Water nonfreezing

The amount of water that does not freeze near 0°C in a protein—water system is a useful measure of the hydration water (Kuntz and Kauz-mann, 1974). Such estimates are self-consistent, in that different methods for determining nonfreezing water give closely similar values, and are reliable, in that they agree with other thermodynamic estimates of hydration. 

in a series of papers (see Kuntz and Kauzmann, 1974), developed the use of magnetic resonance as a probe of the freezing of solvent in protein solutions. The nonfreezing water in a sample measured at - 35°C appears as an NMR signal that is sharp compared to the broadband response for the ice phase. The NMR method gives results for proteins and other macromolecules in close agreement with estimates of 

Measurements of model polypeptides were consistent with the non-freezing water being primarily associated with ionic groups of the protein (Kuntz, 1971). A set of amino acid hydration values, constructed to calculate the amount of nonfreezing water according to the amino acid composition of a protein, gave estimates in close agreement with measurement (Kuntz, 1971). 

Hsi and Bryant (1975) measured NMR reltixation for frozen lysozyme solutions. They found that the nonfreezing water consisted of two components with different T, values The amount of the slower component was 0.06 g, and the amount of the faster component was 0.28 g of water per g of protein. The amount of the slower component, perhaps coincidentally, corresponds to the amount of tight-binding water seen in isotherm measurements. 

Poole et al. (1987) measured powder diffraction for lysozyme samples of varied hydration. Diffraction of ice crystallites was first detected at hydration levels greater than 0.3 h. The point of first appearance of frozen water is also defined by the shift and growth of the O—D stretch band in infrared spectra measured for protein samples variously hydrated with deuterated solvent (Finney et al., 1982). 

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Differential scanning calorimetry measurements have shown a marked cooling/heat-ing cycle hysteresis and that water entrapped in AOT-reversed micelles is only partially freezable. Moreover, the freezable fraction displays strong supercooling behavior as an effect of the very small size of the aqueous micellar core. The nonfreezable water fraction has been recognized as the water located at the water/surfactant interface engaged in solvation of the surfactant head groups [97,98]. 

Other supportive evidence for a specific water-solid interaction is available from thermal studies showing the amount of nonfreezable water [57-59], nuclear magnetic resonance [29,60-66], and diffusion studies [67,68]. The evidence is less clear, however, concerning whether there is distinct binding of water to... 

The nature of the water present within a PEM can also have an effect upon its performance during PEMFC operahon. At /I > 6, water exists in the three forms previously mentioned in Section 3.2.1 free water, loosely bound water, and nonfreezable water. This has been established by Eourier transform infrared (FTIR) studies and more recently by calorimetry and gravimetric analysis. Pulse NMR has also been used in conjunchon with differential scanning calorimetry (DSC) to analyze the contributions of these three different types of water in Nation and BPSH membranes. These values can be seen in Table 3.1. 

Contributions to Total A from Free, Loosely Bound, and Nonfreezable Water for Nafion and BPSH Using a Combination of Pulse NMR and DSC... 

Fig. 3. Phase diagram for the three kinds of water in PVP aqueous solutions (81). A, freezable water B, bound, nonfreezable water (six per repeat unit) C,...

It is well known that at low moisture uptakes, the water associated with the cellulose exhibits properties that differ from those of liquid water and it has been called by such terms as bound water, nonsolvent water, hydrate water, and nonfreezing water. From a review of the literature, which included determinations by such techniques as NMR and calorimetry, Zeronian [303] concluded that between 0.10 and 0.20 g/g of the water present in the fiber cell wall appeared to be bound. Such regains are obtained at RVPs between 0.85 and 0.98. 

Table II compares determinations of the nonfreezing water of lysozyme, measured by scanning calorimetry, NMR, infrared spectroscopy, and X-ray diffraction.

Comparison of Nonfreezing Water, Determined by Several Methods ... 

Measurement Nonfreezing water (grams of water per gram of protein)... 

The determination of nonfreezing water is perhaps the most simple and straightforward way to estimate hydration. Scanning calorimetric and NMR measurements are made with equipment that is commonly available, and these methods should continue to be widely used. 

Harvey and Hoekstra (1972) determined the dielectric constant and loss for lysozyme powders as a function of hydration level in the frequency range 10 —10 Hz. At water contents less than 0.3 h, they found a dispersion at 170 MHz, which increased somewhat with increasing hydration, and a new dispersion at about 10 Hz that develops at high hydration. These dispersions, detected by time-domain techniques, remain measurable down to the lowest temperature studied, — 60°C. Water mobility in the hydration shell below 0 C is in line with other observations of nonfreezing water. Above 0.3 h, in the stage of the hydration process at which condensation completes the surface monolayer, water motion increased strongly with increased hydration (Fig. 11). 

One manifestation of strong solnte-solvent interactions is the inability of affected waters to freeze when the temperature falls well below the freezing point. Nnclear magnetic resonance (NMR), infrared spectroscopy, and low-temperatnre calorimetry have been employed to characterize the number of nonfreezing waters in the hydration shell of DNA (6-9). Based on their infrared measnrements of DNA films, Falk et al. (6) have concluded that about 10 water molecules per nucleotide are incapable of freezing with an additional 3 waters that show... 

Figure 10 shows the variation in the free energy as a function of the nonfreezing water concentration in aqueous suspensions of the starting, reduced, and oxidized carbons. The curves for AG = f C ) exhibit a portion where the nonfreezing water concentration remained constant in a wide range of AG variations. The appearance of such a feature is attributed to the presence of micropores. In... 

FIG. 10 Dependence of the free energy variation on concentration of nonfreezing water for activated carbons produced from fruit. stones (I starting carbon 2 carbon reduced with hydrogen carbon oxidized with hydrogen peroxide at 523 K 4 carbon oxidized with hydrogen peroxide at 623 K). (From Ref. 113.)... 

Figure 18 shows the variation in the free energy of adsorbed water as a function of the concentration of nonfreezing water for all the adsorbents studied. As C is proportional to the average thickness of layers of nonfreezing water, the above-mentioned dependence reflects the shape of the radial function de.scribing... 

FIG. 18 Dependence of the variation of free energy on the thickness of a nonfreezing water layer for carbosil (>1), carbosil modified with titanium dioxide (2). and carbosil modified with zinc silicate (3). For comparison, the corresponding curve for the starting silica gel (4) is also presented. (From Ref. 155.)... 

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