Dielectric characterization of neutralized and nonneutralized chitosan upon drying

Isothermal dielectric loss spectra of neutralized and nonneutralized chitosan were acquired in successive runs from -130 degrees C up to increasing final temperatures, in a frequency range between 20 Hz and 1 MHz. Essentially, three relaxation processes were detected in the temperature range covered: (i) a beta-wet process, detected when the sample has a higher water content that vanishes after heating to 150 degrees C; (ii) a beta process, which is located at temperatures below 0 degrees C, becoming better defined and maintaining its location after annealing at 150 degrees C independently of the protonation state of the amino side group; and (iii) a sigma process that deviates to higher temperatures with drying, being more mobile in the nonneutralized form. Moreover, in dried neutralized chitosan, a fourth process was detected in the low frequency side of the secondary beta process that diminishes after annealing. Whether this process is a distinct relaxation of the dried polymer or a deviated beta-wet process due to the loss of water residues achieved by annealing is not straightforward. Only beta and sigma processes persist after annealing at 150 degrees C. The changes in molecular mobility upon drying of these two relaxation processes were evaluated.     

The effect of different methods of cross-linking of collagen on its dielectric properties

This paper reports on the effect of different methods of collagen cross-linking on its dielectric properties. In order to obtain collagen-hyaluronic acid (HA) scaffolds, collagen was first dehydrated by a combination of thermal and vacuum drying (DHT) and then treated with the chemical reagent carbodiimide (EDC/NHS) for final cross-linking. The measurements of the relative permittivity varepsilon' and the dielectric loss varepsilon' for all materials were carried over the frequency range of 10 Hz-100 kHz and at temperatures from 22 to 260 degrees C. The results for these samples reveal distinct relaxation processes at low temperatures, below 140 degrees C and at higher temperatures as broad peak around 230 degrees C. The first and second relaxation are associated with changes in the secondary structure of collagen accompanied by the release of water and with the denaturation of dry collagen, respectively. The influence of cross-linking on the permittivity of collagen is significant over the entire temperature range.     

Glass transition and enthalpy relaxation of amorphous lactose glass

The glass transition temperature, T(g), and enthalpy relaxation of amorphous lactose glass were investigated by differential scanning calorimetry (DSC) for isothermal aging periods at various temperatures (25, 60, 75, and 90 degrees C) below T(g). Both T(g) and enthalpy relaxation were found to increase with increasing aging time and temperature. The enthalpy relaxation increased approximately exponentially with aging time at a temperature (90 degrees C) close to T(g) (102 degrees C). There was no significant change observed in the enthalpy relaxation around room temperature (25 degrees C) over an aging period of 1month. The Kohlrausch-Williams-Watts (KWW) model was able to fit the experimental enthalpy relaxation data well. The relaxation distribution parameter (beta) was determined to be in the range 0.81-0.89. The enthalpy relaxation time constant (tau) increased with decreasing aging temperature. The observed enthalpy relaxation data showed that molecular mobility in amorphous lactose glass was higher at temperatures closer to T(g). Lactose glass was stable for a long time at 25 degrees C. These findings should be helpful for improving the processing and storage stability of amorphous lactose and lactose containing food and pharmaceutical products.     

Study of polysaccharides by the fluorescence method. III. Chain length dependence of the micro-Brownian motion of amylose in an aqueous solution

The fluorescence polarization method was applied to the investigation of the micro-Brownian motion of amylose chains having a wide range of degree of polymerization (DP). We prepared two types of fluorescent conjugates of amylose: amylose conjugated with fluorescein randomly throughout the chain (F-amylose) and amylose conjugated locally on a terminal segment (t-F-amylose). The degree of fluorescence polarization of these conjugates was measured by changing the solvent viscosity at a constant temperature (25°C). The data obtained were analyzed by a Perrin-type equation to calculate the mean rotational relaxation time, 〈ρ〉. By examination of the plots of 〈ρ〉 vs DP, and by comparison of 〈ρ〉 with the theoretical rotational relaxation time of the whole molecule at a given DP, it was found that 〈ρ〉 mainly reflects the segmental motion of the amylose chain in the high-DP range. Thus, the fact that 〈ρ〉 for t-F-amylose is much smaller than that for F-amylose at a sufficiently high DP shows that a terminal segment undergoes a more rapid micro-Brownian motion than interior segments. In the low-DP range, we felt that the rotational diffusion of the whole molecule contributes significantly to the relaxation process. We also examined, for comparison, the segmental motion of dextran and pullulan in a similar manner and found that these segmental motions are more rapid than those of amylose. Based on the results obtained, the segmental mobility and conformation of the amylose molecule are discussed in relation to its chain length.     

Dielectric relaxation spectroscopy on dimyristoylphosphatidylcholine-packaged gramicidin A

The complex permittivities of aqueous suspensions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and of DMPC packaged gramicidin A' (DMPC-GA) have been determined over the frequency range of 1 MHz to 1 GHz and the temperature range of 0-60 degrees C. A dielectric relaxation/loss has been observed at about 66 MHz for the DMPC suspension (30 degrees C) and at about 57 MHz for the DMPC-GA suspension (30 degrees C). This dielectric relaxation/loss has been attributed to the rotational mobility of the zwitterionic group of DMPC. The temperature dependence (from 60 degrees C to 0 degrees C) of this dispersion/absorption process of the DMPC suspension indicates a sharp reduction of the dielectric relaxation at about 20 degrees C. This dielectric change is related to the conversions of shape and structure of bilayer aggregates. This sharp reduction of the dielectric relaxation disappears or broadens when GA is incorporated into the DMPC aqueous suspension. The interpretation of these results is that the GA addition into the DMPC aqueous suspension induces a small decrease of the rotational mobility of the zwitterionic group above the lipid phase transition, and a small increase of the rotational mobility of the zwitterionic group below the lipid phase transition.     

Freezing of phosphocholine headgroup in fully hydrated sphingomyelin bilayers and its effect on the dynamics of nonfreezable water at subzero temperatures.

Differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) spectroscopy are applied to characterize the nonfreezable water molecules in fully hydrated D2O/sphingomyelin at temperatures below 0 degrees C. Upon cooling, DSC thermogram displays two thermal transitions peaked at -11 and -34 degrees C. The high-temperature exothermic transition corresponds to the freezing of the bulk D2O, and the low-temperature transition, which has not previously been reported, can be ascribed to the freezing of the phosphocholine headgroup in the lipid bilayer. The dynamics of nonfreezable water are also studied by 2H NMR T1 (spin-lattice relaxation time) and T2e (spin-spin relaxation time obtained by two pulse echo) measurements at 30.7 MHz and at temperatures down to -110 degrees C. The temperature dependence of the T1 relaxation time is characterized by a distinct minimum value of 2.1 +/- 0.1 ms at -30 degrees C. T2e is discontinuous at temperature around -70 degrees C, indicating another freezing-like event for the bound water at this temperature. Analysis of the relaxation data suggest that nonfreezable water undergoes both fast and slow motions at characteristic NMR time scales. The slow motions are affected when the lipid headgroup freezes.     

Thermal properties of polysaccharides at low moisture: 1—An endothermic melting process and water-carbohydrate interactions

The thermal properties of a broad range of polysaccharides containing 5–25% w/w water have been studied by differential scanning calorimetry and dynamic mechanical thermal analysis (DMTA). Following room temperature conditioning, an endothermic event accompanied by material softening is observed at 45–80°C for all samples except those above their glass transition temperature. The temperature of the event is determined by thermal history and is apparently independent of polymer type or moisture content. The associated enthalpy increases with water content. Variable frequency DMTA analysis suggests a structural melting event rather than a relaxation process. The endothermic event is recovered over the days timescale after heating, and can be annealed to higher temperatures with increasing holding temperature.

Results are interpreted in terms of a dynamic hydration model in which specific energetic water-carbohydrate interactions occur but with a lifetime defined by their local effective microviscosity. The observation of the endotherm below glass transition temperatures suggests that in aqueous polysaccharide glasses, enthalpic structures involving the solvent can be made and broken.     

Molecular motions in chitosan studied by dielectric relaxation spectroscopy

Neutralized and nonneutralized chitosan films subject to different thermal treatments were studied by dielectric relaxation spectroscopy from -130 to +150 degrees C in the frequency range between 20 Hz and 1 MHz. Two main relaxation processes, both arrhenian type, were detected: process I at temperatures below 0 degrees C with a mean activation energy of 49 +/- 1 kJ mol(-1), which has the characteristics of a secondary relaxation process related with local chain dynamics, and process II observable at higher temperatures with an activation energy of 94 +/- 2 kJ mol(-1), correlated with dc conductivity, which is found in dried polysaccharides systems. Process I is always observed in neutralized chitosan, but it is strongly depleted in the wet nonneutralized form. Although the location of process I is independent of NH2/NH3+ side group, process II deviates to higher temperatures with dryness in both chitosan forms, being located at lower temperatures in nonneutralized chitosan.     

A dielectric analysis of liquid and glassy solid glucose/water solutions

Dielectric relaxation data covering a temperature range from above room temperature to below the glass transition for 40% (w/w) and 75% (w/w) glucose/water solutions in the frequency range between 5 and 13 MHz are presented. These data are used to obtain correlation times for the dielectric relaxation in the viscous liquid and the glass and are compared with correlation times determined from deuterium nuclear spin relaxation times [J. Chem. Phys., 110 (1999) 3472-3483]. The two sets of results have the same temperature dependence, but differ in magnitude by a factor of 3, implying that the relaxation is a small-step rotational diffusion. Both the structural relaxation (alpha process) and the slow beta process are present. In the 40% glucose/water sample, there is a dielectric relaxation attributable to the ice that forms at low temperature. It is shown that the reciprocal of the viscosity, the correlation time derived from the dielectric relaxation, and the dc conductivity have a similar dependence on temperature.     

Water in keratin. Piezoelectric, dielectric, and elastic experiments.

To investigate actions of water in keratin, the piezoelectric, dielectric, and elastic constants are measured at 10 Hz, at temperatures between -160 and 150 degrees C, and at various hydration levels. From changes in the piezoelectric, dielectric, and dynamic mechanical parameters with moisture content (m.c.), we have identified three regimes (I, II, and III) in the hydration of water for keratin. At high hydration (21% m.c.) around 0 degree C, the piezoelectric constants for keratin steeply decrease with increasing temperature. This may be attributed to interfacial polarization which is strongly related to self-associated water molecules (particularly regime III water) just around crystalline helical regions which can exhibit the stress-induced, i.e., piezoelectric, polarization and may be attributed to electrode polarization induced by the increase of mobile ions in the amorphous matrix region, some of which would be released from their trapped states just around the piezoelectric phase by the regime III water. With increasing hydration, the elastic constants for keratin are found to increase below -70 degrees C and decrease above -70 degrees C. This suggests a viscoelastic transition of the keratin structure due to bound water (regime II water). The piezoelectric, dielectric, and elastic loss peaks are found at around -120 degrees C for hydrated keratin, believed to be due to tightly bound water (regime I water), which acts only to stiffen the keratin structure. The adsorption regions of water in keratin are discussed by a piezoelectric two-phase model, which consists of piezoelectric and nonpiezoelectric phases. It is proposed that water molecule would at least adsorb in the nonpiezoelectric phase.     

Far-ultraviolet optical activity of saccharide derivatives. II. Dextran,amylose, and mycodextran acetes; dextran and amylose xanthates

The ultraviolet ORD and CD spectra of amylose, dextran, and mycodextran acetates and some of thier oligomers were recorded in trifluoroethanol solution in the 300–185nm wavelength range. Similarly, the spectra of amylose and dextran xanthates in water solution were obtained in the 400–200 nm range. In the amylose acetate series, the monomer and dimer both show a normal acetyl n → π* transition in CD, while the trimer and the polymer both exhibit an additional, shorter wavelength peak. The latter is presumed to arise from a helical conformation of the amylose chain. This interpretation is substantiated by a helix–coil type transition of the CD spectra of amylose triacetate at elevated temperatures and a reversion of the anomalous CD to the normal CD upon partial deacetylation. By contrast, neither dextran acetates nor mycodextran acetate exhibit any conformational effects. The CD of dextran acetates is quite sensitive to β-1,6 and branch linkages. The ORD and CD of amylose xanthate are complex, suggesting the presence of organized structure in solution. The dextran xanthate shows only a simple ORD spectrum and no observable CD.     

Temperature dependency of molecular mobility in preserved seeds

Although cryogenic storage is presumed to provide nearly infinite longevity to cells, the actual timescale for changes in viability has not been addressed theoretically or empirically. Molecular mobility within preserved biological materials provides a first approximation of the rate of deteriorative reactions that ultimately affect shelf-life. Here, temperature effects on molecular mobility in partially dried seeds are calculated from heat capacities, measured using differential scanning calorimetry, and models for relaxation of glasses based on configurational entropy. Based on these analyses, glassy behavior in seeds containing 0.07 g H(2)O/g dm followed strict Vogel-Tamman-Fulcher (VTF) behavior at temperatures above and just below the glass transition temperature (Tg) at 28 degrees C. Temperature dependency of relaxation times followed Arrhenius kinetics as temperatures decreased well below Tg. The transition from VTF to Arrhenius kinetics occurred between approximately 5 and -10 degrees C. Overall, relaxation times calculated for seeds containing 0.07 g H(2)O/g dm decreased by approximately eight orders of magnitude when seeds were cooled from 60 to -60 degrees C, comparable to the magnitude of change in aging kinetics reported for seeds and pollen stored at a similar temperature range. The Kauzmann temperature (T(K)), often considered the point at which molecular mobility of glasses is practically nil, was calculated as -42 degrees C. Calculated relaxation times, temperature coefficients lower than expected from VTF kinetics, and T(K) that is 70 degrees C below Tg suggest there is molecular mobility, albeit limited, at cryogenic temperatures.     

Zwitterionic dipoles as a dielectric probe for investigating head group mobility in phospholipid membranes

For phospholipid membranes with zwitterionic head groups, the dipole can be considered as a specific label for tracing the changes in the dynamic behaviour of this region of the bilayer in its various phases. Measurements of the dielectric properties of fully hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers in the frequency range 1--50 MHz show a dispersion which is attributed to the motion of the phosphocholine dipoles in the plane of the bilayers. When the temperature is varied, both the permittivity and loss factor increase sharply at the pretransition (35 degrees C) and the main transition (42 degress C). The relaxation time and amplitude were also determined for this dispersion and these further reflect the structural changes occurring with temperature. The relaxation times varied between 4 ns at 30 degrees C and 2.3 ns at 50 degrees C. Due to steric hindrances a restriction in the angle of head group rotation occurs at lower temperatures but is greatly reduced above the main transition.     

Enzyme activity down to -100 degrees C

The activities of two enzymes, beef liver catalase (EC 1.11.1.6) and calf intestine alkaline phosphatase (EC 3.1.3.1), have been measured down to -97 degrees C and -100 degrees C, respectively. Enzyme activity has not previously been measured at such low temperatures. For catalase, the cryosolvents used were methanol:ethylene glycol:water (70:10:20) and DMSO:ethylene glycol:water (60:20:20). For alkaline phosphatase, methanol:ethylene glycol:water (70:10:20) was used. All of the Arrhenius plots were linear over the whole of the temperature range examined. Since the lowest temperatures at which activity was measured are well below the dynamic transition observed for proteins, the results indicate that the motions which cease below the dynamic transition are not essential for enzyme activity. In all cases the use of cryosolvent led to substantial increases in Arrhenius activation energies, and this imposed practical limitations on the measurement of enzyme activity below -100 degrees C. At even lower temperatures, enzyme activity may be limited by the effect of solvent fluidity on substrate/product diffusion, but overall there is no evidence that any intrinsic enzyme property imposes a lower temperature limit for enzyme activity.     

Dielectric spectroscopy of L-alpha-lysolecithin-packaged gramicidin A

The complex permittivities of L-alpha-lysolecithin in the absence and presence of the gramicidin A ion channel were measured over the temperature range 0-60 degrees C and over the frequency range 1-1000 MHz. One dielectric relaxation/loss has been observed. It is located at 103.3 MHz (1.54 ns) for a micellar 0.4 M L-alpha-lysolecithin solution at 20 degrees C, whereas it is shifted to 71.7 MHz (2.22 ns) for a lamellar L-alpha-lysolecithin-gramicidin A aqueous solution (0.4 M L-alpha-lysolecithin, 0.0308 M gramicidin A) at 20 degrees C. The dielectric relaxation decreases and the relaxation time increases when gramicidin A is incorporated into L-alpha-lysolecithin. These dielectric changes are related, in part, to the micellar-to-lamellar lipid phase transition induced by the incorporation of gramicidin A into lysolecithin. We suggest that the diffuse rotational motion of the polar head group of L-alpha-lysolecithin contributes to the dielectric relaxation/loss at around 100 MHz.     

Domain specificity in metal binding to metallothionein. A circular dichroism and magnetic circular dichroism study of cadmium and zinc binding at temperature extremes

Rabbit liver Zn metallothionein-(MT) will bind cadmium readily between -26 degrees C and 70 degrees C. The binding reaction was monitored by recording the circular dichroism and magnetic circular dichroism spectra, in the region of the RS(-)----Cd2+ charge transfer transition at 250 nm, at intervals as aliquots of cadmium were added. For all temperatures, these data can be analyzed in terms of a distributed mechanism for cadmium binding when Zn-MT is used, and a domain-specific mechanism when apo-MT is used. The CD spectrum measured at -26 degrees C for Cd,Zn-MT, which was made by adding excess cadmium directly to Zn7-MT at -26 degrees C, is not the same as the CD spectrum of Cd-MT prepared at room temperature from the same Zn7-MT. Measurements of the stoichiometry of the cadmium and zinc bound to MT in the presence of excess cadmium at different temperatures indicates that below 5 degrees C at least one zinc atom remains bound to the protein. The mixed metal metallothionein, Cd/Zn-MT, that always forms below 5 degrees C, is characterized by a single maximum near 250 nm in the CD spectrum, rather than the derivative-shaped CD envelope that is diagnostic of the (Cd4-S11)alpha cluster, which indicates that the zinc occupies a site in the alpha domain. Rearrangement of the bound metals to the domain-specific distribution takes place if Cd,Zn-MT, prepared at subzero temperatures, is warmed above 30 degrees C.     

Volume and density changes of biological fluids with temperature

High-precision (10(-5) g/ml) mass density measurements on human blood, plasma, plasma ultrafiltrate (using PM-10 membranes), and erythrocyte concentrate samples were performed with the mechanical oscillator technique. Measurement temperatures varied between 4 and 48 degrees C and were accurate to +/- 1 X 10(-2) K. The coefficient of thermal expansion (beta), defined as relative volume change with temperature, was calculated. It was shown that beta increases with temperature in these fluid samples over the entire temperature range investigated; the magnitude of this increase declines with increasing temperature; beta increases with density at temperatures below 40 degrees C but is independent of density above 40 degrees C; and the beta of the intracellular fluid has about twice the value of the beta for extracellular fluid at low (4-10 degrees C) temperatures but is equal for both fluids at greater than or equal to 40 degrees C. The mechanical oscillator technique provides data with an accuracy sufficient to perform precise (10(-5) K) calculations of beta of small volumes of biological fluids.     

Study of anisotropy in nuclear magnetic resonance relaxation times of water protons in skeletal muscle.

The anisotropy of the spin-lattice relaxation time (T1) and the spin-spin relaxation times (T2) of water protons in skeletal muscle tissue have been studied by the spin-echo technique. Both T1 and T2 have been measured for the water protons of the tibialis anterior muscle of mature male rats for theta = 0, 55, and 90 degrees, where theta is the orientation of the muscle fiber with respect to the static field. The anisotropy in T1 and T2 has been measured at temperatures of 28, -5 and -10 degrees C. No significant anisotropy was observed in the T1 of the tissue water, while an average anisotropy of approximately 5% was observed in T2 at room temperature. The average anisotropy of T2 at -5 and -10 degrees C was found to be approximately 2 and 1.3%, respectively.     

Oxygen-17 NMR relaxation studies on gelatinization temperature and water mobility in maize starches

The effects of starch/water ratio, amylose content, degree of phosphorylation, and added KI on water mobility in maize starch-water dispersions were studied by oxygen-17 spin-spin relaxation time measurements over a range of temperatures. The results demonstrate that: (i) the changes in spin-spin relaxation time (ΔT₂) reflect the degree of starch-water interaction at different stages of the heating process; (ii) the amount of added water affects the initial T₂ and ΔT₂ during gelatinization; (iii) higher amylose contents result in lower water mobility in starch-water systems; (iv) higher degrees of phosphorylation lead to a decrease in water mobility, accompanied by a decrease in gelatinization temperature; and (v) added KI effectively decreases water mobility and gelatinization temperature in the starch-water systems studied.     

Effect of water on the molecular mobility of elastin

Purified and hydrated elastin is studied by both thermal and dielectric techniques to have insight into the chain dynamics of this protein. By differential scanning calorimetry, the glassy behavior of elastin is highlighted; the glass transition temperature (T(g)) of elastin is found to be widely dependent on hydration, falling from 200 degrees C in the dehydrated state to 30 degrees C for 30% hydration. A limit of T(g) at around 0 degrees C is found when crystallizable water is present in the system, that is, when the formation of ice prevents motions of some 10 nm along the polypeptidic chains. The technique of thermally stimulated currents, carried out in the -180 to 0 degrees C temperature range, is useful to detect localized motions. In this case, too, the localized motions vary considerably according to hydration: a first relaxation mode is observed at -145 degrees C and it is associated with the reorientation of crystallizable water in ice I; a second relaxation mode, more complex and cooperative, occurs at around -80 degrees C and could be attributed to the complex constituted by the dipolar groups of the polypeptidic chain and noncrystallizable water, behaving as a glassy system.