Methods of Structure Analysis

A number of methods have been described to supply information during freezing. Electrical resistance during cooling and warming (ER) is measured in a test vial at different freezing and warming rates. For a more accurate interpretation of the function log (ER) = f(T) the first derivative of the plot is calculated as shown in Figure 2. The advantages of the method are: sample size is of the order of a product in

-80 -60 -40 -20 0 20 (A) Temperature (°C)

Figure 2 (A) Electrical resistance (ER) of a pharmaceutical product as a function of temperature during cooling at 1 °C min-and warming at 3°C min-1. Heat transfer medium and product are approximately uniformly heated. (B) Measurement of the electrical resistance as in A, but with the wall of the vial insulated by a plastic tape up to the filling height of the product. Heat is therefore mostly removed through the bottom of the vial. (A and B from Willemer, H., Koln, unpublished measurements.)

a vial; it simulates heat transfer from the shelf to the vial/product; and the equipment is relatively inexpensive and easy to operate. The disadvantages are that interpretation needs some experience, the measured data reflect the mobility of ions and the amount of energy used or freed during an event cannot be calculated. In Figure 2A, the heat transfer medium and product are at a similar temperature; in Figure 2B, the wall of the test vial is isolated from the heat transfer medium to simulate the freezing of a product in a vial on the shelf. In Figure 2A, the effect of sub-cooling during freezing can be seen at about — 10°C, but the derivative shows it more clearly between

— 3°C and — 10°C. During warming at event 1 ( — 12°C) the structure softens, allowing unfrozen water to crystallize, represented by the increase in resistance. In Figure 2B the crystallization energy cannot be quickly removed: freezing occurs in two steps. During warming, events 1 and 2 are not found, all freezable water is crystallized during cooling.

Differential scanning calorimetry (DSC) compares heat flows, one to and from the sample and the other to and from a substance with no transitions in the measuring range. Roos and Karel showed by DSC (Figure 3) the influence of unfrozen water on Tg of fructose (1) and glucose solutions (2). After rapid freezing (30°C min-1) to — 100°C Tg of fructose and glucose is at — 88°C and — 84°C respectively; at

— 48°C and —44°C respectively the unfrozen water crystallizes, followed by the melting of ice. If the products are thermally treated or annealed (after freezing the product is warmed to —48°Cfor 15 min and then cooled again to — 100°C), Tg, called Tg if all freezable water is frozen, is raised to —58°C and

— 57°C and no crystallization event is measurable. Time and temperature of annealing must be carefully determined to achieve a certain mobility of the molecules without collapse of the structure (see Figure 9B). The advantages of DSC are the quantitative measurement of the changes in the heat capacity of the sample and the energy freed or used in an event. The disadvantage is the small sample (milligrams), which can behave differently from a product in vials (grams) and the cost of the equipment.

In a cryomicroscope the sample can be optically observed during cooling and warming at different rates. Some models also permit freeze-drying of the sample. Willemer has shown (Figure 4) the structure of a Factor VIII solution during warming after quick freezing. This product must be freeze-dried at a temperature of the sublimation front of the ice (Tice) below — 44°C and if annealing is necessary it may be possible at —43°Cto —42°C for several minutes but a longer time at —45°C is recommended. The advantage is the visual confirmation of data gained by

Figure 3 Results of annealing (thermal treatment) on the formation of ice in a 60% fructose solution (1) and in a 60% glucose solution (2). Curve A: after cooling at 30°C min~1 down to -100°C, the DSC plots have been recorded during warming at 5°Cmin~1. Tg approximately -85°C and -88°C, respectively, for fructose and glucose. At approximately -48°C and -44°C respectively, ice crystallization starts clearly, followed by the beginning of the melting of ice. (During freezing only a part of the water has been crystallized.) Curve B: after cooling down to - 100°C, the product has been warmed at 10°C min~1 to -48°C, kept for 15 min at this temperature (thermal treatment), cooled down again at 10°Cmin~1 to -100°C, and the DSC plot (B) measured during rewarming. During thermal treatment all freezable water is crystallized, and Tg. is increased to -58°C and -57°C, respectively. During warming, no crystallization can be detected. (Reproduced with permission from Roos and Karel, 1991.)

other methods, e.g. ER or DSC, and the possibility to analyse the image quantitatively by computer. The disadvantages are high cost and the relatively small region of the sample that can be observed.

Nuclear magnetic resonance (NMR) provides information, among other things about free or bound water (e.g. to protein molecules), the influence of unfrozen water on the collapse temperature and the crystallization of amorphous dry products. Hanafusa has shown by NMR (Figure 5) how the amount of unfrozen (bound) water in a 0.57% ovalbumin solution is reduced by the addition of a 0.01 M solution of sucrose or glycerol. Similar information can be gained for a coffee extract with 25% solids: during freezing and rewarming at —70°C 0.01 g water per gram solid are unfrozen, at — 40°C 0.1 g per gram and at — 20°C approximately 30%; thereafter the amount increases rapidly. This extract has to be freeze-dried at a Tico < - 20°C, otherwise the structure would collapse. The unique advantage of NMR is the ability to discriminate between free, crystallized and bound water.

More details concerning these methods and additional procedures have been reported by Oetjen.

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Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

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