Molecular mobility studies on the amorphous state of disaccharides. I—thermally stimulated currents and differential scanning calorimetry

The relaxational processes in amorphous solid gentiobiose and cellobiose are studied by thermally stimulated depolarization currents (TSDC) in the temperature region from 108 K up to 423 K. The slow molecular mobility was characterized in the crystal and in the glassy state. The features of different motional components of the secondary relaxation have been monitored as a function of time as the glass structurally relaxes on aging. It is concluded that some modes of motion of this mobility are aging independent, while others are affected by aging. The value of the steepness index or fragility (T_g-normalized temperature dependence of the relaxation time) was obtained by differential scanning calorimetry (DSC) from the analysis of the scanning rate dependency of T_g.     

Slow molecular mobility in the crystalline and amorphous solid states of glucose as studied by Thermally Stimulated Depolarization Currents (TSDC)

Thermally Stimulated Depolarization Currents (TSDC) measurements on α-d-glucose have been carried out in the temperature region from −165 °C (108 K) to 120 °C (393 K). The slow molecular mobility was characterized in the crystalline and in the glassy states, as well as in the glass transition region. The influence of aging on the measured TSDC peaks of the secondary relaxation has been discussed and it was concluded that there are motional modes that are aging independent while others are affected by aging. Important discrepancies were reported in the value of the steepness index or fragility (T_g—normalized temperature dependence of the relaxation time) obtained by different, and well-established, experimental techniques. A careful discussion of the possible origins of these discrepancies is presented.     

Dynamics of uncrystallized water and protein in hydrated elastin studied by thermal and dielectric techniques

Dynamics of uncrystallized water and protein was studied in hydrated pellets of the fibrous protein elastin in a wide hydration range (0 to 23 wt.%), by differential scanning calorimetry (DSC), thermally stimulated depolarization current technique (TSDC) and dielectric relaxation spectroscopy (DRS). Additionally, water equilibrium sorption–desorption measurements (ESI) were performed at room temperature. The glass transition of the system was studied by DSC and its complex dependence on hydration water was verified. A critical water fraction of about 18 wt.% was found, associated with a reorganization of water in the material. Three dielectric relaxations, associated to dynamics related to distinct uncrystallized water populations, were recorded by TSDC and DRS. The low temperature secondary relaxation of hydrophilic polar groups on the protein surface triggered by hydration water for almost dry samples contains contributions from water molecules themselves at higher water fractions (ν relaxation). This particular relaxation is attributed to water molecules in the primary and secondary hydration shells of the protein fibers. At higher temperatures and for water fraction values equal to or higher than 10 wt.%, a local relaxation of water molecules condensed within small openings in the interior of the protein fibers was recorded. The evolution of this relaxation (w relaxation) with hydration level results in enhanced cooperativity at high water fraction values, implying the existence of “internal” water confined within the protein structure. At higher temperatures a relaxation associated with water dynamics within clusters between fibers (p relaxation) was also recorded, in the same hydration range.     

Crystalline/amorphous phase structure and molecular mobility of biodegradable poly(butylene adipate-co-butylene terephthalate) and related polyesters

Differential scanning calorimetry (DSC), atomic force microscopy (AFM), wide-angle X-ray scattering (WAXD), and solid-state (13)C NMR have been used to investigate the crystalline/amorphous structure and molecular mobility of biodegradable poly(butylene adipate-co-44 mol % butylene terephthalate) [P(BA-co-44 mol % BT)] copolyester sample crystallized from the melt. The DSC endothermic peak, which is ascribed to the melting of the crystalline region, was broad relative to those reported for conventional partially crystalline polyesters. In AFM observation, spherulitic morphology was not observed while small particles with a size of about 100 nm were detected. The WAXD pattern of the sample was very broad. These results have indicated that a melt-crystallized P(BA-co-44 mol % BT) sample contains small crystals with a wide distribution in size. A solid-state (13)C NMR technique was also used to perform molecular-level and selective analyses for both butylene terephthalate and butylene adipate units. For the butylene terephthalate units, the existence of two components with different microstructure and molecular mobility was detected: one component was assigned to the alpha-form crystal of poly(butylene terephthalate) homopolymer (PBT) and the other was in amorphous regions. In contrast, all of butylene adipate units were located in amorphous regions. Solid-state NMR data have suggested that sizes of crystalline regions are less than 3 nm.     

Cloning and expression of a fungal L-arabinitol 4-dehydrogenase gene

L-Arabinitol 4-dehydrogenase (EC ) was purified from the filamentous fungus Trichoderma reesei (Hypocrea jecorina). It is an enzyme in the L-arabinose catabolic pathway of fungi catalyzing the reaction from L-arabinitol to L-xylulose. The amino acid sequence of peptide fragments was determined and used to identify the corresponding gene. We named the gene lad1. It is not constitutively expressed. In a Northern analysis we found it only after growth on L-arabinose. The gene was cloned and overexpressed in Saccharomyces cerevisiae, and the enzyme activity was confirmed in a cell extract. The enzyme consists of 377 amino acids and has a calculated molecular mass of 39,822 Da. It belongs to the family of zinc-binding dehydrogenases and has some amino acid sequence similarity to sorbitol dehydrogenases. It shows activity toward L-arabinitol, adonitol (ribitol), and xylitol with K(m) values of about 40 mM toward L-arabinitol and adonitol and about 180 mM toward xylitol. No activity was observed with D-sorbitol, D-arabinitol, and D-mannitol. NAD is the required cofactor with a K(m) of 180 microM. No activity was observed with NADP.     

Molecular mobility and phase structure of biodegradable poly(butylene succinate) and poly(butylene succinate-co-butylene adipate)

Molecular mobility and phase structure of biodegradable poly(butylene succinate) (PBS) and poly(butylene succinate-co-20 mol % butylene adipate) [P(BS-co-20 mol % BA)] have been investigated by high-resolution solid-state (13)C NMR. For both samples, two components with different (13)C spin-lattice relaxation time (T(1C)) values have been observed in the crystalline region. The crystalline component with shorter T(1C) value is assignable to the interface near amorphous phase. The crystalline component with longer T(1C) value is ascribed to the inside of the crystalline region. On the basis of T(1C), it has been concluded that the BA units are not included in the crystalline region of P(BS-co-20 mol % BA). Molecular mobility and higher-ordered structure of amorphous phase have been also compared between the melt and solid state. Variable-temperature high-resolution (13)C NMR measurements for the amorphous phase have revealed the remarkable difference in dynamics and structure between the melt and solid state.     

Interaction between dry starch and plasticisers glycerol or ethylene glycol, measured by differential scanning calorimetry and solid state NMR spectroscopy

The interaction of crystalline amylose and of crystalline and amorphous amylopectin with the plasticisers glycerol or ethylene glycol in the absence of water was studied, by using differential scanning calorimetry (DSC) and solid state nuclear magnetic resonance (NMR) spectroscopy. Upon heating starch freshly mixed with plasticisers, a strong exothermal interaction enthalpy of ΔH−35 J/g was detected by DSC. At room temperature glycerol interacts mainly with the amorphous starch regions, the interaction taking 8 days to reach equilibrium. For ethylene glycol the interaction is faster, taking four days to reach equilibrium, and the rate is not affected by crystallinity. Ethylene glycol interacts in a more ordered manner with amorphous than with crystalline material, resulting in a narrower ethylene glycol cross-polarisation magic angle spinning (CP/MAS) signal when equilibrium is reached at room temperature. Upon heating, more glycerol or ethylene glycol is immobilised, but in a less ordered manner than upon storage at room temperature. This results in a more intense, but broader plasticiser CP/MAS signal upon heating. Interaction in a more ordered manner probably implies interaction with more of the hydroxy groups of the plasticiser. The polysaccharide mobility is increased more when the plasticiser interacts in a more ordered manner, as observed by small starch signals in HP/DEC spectra.     

In vitro deposition of hydroxyapatite on cortical bone collagen stimulated by deformation-induced piezoelectricity

In the present work, we have studied the effect of the piezoelectricity of elastically deformed cortical bone collagen on surface using a biomimetic approach. The mineralization process induced as a consequence of the piezoelectricity effect was evaluated using scanning electron microscopy (SEM), thermally stimulated depolarization current (TSDC), and differential scanning calorimetry (DSC). SEM micrographs showed that mineralization occurred predominantly over the compressed side of bone collagen, due to the effect of piezoelectricity, when the sample was immersed in the simulated body fluid (SBF) in a cell-free system. The TSDC method was used to examine the complex collagen dielectric response. The dielectric spectra of deformed and undeformed collagen samples with different hydration levels were compared and correlated with the mineralization process followed by SEM. The dielectric measurements showed that the mineralization induced significant changes in the dielectric spectra of the deformed sample. DSC and TSDC results demonstrated a reduction of the collagen glass transition as the mineralization process advanced. The combined use of SEM, TSDC, and DSC showed that, even without osteoblasts present, the piezoelectric dipoles produced by deformed collagen can produce the precipitation of hydroxyapatite by electrochemical means, without a catalytic converter as occurs in classical biomimetic deposition.     

Solid State Characterization of Commercial Crystalline and Amorphous Atorvastatin Calcium Samples

Atorvastatin calcium (ATC), an anti-lipid BCS class II drug, is marketed in crystalline and amorphous solid forms. The objective of this study was to perform solid state characterization of commercial crystalline and amorphous ATC drug samples available in the Indian market. Six samples each of crystalline and amorphous ATC were characterized using X-ray powder diffractometry (XRPD), differential scanning calorimetry (DSC), thermogravimetric analysis, Karl Fisher titrimetry, microscopy (hot stage microscopy, scanning electron microscopy), contact angle, and intrinsic dissolution rate (IDR). All crystalline ATC samples were found to be stable form I, however one sample possessed polymorphic impurity, evidenced in XRPD and DSC analysis. Amongst the amorphous ATC samples, XRPD demonstrated five samples to be amorphous ‘form 27’, while, one matched amorphous ‘form 23’. Thermal behavior of amorphous ATC samples was compared to amorphous ATC generated by melt quenching in DSC. ATC was found to be an excellent glass former with T_g/T_m of 0.95. Residual crystallinity was detected in two of the amorphous samples by complementary use of conventional and modulated DSC techniques. The wettability and IDR of all amorphous samples was found to be higher than the crystalline samples. In conclusion, commercial ATC samples exhibited diverse solid state behavior that can impact the performance and stability of the dosage forms.     

Effect of montmorillonite on morphology,glass transition and crystallinity of the xylitol-plasticized bionanocomposites

High amylose based nanocomposites plasticized by xylitol were prepared via twin-screw extrusion. The synergistic interaction in the xylitol-plasticized nanocomposite was studied via various characterization methods and the unique behavior of the xylitol-plasticized nanocomposite had been discussed. As revealed in the XRD and TEM results, good intercalated/exfoliated morphology had been achieved in all the nanocomposites. Furthermore, the expansion of nanoclay basal spacing was related to the xylitol/nanoclay ratio. DSC analysis clearly proved the unique crystallization process of xylitol-plasticized samples. Moreover, in the crystallization domain results, two domains sized at approximately 93.7 Å and 346 Å were found. This observation points to a two-level complex effect from two aggregate domains; one, the re-aggregation of certain number of silicate layers into domains which trap some of the amylose polymer chains, and two, the bulk drying process which combines smaller amylose crystalline domains within a larger amorphous high amylose matrix.     

Citrate increases glass transition temperature of vitrified sucrose preparations

The aim of this study was to investigate the effect of sodium citrate on the properties of dried amorphous sucrose glasses. Addition of sodium citrate to a sucrose solution followed by freeze-drying or convective drying resulted in a glass transition temperature (Tg) that was higher than the well-studied sucrose Tg. This result was obtained either at reduced water content of the analysed sample or by removal of water during Modulated DSC analysis. After removal of the remaining water ( < 3.5% w/w), a Tg of approximately 105 degrees C was obtained at a mass ratio of sodium citrate to sucrose of 0.3. FTIR analysis showed a similar increase in Tg as was found with Modulated DSC analysis. The Tg values were derived from breaks in the vibrational frequency vs. temperature plots in the OH stretching and bending regions. Elevated average strength of hydrogen bonding in the sucrose/citrate glass was concluded from the downshift of the OH stretching band of 25 cm(-1) and from the reduced wavenumber temperature coefficient (WTC). The antisymmetric carboxylate stretch of citrate sensed the glass transition of the mixture, from which we conclude that citrate interacts with the sucrose OH via its carboxylate groups.     

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.     

In vitro hydrolysis of poly(l-lactide) crystalline residues as extended-chain crystallites: II. Effects of hydrolysis temperature

The effects of hydrolysis temperature on the hydrolysis behavior and mechanism of poly(l-lactide) crystalline residues or extended-chain crystallites were investigated in phosphate-buffered solution (50-97 degrees C), using gel permeation chromatography and differential scanning calorimetry (DSC). The hydrolysis of the crystalline residues proceeded from their surface composed of very short chains with a free end along the chain direction, irrespective of hydrolysis temperature, but the hydrolysis from their lateral surface could not be traced. The activation energy of hydrolysis for the crystalline residues (extended-chain crystallites) was evaluated to be 18.0 kcal mol(-1) (75.2 kJ mol(-1)). The monotonic melting temperature (T(m)) and crystallinity decreases occurred after their initial very small increases, excluding the monotonic crystallinity decrease at 97 degrees C with no initial increase. The T(m) decrease reflects the decreased thickness of the crystalline residues. The equilibrium T(m) of the crystalline residues (extended-chain crystallites) was estimated to be 464.5-464.9 K. The free energy values for the surface composed of very short chains with a free end, which are neighboring the air (or nitrogen during DSC scanning), were calculated to be 55.6-56.4 erg cm(-2) for heat of fusion per unit mass = 135 J g(-1). The obtained surface free energy values are significantly higher than that for the surface composed of folding chains, tie chains, and the chains with a free end, which are neighboring the same kind of amorphous chains (39.9 erg cm(-2)).     

The glass transition temperatures of amorphous trehalose-water mixtures and the mobility of water: an experimental and in silico study

Isothermal-isobaric molecular dynamics simulations are used to calculate the specific volume of models of trehalose and three amorphous trehalose-water mixtures (2.9%, 4.5% and 5.3% (w/w) water, respectively) as a function of temperature. Plots of specific volume versus temperature exhibit a characteristic change in slope when the amorphous systems change from the glassy to the rubbery state and the intersection of the two regression lines provides an estimate of the glass transition temperature T(g). A comparison of the calculated and experimental T(g) values, as obtained from differential scanning calorimetry, shows that despite the predicted values being systematically higher (about 21-26K), the trend and the incremental differences between the T(g) values have been computed correctly: T(g)(5.3%(w/w))相似文献   

The Effect of Glycerol on Molecular Mobility in Amorphous Sucrose Detected by Phosphorescence of Erythrosin B

Luminescence from the triplet probe erythrosin B provides spectroscopic characteristics such as emission energy and lifetime that are sensitive to molecular mobility of the local solvent environment. This study investigated how variation in glycerol content influences the properties of an amorphous sucrose matrix by monitoring phosphorescence of erythrosin B over the temperature range from 5 to 100°C. Emission energy, lifetime, and red-edge excitation data revealed that glycerol affected the mobility of amorphous sucrose matrix primarily through plasticization when the mole ratio of glycerol/sucrose was above 0.27, resulting in a decrease in emission energy (ν _p), lifetime (τ) and energy difference (Δν _P) with excitation at the absorption peak and red edge and an increase in the nonradiative, collisional quenching rate k _TS0. At mole ratios ≤0.27 and at temperatures below the matrix T _g, glycerol exhibited an “antiplasticization” effect, as indicated by higher values of emission energy, lifetime, and energy difference and lower values of the matrix collisional quenching rate. Changes in the distribution of emission energy (emission bandwidth) and lifetime, and variation in the emission lifetime vs wavelength showed that glycerol reduced the spectral heterogeneity. These data illustrate the complex effect of a hydrogen bonding solute on the mobility of an amorphous, hydrogen bonded sugar matrix.     

Melting, glass transition, and apparent heat capacity of α-d-glucose by thermal analysis

The thermal behaviors of α-d-glucose in the melting and glass transition regions were examined utilizing the calorimetric methods of standard differential scanning calorimetry (DSC), standard temperature-modulated differential scanning calorimetry (TMDSC), quasi-isothermal temperature-modulated differential scanning calorimetry (quasi-TMDSC), and thermogravimetric analysis (TGA). The quantitative thermal analyses of experimental data of crystalline and amorphous α-d-glucose were performed based on heat capacities. The total, apparent and reversing heat capacities, and phase transitions were evaluated on heating and cooling. The melting temperature (T_m) of a crystalline carbohydrate such as α-d-glucose, shows a heating rate dependence, with the melting peak shifted to lower temperature for a lower heating rate, and with superheating of around 25 K. The superheating of crystalline α-d-glucose is observed as shifting the melting peak for higher heating rates, above the equilibrium melting temperature due to of the slow melting process. The equilibrium melting temperature and heat of fusion of crystalline α-d-glucose were estimated. Changes of reversing heat capacity evaluated by TMDSC at glass transition (T_g) of amorphous and melting process at T_m of fully crystalline α-d-glucose are similar. In both, the amorphous and crystalline phases, the same origin of heat capacity changes, in the T_g and T_m area, are attributable to molecular rotational motion. Degradation occurs simultaneously with the melting process of the crystalline phase. The stability of crystalline α-d-glucose was examined by TGA and TMDSC in the melting region, with the degradation shown to be resulting from changes of mass with temperature and time. The experimental heat capacities of fully crystalline and amorphous α-d-glucose were analyzed in reference to the solid, vibrational, and liquid heat capacities, which were approximated based on the ATHAS scheme and Data Bank.     

Effect of polyols on the post-thawing motility of pellet-frozen ram spermatozoa

The cryoprotective effects of polyols in the absence and presence of glycerol in Tris-glucose-egg yolk based diluents on the post-thawing motility and acrosome integrity of pellet-frozen ram spermatozoa were examined. Incorporation of adonitol or xylitol (low molecular weight polyols; LMWPs) in diluents improved motility of spermatozoa in the absence of glycerol with maximum motility at 0.3 M (26.9 vs 13.3% mean motile spermatozoa; P < 0.001). Five concentrations of adonitol (0, 150, 300, 450, 600 mM) were examined in diluents containing 5 concentrations of glycerol (0, 1.5, 3.0, 4.5, 6.0% v/v). There was an additive effect of incorporation of 1.5% v/v glycerol with up to 450 mM adonitol (P < 0.001), but at higher levels of glycerol the incorporation of adonitol was detrimental to motility. The acrosome integrity of spermatozoa in diluents containing 0, 150 and 300 mM adonitol was superior to those containing 450 and 600 mM adonitol (46.1 vs 35.1% mean intact acrosomes; P < 0.05). Among the high molecular weight polyols (HMWPs) examined, better recovery of spermatozoa was obtained in diluents containing sorbitol than mannitol or inositol (P < 0.001). Sorbitol or mannitol (300 mM) improved the motility of spermatozoa in diluents without glycerol (P < 0.001), but the incorporation of HMWPs was detrimental in diluents containing glycerol. All five polyols were examined in isotonic diluents containing 360:0, 300:55, 240:110, 180:165, 120:220mM (Tris:polyol; 360 mosmol) and 6.0% v/v glycerol. There was a linear decrease in motility and acrosome integrity of spermatozoa with increasing polyol concentration in the diluent (P < 0.001) except for inositol, which was not detrimental. We conclude that the polyols examined have a cryoprotective effect on pellet-frozen ram spermatozoa except for inositol. However, in our study, no combination of polyols and glycerol was superior in terms of post-thawing motility and acrosome integrity of spermatozoa to 6.0% v/v glycerol alone in Tris-glucose-egg yolk diluents.     

Predict the glass transition temperature of glycerol-water binary cryoprotectant by molecular dynamic simulation

Vitrification is proposed to be the best way for the cryopreservation of organs. The glass transition temperature (T_g) of vitrification solutions is a critical parameter of fundamental importance for cryopreservation by vitrification. The instruments that can detect the thermodynamic, mechanical and dielectric changes of a substance may be used to determine the glass transition temperature. T_g is usually measured by using differential scanning calorimetry (DSC). In this study, the T_g of the glycerol-aqueous solution (60%, wt/%) was determined by isothermal-isobaric molecular dynamic simulation (NPT-MD). The software package Discover in Material Studio with the Polymer Consortium Force Field (PCFF) was used for the simulation. The state parameters of heat capacity at constant pressure (C_p), density (ρ), amorphous cell volume (V_cell) and specific volume (V_specific) and radial distribution function (rdf) were obtained by NPT-MD in the temperature range of 90–270 K. These parameters showed a discontinuity at a specific temperature in the plot of state parameter versus temperature. The temperature at the discontinuity is taken as the simulated T_g value for glycerol–water binary solution. The T_g values determined by simulation method were compared with the values in the literatures. The simulation values of T_g (160.06–167.51 K) agree well with the DSC results (163.60–167.10 K) and the DMA results (159.00 K). We drew the conclusion that molecular dynamic simulation (MDS) is a potential method for investigating the glass transition temperature (T_g) of glycerol–water binary cryoprotectants and may be used for other vitrification solutions.     

Cyclodextrins micrometric powders obtained by supercritical fluid processing

Supercritical fluid technology offers the possibility to produce dry powder formulations of biocompatible materials, overcoming the drawbacks of classical micronization processes. In this work, Supercritical Assisted Atomization (SAA) has been used to micronize alpha-cyclodextrin (alpha-CD) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD). Some process parameters, such as precipitation temperature and solute concentration in the liquid solution, have been studied to evaluate their influence on morphology and size of precipitated particles. Cyclodextrins (CDs) micronization has been successful: well-defined spherical microparticles of alpha-CD and HP-beta-CD have been produced. Particle size analysis revealed that sharp distributions have been obtained: 95% of particles have diameters ranging between 0.1 and 5 microm for both CDs. X-ray and DSC analyses have been also performed to investigate CDs modifications induced by SAA processing: amorphous particles have been obtained in both cases, whereas raw alpha-CD was crystalline and raw HP-beta-CD was amorphous.     

Glass transition and dynamics in BSA-water mixtures over wide ranges of composition studied by thermal and dielectric techniques

Protein-water dynamics in mixtures of water and a globular protein, bovine serum albumin (BSA), was studied over wide ranges of composition, in the form of solutions or hydrated solid pellets, by differential scanning calorimetry (DSC), thermally stimulated depolarization current technique (TSDC) and dielectric relaxation spectroscopy (DRS). Additionally, water equilibrium sorption isotherm (ESI) measurements were performed at room temperature. The crystallization and melting events were studied by DSC and the amount of uncrystallized water was calculated by the enthalpy of melting during heating. The glass transition of the system was detected by DSC for water contents higher than the critical water content corresponding to the formation of the first sorption layer of water molecules directly bound to primary hydration sites, namely 0.073 (grams of water per grams of dry protein), estimated by ESI. A strong plasticization of the T(g) was observed by DSC for hydration levels lower than those necessary for crystallization of water during cooling, i.e. lower than about 0.3 (grams of water per grams of hydrated protein) followed by a stabilization of T(g) at about -80°C for higher water contents. The α relaxation associated with the glass transition was also observed in dielectric measurements. In TSDC a microphase separation could be detected resulting in double T(g) for some hydration levels. A dielectric relaxation of small polar groups of the protein plasticized by water, overlapped by relaxations of uncrystallized water molecules, and a separate relaxation of water in the crystallized water phase (bulk ice crystals) were also recorded.