Labconco 7949030, 7948020, 7948030, 7949020, 7754030 Data Sheet

A

Guide

To

Freeze

Drying

for the

Laboratory

An Industry Service Publication

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Foreword

This booklet has been developed to serve as a basic guide to the freeze drying process. The information presented is generic in nature and is the result of research and experience by Labconco personnel and users of freeze drying equipment. It is our intention to provide a non-biased review of preparation techniques and freeze drying methods. The purpose of this booklet is to help you make an informed choice of equipment for your laboratory applications.

Our Method

We begin our discussion of freeze drying for the laboratory by examining the three steps in the process: prefreezing, primary drying and secondary drying. Next, we examine a typical freeze drying cycle and the methods available to facilitate the freeze drying process using equipment designed for use by laboratories. Finally, suggestions to optimize successful results are discussed, including determination of end point, contamination, backfilling of dried samples and product stability.

A glossary of terms used throughout this booklet to explain the freeze drying process follows the text, along with a bibliography.

Introduction

Freeze drying has been used in a number of applications for many years, most commonly in the food and pharmaceutical industries. There are, however, many other uses for the process including heat-sensitive sample preparation, plant material research, the stabilization of living materials such as microbial cultures, long term storage of HPLC samples, preservation of whole animal specimens for museum display, restoration of books and other items damaged by water, and the concentration and recovery of reaction products.

Specialized equipment is required to create the conditions conducive to the freeze drying process. This equipment is currently available and can accommodate freeze drying of materials from laboratory scale projects to industrial production.

Freeze drying involves the removal of water or other solvent from a frozen product by a process called sublimation. Sublimation occurs when a frozen liquid goes directly to the gaseous state without passing through the liquid phase. In contrast, drying at ambient temperatures from the liquid phase usually results in changes in the product, and may be suitable only for some materials. However, in freeze drying, the material does not go through the liquid phase, and it allows the preparation of a stable product that is easy to use and aesthetic in appearance.

The advantages of freeze drying are obvious. Properly freeze dried products do not need refrigeration, and can be stored at ambient temperatures. Because the cost of the specialized equipment required for freeze drying can be substantial, the process may appear to be an expensive undertaking. However, savings realized by stabilizing an otherwise unstable product at ambient temperatures, thus eliminating the need for refrigeration, more than compensate for the investment in freeze drying equipment.

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Principles of Freeze Drying

The freeze drying process consists of three stages: prefreezing, primary drying, and secondary drying.

Prefreezing: Since freeze drying is a change in state from the solid phase to the gaseous phase, material to be freeze dried must first be adequately prefrozen. The method of prefreezing and the final temperature of the frozen product can affect the ability to successfully freeze dry the material.

Rapid cooling results in small ice crystals, useful in preserving structures to be examined microscopically, but resulting in a product that is more difficult to freeze dry. Slower cooling results in larger ice crystals and less restrictive channels in the matrix during the drying process.

Products freeze in two ways, depending on the makeup of the product. The majority of products that are subjected to freeze drying consist primarily of water, the solvent, and the materials dissolved or suspended in the water, the solute. Most samples that are to be freeze dried are eutectics which are a mixture of substances that freeze at lower temperatures than the surrounding water. When the aqueous suspension is cooled, changes occur in the solute concentrations of the product matrix. And as cooling proceeds, the water is separated from the solutes as it changes to ice, creating more concentrated areas of solute. These pockets of concentrated materials have a lower freezing temperature than the water. Although a product may appear to be frozen because of all the ice present, in actuality it is not completely frozen until all of the solute in the suspension is frozen. The mixture of various concentration of solutes with the solvent constitutes the eutectic of the suspension. Only when all of the eutectic mixture is frozen is the suspension properly frozen. This is called the eutectic temperature.

It is very important in freeze drying to prefreeze the product to below the eutectic temperature before beginning the freeze drying process. Small pockets of unfrozen material remaining in the product expand and compromise the structural stability of the freeze dried product.

The second type of frozen product is a suspension that undergoes glass formation during the freezing process. Instead of forming eutectics, the entire suspension becomes increasingly viscous as the temperature is lowered. Finally the product freezes at the glass transition point forming a vitreous solid. This type of product is extremely difficult to freeze dry.

Primary drying: Several factors can affect the ability to freeze dry a frozen suspension. While these factors can be discussed independently, it must be remembered that they interact in a dynamic system, and it is this delicate balance between these factors that results in a properly freeze dried product.

After prefreezing the product, conditions must be established in which ice can be removed from the frozen product via sublimation, resulting in a dry, structurally intact product. This requires very careful control of the two parameters, temperature and pressure, involved in the freeze drying system. The rate of sublimation of ice from a frozen product depends upon the difference in

vapor pressure of the product compared to the vapor pressure of the ice collector. Molecules migrate from the higher pressure sample to a lower pressure area. Since vapor pressure is related to temperature, it is necessary that the product temperature is warmer than the cold trap (ice collector) temperature. It is extremely important that the temperature at which a product is freeze dried is balanced between the temperature that maintains the frozen integrity of the product and the temperature that maximizes the vapor pressure of the product. This balance is key to optimum drying. The typical phase diagram shown in Figure 1 illustrates this point. Most products are frozen well below their eutectic or glass transition point (Point A), and then the temperature is raised to just below this critical temperature (Point B) and they are subjected to a reduced pressure. At this point the freeze drying process is started.

Figure 1

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<![endif]>PRESSURE

F

U

S

I

Liquid

L

O

N

Phase

E

I

N

IO

A

T

IZ

A

O

R

P

Solid

A

V

Phase

B TRIPLE

E

POINT

IN

L

N

IO

T

SUB

A

D

C

M

LI

N

LIN

E

CRITICAL POINT

Vapor

Phase

TEMPERATURE

A typical phase diagram.

Some products such as aqueous sucrose solutions can undergo structural changes during the drying process resulting in a phenomenon known as collapse. Although the product is frozen below its eutectic temperature, warming during the freeze drying process can affect the structure of the frozen matrix at the boundary of the drying front. This results in a collapse of the structural matrix. To prevent collapse of products containing

A vacuum pump is essential to evacuate the environment around the product to be freeze dried.

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