increase with an increasing temperature and generally decrease with a decreasing mois- ture content. (A few exceptions to the moisture content »rule« are observed for lipid oxidation and Maillard browning reactions).

In freeze drying, we have widely varying moisture contents and tem- peratures in the sample, these changing with time and location. This means that the potential for chemical changes to occur within the sam- ple will vary with time and location. Our choice of processing condi- tions will thus have to consider the temperature and moisture gradi- ents in the sample and how they change as drying proceeds.

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Non-enzymatic browning: There are a number of reaction mechanisms which are grouped under the heading non-enzymatic browning, because they result in brown products and are not enzymatic in nature. The Mail- lard reaction (a carbonyl-amine reaction) and high temperature car- bohydrate dehydration (called caramelisation) are most likely to be problems in freeze drying. The Maillard reaction generally involves reducing carbohydrates and amino acids (or proteins), both of which are present in biological materials. The caramelisation reaction appears to occur at lower temperatures in freeze drying than is required in liquid systems, perhaps due to the removal of the water produced in the reaction when freeze drying. This is especially noted in samples hav- ing relatively high or low pH values (ie. highly acid or basic solutions).

Besides the brown colour developed, these reactions also result in many breakdown compounds having a strong aroma, and which can serve as reactants for other reactions. In addition, Maillard browning involving proteins can give a loss of biological activity.

Protein denaturation: Proteins are important components in the biological system, acting as structure material and as catalysts of chem- ical reactions (enzymes). The ability of proteins to function depends on many factors, among them the geometrical structure. Protein struc- ture is stabilized by the presence of solvent (liquid water). Removing this solvent can lead to structural changes. Exposed amino acid side chains become available for reactions (both with other side chains on the same molecule, and with side chains of other molecules) which change the characteristics of the protein molecule. When water is returned to the sample, these protein molecules may be unable to return to their original condition.

Factors influencing the potential for protein changes are the own internal bonding of the protein, the side chain reactivity, and the tem- perature and moisture content in freeze drying.

Nutrient destruction: A number of nutrients in foodstuffs (especially Vitamin C) are labile when subjected to heat, and as such their reten- tion depends on the temperature conditions in the freeze drying process. The role of moisture is less well understood, but it appears that as the moisture level falls, the other temperature sensitivity of the reaction also decreases.

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Lipid oxidation: As the most important reactant, oxygen, is not pres- ent in the chamber during freeze drying, this reaction is most impor- tant during storage. Freeze dried material containing unsaturated lipids is especially susceptible, as the porous structure of the freeze dried cake gives oxygen a good access to the internal pore surface of the materi- al. This means that lipid-containing materials must be packaged so as to hinder the contact of lipid and oxygen, in chosen high-quality, low transmittance packaging materials, and by closing with either vacuum or inert gases in the package headspace.

There are also indications that free radicals, which can initiate lipid oxidation, may be generated in the freeze drying process. In the dry state the free radicals are quite stable, and thus their presence places even higher requirements on the fulfillment of the packaging goals.

Nucleic acid breaks: There have been indications in the scientific lit- erature that genetic mutations can occur in freeze dried microbial cul- tures. It is proposed that the genetic changes result from nucleic acid breaks. At this stage in time, it is necessary to note that many micro- bial species can be dried successfully without undergoing genetic changes, and thus genetic mutation is not a necessary consequence of freeze drying. At present not much is known about the cause of genetic mutations or about the influence of freeze drying parameters.

Summary of chemical stability: We have noted that reaction rates in freeze dried materials will depend on the temperature and moisture levels in the sample, with an interactive relationship between these parameters. Thus, depending on the sensitivity of the material, it will be necessary to restrict the sample temperature to particular maximum values. In most cases, the moisture content in the sample will be related to the temperature gradient in the dry layer and thus cannot be controlled independently.

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Supplement C:

Mass transport

Mass transport (or mass transfer) is the movement of matter from one location to another. In freeze drying we are most interested in the move- ment of water, especially from our sample to some other location. The driving force for mass transport in freeze drying is pressure difference (actually, in the most general case, it is a concept called chemical potential, which in our case can be approximated by pressure), while the resistances are a number of friction flow elements. A simple example can be used to clarify the relationship.

Figure C 1 Basic system for mass transport discussion.

Figure C 1 Basic system for mass transport discussion. Figure C 2 Basic system with unequal

Figure C 2 Basic system with unequal distribution of molecules before mass transport.

unequal distribution of molecules before mass transport. In Figure C 1, we have 2 equal volume

In Figure C 1, we have 2 equal volume chambers, left (L) and right (R) connected by a tube (T). To separate the two chambers, we place a

69

valve (V) in the tube line. If we have an empty chamber, then we will have no pressure in the chamber since pressure is a measure of the number of molecules present in a given volume. The more molecules in a fixed vol- ume, the higher the pressure. Temperature is also important; higher temperatures give increased pressures since each molecule has more energy, but for our discussion here, we will let the temperature be con- stant. If in Figure C 1, we put (with the valve closed) 100 units of water molecules in the left chamber (NL=100) and 50 units in the right chamber (NR= 50), we will find that each chamber shows its respec- tive pressure, PL and PR. This is shown in Figure C 2. If we now open the valve, the system will tend to equalise itself so that the number of molecules is the same in each chamber (ie. the pressure will become equal in each side). This equalization is accomplished by the net phys- ical movement of 25 units of water molecules from the left chamber,

Figure C 3 Basic system with equal distribution of molecules after mass transport.

with equal distribution of molecules after mass transport. Figure C 4 Variant of basic system with

Figure C 4 Variant of basic system with higher mass transport resistances.

of molecules after mass transport. Figure C 4 Variant of basic system with higher mass transport

70

through the tube and valve to the right chamber (Figure C 3). Thus we have conducted a mass transport due to the pressure difference driving force.

The resistances to the movement of these water molecules will deter- mine how fast the pressure in the two chambers can equalise. By resistances we can mean a number of frictions and hindrances in the tube and valve, such as the friction which occurs when a water mole- cule hits the wall of the tube, or hindrances which can result if there were a plate with a small opening in the tube, or if there were air mol- ecules through which the water molecules must move. Since the water molecules move in straight lines, if the tube is not straight, one can imagine that the water molecules will hit the walls more times and thus move more slowly through the tube.

Figure C 4 shows a system in which we could expect the mass transport to occur more slowly than for the system in Figure C 2, but in all cases the pressure will be the same in the end.

Figure C 5. Dynamic mass transport in the basic system.

Figure C 5. Dynamic mass transport in the basic system. Up till now, we have considered

Up till now, we have considered a simple closed system in which the system is allowed to reach equilibrium. Of more interest in freeze dry- ing is the dynamic case where water molecules are continuously sup- plied at chamber L (called a source) and continuously removed at chamber R (called a sink) so that the values of NL and NR (and hence PL and PR) remain unchanged. In this case we get a continuous move- ment of water molecules from L to R, the rate of this movement depending on the resistances. To consider this dynamic situation fur- ther, we must establish a water molecule source and a water molecule

71

sink. Since we are interested in freeze drying, we will base our next dis- cussion on conditions related to freeze drying.

If we refer to Figure A 1, we can see that if we have ice (solid water) at -20°C (point J), it will exert a pressure of about 1 torr, while ice at -40°C will exert a pressure about 0.1 torr (point K). Thus, if we go to our two chamber systems of Figure C 1, put ice in the two chambers, and then control the temperatures so that TL=-20°C and TR=-40°C, we will have our requirement of a water pressure difference between the two chambers (Figure C 5). (How we control the temperatures is discussed in Supplement D.) Under these conditions, water molecules will move (just as in our earlier example) from the left chamber to the right chamber. If we supply the energy required to convert solid water to vaporous water (sublimation) in the left chamber, then as water vapour molecules leave the left chamber, new ones are formed from the ice. In the right chamber, the opposite action is occurring; energy is being removed and the newly arriving water vapour molecules are being converted (condensed) from vapour to solid. Thus the net effect has been a reduction of solid water in the left chamber and a corre- sponding increase of the amount of solid water in the right chamber. This is the basic mass transport occurring in freeze drying.

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Supplement D:

Heat transport

DryPro 9000 freeze dryer with chamber and built-in HSC 500 controller

DryPro 9000 freeze dryer with chamber and built-in HSC 500 controller

The movement of energy occurs through a process called heat transport. Energy movement is important in freeze drying because each time we have a change in the state of water (from liquid to solid, solid to vapour or vapour to solid), it is necessary to remove or supply energy to the water molecules. Thus an essential part of the freeze drying process is heat transport.

As an introduction, we can note 3 modes of heat transport, which are shown in Figure D 1. We can see an energy source (here depicted by a flame - we could have used an electric heater) at the bottom. This energy source is used to raise the energy of the molecules in the sur- face at the bottom. The energy content of a material can be measured by its temperature, a higher temperature indicating a higher energy lev- el. For our examples, we assume that the energy of the lower surface is the same, as noted by the equal temperature (TL). To transfer this energy from location L to location U requires the existence of an energy (ie. temperature) difference between these locations; that is, the driving force for heat transport is a temperature difference.

73

Figure D 1. Three modes of heat transport

Figure D 1. Three modes of heat transport The resistance to this energy transfer depends on

The resistance to this energy transfer depends on the physical system, and for simplicity, these have been divided into 3 basic types, conduc- tion, convection and radiation, shown in Figure D 1. Conduction is the mode of energy transport in solids where there is no physical movement of matter possible. Convection is the mode of energy transport in liquids, vapours and gasses where the energy is transported by the actual phys- ical movement of molecules in a space. The effectiveness of convec- tion partly depends on the number of molecules available in the space which act as the energy »carriers«. If we empty the space (by having a vacuum, for example), there can be no convection. Heat transport between independent locations can occur by a wave phenomenon called radiation. Since radiation is relatively independent of an entervening gas media (conditions of interest for freeze drying), we can still have heat transport between 2 independent locations through radiation.

To first consider the static case where the energy from the lower sur- face is not allowed to escape at the upper surface, then an energy trans- fer occurs only until the upper surface reaches the same temperature as the lower surface. Thus, when the driving force disappears, there is no further energy transport. The rate at which this occurs will depend on the resistances to the heat transport, and we can generally say that the resistances increase in the order conduction, convection and radiation.

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Figure D 2. Boiling system for heat transport discussion.

Figure D 2. Boiling system for heat transport discussion. The dynamic situation is of more practical

The dynamic situation is of more practical interest and Figure D 2 shows the well-known process of heating liquid water to boiling (vaporisation). While we could have described the transitions for solid water (ice) (melting/sublimation), since boiling is so well known it is an obvious example.

In Figure D 2, we have a pot of water sitting on an electric heating plate. If the physical contact of the pot and heating plate is good, we have heat transfer by conduction; if contact is not good, we will probably have a combination of conduction and convection.

Turning on the heating plate allows electrical energy to be converted to heat energy. This raises the energy level of the heating plate, which we observe as a change in temperature. We have now established a temperature gradient between the heating plate and the pot of water and thus have a driving force for transport of the heat energy.

The energy transported to the water molecules in the pot raises their energy and thus we note a rise in the water temperature. The rate at which the temperature rises partly depends on the size of the resist- ances to heat transfer which exist. If we have our pot open so that the external pressure is 1013 mbar (1 atm) we can see, from Figure A 1, that when the water reaches a temperature of +100°C (ie. the water molecules reach the corresponding energy level), further transfer of energy to the water molecules results in a change in state from liquid to vapour, which we call boiling (ie. we cross the line at point E).

To achieve this change in state requires an energy transfer to the water molecules. In the boiling process, it takes much more energy to

75

cause the change in state of the water molecules than to heat them to the boiling temperature.

Every time we cross a state line in Figure A 1, we require the input or removal of energy. Moving from left to right in Figure A 1 requires the input of energy, while moving from right to left requires the removal of energy. Similarly, in Figure A 2 or A 3, moving up or down respectively requires the input or removal of energy.

We can now return to Figure C 5, in which ice was transported from the left chamber to the right chamber. We had assumed that T L stayed at -20°C, while T R stayed at -40°C through the proper transport of energy (ie. heat transport). To achieve this, we can envision an electrical heater in the left chamber to supply the heat required to generate water vapour molecules from ice (sublimation), while in the right cham- ber, we can have a freezer coil to remove heat so these water molecules will condense to ice (condensation) and be thus removed from the vapour space.

Through Supplements C and D we have developed the basis for the freeze drying process - a combination of mass transport and heat trans- port.

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General references on freeze drying

In this booklet, we have started our discussion on freeze drying at a very basic level and then, from this base, we have developed the sub- ject through more detailed examinations of the properties of materi- als and the steps of the freeze drying process. It is, of course, not pos- sible in a booklet such as this to cover all the possible freeze drying- related subjects (such as engineering calculations, or additive use when freeze drying microbial cultures, to name two topics which could not be covered). We therefore give here a limited list of general references on freeze drying which can be used to supplement the information pre- sented in this booklet.

It should perhaps be mentioned that much of the earlier literature not- ed below refers to freeze drying and foods. This orientation arose because food preservation was originally considered to be the major potential application for freeze drying. Interest in freeze drying later also expanded to include preservation of biologically active materials by freeze drying. In any case, it is important to emphasise that many of the references listed below which have food in the title give an ex- cellent coverage of the freeze drying process in general, and will thus also be of interest to the more biologically oriented user. Furthermore, many of these references contain sections on freeze drying of biologi- cal preparations, as well.

As this is a limited list, no individual research articles have been pre- sented. References to research articles can be found in the reference lists of the books noted below. Beyond these, other good sources for locating research articles with information on freeze drying are science citation index and chemical abstracts (or their associated computer data bases); consult under the headings »freeze drying, freeze dried, lyophili- sation, or lyophilised«.

Harper, J. C. and Tappel, A. L. Freeze Drying of Food Products Advances in Food Research, Vol. 7 Academic Press

1957.

77

Rey, L.

1960.

Traité de Lyophilisation Herman, Paris (French and English Articles)

Fisher, F. J.

Freeze Drying of Foods National Academy of Science - National Research Council

Washington, D. C.

1962.

Rey, L.

1964.

Aspects théoriques et industriels de la lyophilisation (Research and Developments in Freeze Drying) Herman, Paris (French and English Articles)

Burke, R. F. and Decareau, R. V. Freeze Dehydration of Foods Advances in Food Research, Vol. 13 Academic Press

1964.

Rey, L.

1966.

Advances in Freeze Drying (Lyophilisation. Recherches et Applications Nouvelles) Herman, Paris (French and English Articles)

King, C. J.

Freeze Drying of Foods CRC Press

1971.

Goldblith, S. A., Rey, L., and Rothmayr, W. W. Freeze Drying and Advanced Food Technology Academic Press

1975.

Cabasso, V. J. and Regamey, R. H.

Freeze Drying of Biological Products Developments in Biological Standardization, Vol. 36 S. Karger

1977.

Mellor, J. D.

Fundamentals of Freeze Drying

Academic Press

78

1978.

In addition to the above books, there are the Proceeding of Congress- es and Meetings of the International Institute of Refrigeration (espe- cially Commission 10 (now C1)-Freeze Drying, Cryobiology, Medical Applications). These are generally referred to as bulletins, Proceedings of Annex of the IIR.

1969

- Lausanne -Thermodynamic Aspects of Freeze Drying

1969

- Paris - Surface Reactions in Freeze Dried Systems

1971

- Washington - Progress in Refrigeration Science and Technology (Freeze Drying)

1973

- Sapporo - Freeze Drying of Biological Materials

1974

- Bressanone - Heat and Mass Transport during Freeze Drying of Food Stuffs

1975

- Moscow - Freeze Drying

1979

- Venice - Freeze Drying, Cryobiology, Medical Applications

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