Changes in Natural Silk Fibres by Hydration, Tensile Loading and Heating as Studied by 1H NMR: Anisotropy in NMR Relaxation Times.

B. mori silkworm natural silk is a fibrous biopolymer with a block copolymer design containing both hydrophobic and hydrophilic regions. Using 1H NMR relaxation, this work studied B. mori natural silk fibres oriented at 0° and 90° to the static magnetic field B0 to clarify how measured NMR parameters reflect the structure and anisotropic properties of hydrated silk fibres. The FTIR method was applied to monitor the changes in the silk I and ß-sheet conformations. Unloaded B. mori silk fibres at different hydration levels (HL), the silk threads before and after tensile loading in water, and fibres after a stepped increase in temperature have been explored. NMR data discovered two components in T1 and T2 relaxations for both orientations of silk fibres (0° and 90°). For the slower T2 component, the results showed an obvious anisotropic effect with higher relaxation times for the silk fibres oriented at 90° to B0. The T1 component (water protons, HL = 0.11) was sequentially decreased over a range of fibres: 0° oriented, randomly oriented, silk B. mori cocoon, 90° oriented. The degree of anisotropy in T2 relaxation was decreasing with increasing HL. The T2 in silk threads oriented at 0° and 90° also showed anisotropy in increased HL (to 0.42 g H2O/g dry matter), at tensile loading, and at an increasing temperature towards 320 K. The changes in NMR parameters and different relaxation mechanisms affecting water molecular interactions and silk properties have been discussed. The findings provide new insights relating to the water anisotropy in hydrated Bombyx mori silk fibres at tensile loading and under a changing HL and temperature.

Analysis of single particle photodegradation using photothermal infrared microspectroscopy.

The increasing use of high throughput methods, coupled with the need to develop approaches to anticipate long term stability issues, has necessitated the introduction of testing approaches whereby extremely small samples may be rapidly analysed. A novel method is described whereby the UV light-induced degradation of single particles of a model drug, nifedipine, may be rapidly and simply monitored using photothermal infrared microspectroscopy (PTMS). The technique involves the contact attachment of individual particles to a heated probe tip composed of a modified Wollaston wire which enables temperature fluctuations to be measured. Application of a focused IR beam to excite the sample allows measurement and subsequent Fourier transformation of the resultant interferogram to produce an IR spectrum which is in good agreement with that obtained from conventional IR methods. By application of a UV source to the assembly for specified time periods, we demonstrate that it is possible to monitor the appearance of peaks associated with degradation products as a function of time. The technique is critically evaluated in terms of practical issues associated with volatilization, particle size effects and orientation to the light source as well as more general issues associated with the sensitivity, resolution and quantitative interpretation of data from the PTMS technique. Overall the method has been shown to be capable of rapid measurement of photo-instability on individual particles, with important implications for development of the approach as a rapid screening or high throughput technique, although there are practical and theoretical limitations to reliable quantitative analysis at the present time.

Development of photothermal FTIR microspectroscopy as a novel means of spatially identifying amorphous and crystalline salbutamol sulfate on composite surfaces.

Photothermal Fourier transform infrared (FTIR) microspectroscopy (PTMS), involving the combination of FTIR spectroscopy with atomic force microscopy, has been used to examine compacts of amorphous and crystalline salbutamol sulfate in order to assess the ability of the technique to distinguish between different physical forms in a multicomponent material. Samples of amorphous and crystalline material were assessed using modulated temperature differential scanning calorimetry (DSC), atomic force microscopy, microthermal analysis, and conventional FTIR. Mixed compacts were then prepared such that verification of the location of the forms present was possible via topography and localized thermal analysis. PTMS studies were then performed on selected interrogation points, with spectra obtained which were largely intermediate between those corresponding to the two individual forms. Calculation of the thermal diffusivity indicated a resolution for the technique corresponding to a hemisphere of a major diameter in the region of 40 µm, which is large in relation to the particle sizes involved. However, distinction into amorphous, crystalline, and indeterminate categories was possible using chemometric analysis (hierarchical cluster analysis and principal component analysis). Good agreement was found between the identification methods for the mixed systems. The study has therefore shown the potential, as well as identifying the limitations, of using PTMS as a means of spatially identifying components in complex materials.

Development of fully amorphous dispersions of a low T(g) drug via co-spray drying with hydrophilic polymers.

The aim of the study was to prepare molecular dispersions of a physically highly unstable amorphous drug, paracetamol (acetaminophen with a T(g) of ca. 25°C) via co-spray drying with a variety of polymers. Solid dispersions at a range of drug loadings (10-90%w/w) using hydroxypropyl methylcellulose/acetate succinate (HPMC/HPMC AS), polyvinylpyrrolidone (PVP) and copovidone were produced and characterised by modulated temperature differential scanning calorimetry (MTDSC), thermogravimetric analysis (TGA), X-ray powder diffraction (XRPD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). PVP-based polymers showed a greater tendency than the HPMC-based group to generate temperature-stable dispersions. In particular, copovidone (Plasdone® S-630) was found to be the most effective of the polymers studied and could formulate molecular dispersions at drug loadings up to and including 40%w/w. However, no evidence for direct drug-polymer interactions was found for such systems as a possible stabilising mechanism. The expected relationship of a higher T(g) of the polymer leading to greater stabilisation was not observed, while there was an inverse relationship between viscosity grade and amorphous phase generation. The study has therefore shown that temperature-stable amorphous dispersions of a low T(g) drug may be prepared by co-spray drying, particularly using PVP-based polymers.

An investigation into water interactions with amorphous and milled salbutamol sulphate: the development of predictive models for uptake and recrystallization.

An investigation into the effect of water uptake on the glass transition of spray dried and milled salbutamol sulphate has been performed, with a particular view to exploring how the water uptake, T(g) value and recrystallization behaviour correlate. Samples of milled and spray dried drug were stored under controlled humidity conditions and the T(g) measured as a function of time. The T(g) was measured using modulated temperature differential scanning calorimetry (MTDSC) while the water content was measured using thermogravimetric analysis (TGA). A correlation was found between time of storage, water content and T(g) in that the samples showed time dependent equilibration with the storage environment (either gaining or losing water depending on the RH). The relationship between water content and stability, based on the concept of T(g) lowering, was modelled using the semi-empirical approach of Royall et al. (1999) as well as a derivation of the Kwei equation which allowed the interaction between the water and substrate to be accounted for. A method for predicting stability based on two simple DSC runs is proposed. In addition, we discuss the observation of a double glass transition for the spray dried samples.

NMR studies of hydration in low water content biopolymer systems.

The problem of characterising the behaviour of water and biopolymers by NMR in low water content biopolymer systems is discussed. A low water content system is defined, and the problems of characterising the relaxation behaviour of the water are analysed. In the case of protons, the types of protons contributing to the signal and the exchange mechanism between them cannot be systematised in terms of existing models that have been developed for high water content systems. It is suggested that any successful model must take account of at least three separate pools of water including water vapour. Experimental results indicate that although the motion of the biopolymer is radically affected by water, the reverse is not necessarily true. It is concluded that the use of nuclei such as (13)C and (15)N may be very effectively used to characterise biopolymer motion, but the use of both (1)H and (2)H for characterising water is still problematic. Despite the formidable experimental and theoretical difficulties, (17)O NMR may be the only way to finally to untangle the problem.

The use of dynamic vapour sorption methods for the characterisation of water uptake in amorphous trehalose.

Water uptake by amorphous sugars is an issue of high importance for the food and pharmaceutical industries. However, while the processes associated with sorption-induced crystallisation have been widely studied, little is known regarding the uptake mechanisms associated with pre-crystallisation water levels. In the present investigation we use dynamic vapour sorption to study the water uptake mechanisms associated with amorphous trehalose. More specifically, we have prepared spray-dried amorphous trehalose with three initial water contents and studied water uptake as a function of time and relative humidity. We model the data obtained prior to crystallisation and suggest two mechanisms that are based on Type II diffusion (using the Peleg equation), which predominates under high humidity conditions (50% RH and above), while we use a Type I Fickian diffusion expression to model uptake under low relative humidity conditions (40% RH and below). The model allows prediction of equilibrium sorption values which correlate well with previously published data calculated from equilibrium vapour pressure data. We also note that the water content following recrystallisation is greater than that predicted by the stoichiometric ratio. A novel model is suggested whereby recrystallisation onset times may be estimated from the vapour sorption data. In conclusion the study has demonstrated that pre-crystallisation water sorption may be associated with two mechanisms depending on the humidity conditions and that such modelling allows insights into both the mechanisms of uptake and the storage behaviour of amorphous sugar samples.

Compositional analysis of metal chelating materials using near-field photothermal Fourier transform infrared microspectroscopy.

Photothermal-Fourier transform-infrared (PT-FT-IR) microspectroscopy employs a thermal probe mounted in a scanning probe microscope (SPM). By placement of the tip of the probe on the surface of a solid sample, it can obtain localized IR spectra of a wide range of samples. A second mode of analysis is also available; a sample can be taken from the selected location using a technique called thermally assisted nanosampling (TAN), then a spectrum can be obtained of the nanosample while the probe is remote from the surface. We report a novel method of local compositional analysis that combines both of these types of measurement; a reagent is attached to the tip using TAN, then the reagent is placed in contact with analyte. IR spectroscopy can then be used to analyze any interaction between the reagent and surface it is placed in contact with. All of these modes of analysis were illustrated using a metal chelating agent. In the surface mode, changes to a solid bead of a chelating resin were measured using standard PT-FT-IR. In the nanosampling mode of analysis, a particle of a chelating polymer was attached to the tip of the probe using TAN and this was placed in contact with a concentrated calcium solution. Strong spectral changes were observed that mirrored those found when exposing the surface bound chelating resin bead to a solution of the same ion. A semiquantitative simulation of the PT spectrum for a chelating resin bead was achieved using a thermal diffusion model derived from photoacoustic spectroscopy indicating that semiquantitative or quantitative measurements will be possible in such a system.

Kafirin microparticle encapsulation of catechin and sorghum condensed tannins.

To exploit the porous nature of previously developed kafirin microparticles, encapsulation of the bioactive polyphenols, catechin and sorghum condensed tannins, was investigated. The antioxidant release profiles of the encapsulated substances were studied under simulated gastric conditions. Kafirin microparticles encapsulating catechin or sorghum condensed tannins were similar in size to control kafirin microparticles (5-6 mum). TEM showed that kafirin microparticles encapsulating catechin had a rough porous surface. Microparticles encapsulating sorghum condensed tannins were irregular in shape, some apparently joined together, with a mixture of rough and smooth surfaces. Over a period of 4 h, catechin and sorghum condensed tannin encapsulated kafirin microparticles showed virtually no protein digestion but released approximately 70 and 50%, respectively, of total antioxidant activity. Thus, the use of kafirin microparticles to encapsulate catechin and sorghum condensed tannins has potential as an effective method of controlled release of dietary antioxidants.

Preparation of free-standing films from kafirin protein microparticles: mechanism of formation and functional properties.

A method of preparing free-standing films using kafirin microparticles made by phase separation from acetic acid is described. Film preparation involved the suspension of the microparticles in acetic acid solution containing plasticizer. On evaporation of the acetic acid, a complete, smooth, flexible, transparent film was formed. A minimum concentration of acid was required to form a cohesive film relative to the concentration of kafirin. This was approximately 10.8:1, percent acetic acid to percent kafirin. Film formation appears to be by controlled aggregation of kafirin microparticles, followed by dissolution of the microparticles in acetic acid and drying into a cohesive film. The functional properties of microparticle films were generally superior to films cast directly from a solution of kafirin, at the same protein content. Kafirin microparticle films were very thin (<15 microm), relatively strong but not very extensible, with better water barrier properties and lower protein digestibility than conventionally cast kafirin films.

Zein-iodine complex studied by FTIR spectroscopy and dielectric and dynamic rheometry in films and precipitates.

In the present study the effect of iodine on properties of zein films and zein precipitates obtained after hydrophobic aggregation was evaluated. Zein films were cast with and without glycerol (as plasticizer) after incorporation of iodine at different levels (2-8%, zein wt basis). Zein films were characterized by secondary structure (determined by infrared spectroscopy) and dielectric and mechanical properties. The rheological properties of zein precipitates as a function of frequency and temperature were evaluated using a dynamic rheometer. Inclusion of iodine changed the secondary structure of zein films and decreased their tensile strength as well as strain at failure. In aggregates, changes in G' (elastic modulus) and G'' (viscous modulus) during heating were affected by the presence of iodine due to the inhibition of aggregation. The water-holding capacity of precipitates precipitated in the presence of iodine was higher than that of those without iodine.

The effects of iodine on kidney bean starch: films and pasting properties.

In the present study the effect of iodine on the structural characteristics (by infrared spectroscopy and X-ray) of films made from kidney bean starch was evaluated. The pasting properties as affected by iodine and glycerol were also evaluated. Kidney bean starch showed C-type (mixture of A- and B-type) crystalline structure, the conversion of starch into films resulted into reduction in intensity of diffractograms. The starch powder FTIR spectra had peaks centered at 1020 and 995 cm(-1) with a higher intensity at 1020 cm(-1), which is consistent with a partially crystalline material since fully crystalline material show similar intensity peaks centered around 1020 and 1006 cm(-1). Films without iodine showed one main peak centered around 1000 cm(-1) consistent with a disordered state similar to that in gelatinized starch. Iodine addition gradually increased the intensity of the bands around 1020 cm(-1) consistent with the formation of more ordered conformation similar to that in the crystalline material. Iodine encourages the formation of helical structures, however, the formation of crystalline material cannot be inferred. The increasing amounts of iodine up to 0.33% level progressively increased the peak-, through- and breakdown-viscosity. Iodine beyond 0.33% level gradually decreased peak-, trough-, breakdown- and setback-viscosity. Pasting temperature gradually increased with the increase in iodine.

Plasticization of zein: a thermomechanical, FTIR, and dielectric study.

Zein, the main seed storage protein of maize, has been widely studied as a possible source of material for the production of biodegradable plastic films. Plasticization of zein is critical to make functional films. While there have been a number of publications which report the behavior of systems with a wide variety of plasticizers, there have been few which attempt to examine the interactions of protein and plasticizer at the molecular level. In this paper, we report on the plasticizing effects of water, glycerol, and 2-mercaptoethanol, which were examined by a combination of spectroscopy (FTIR and dielectric) and thermomechanical methods. The results suggest that both water and glycerol are adsorbed onto the protein and form hydrogen bonds with the amide groups. The plasticizer then builds up in patches on the protein surface. 2-Mercaptoethanol only exhibited a weak plasticizing effect due probably to disulfide bond breaking.

Thermal probe based analytical microscopy: thermal analysis and photothermal Fourier-transform infrared microspectroscopy together with thermally assisted nanosampling coupled with capillary electrophoresis.

In this study, we have demonstrated that a scanning probe microscope (SPM) can be used for thermally assisted nanosampling (TAN) with subsequent analysis by capillary electrophoresis (CE). Localized thermomechanical analysis (L-TMA) and photothermal Fourier-transform infrared (PT-FTIR) microspectroscopy can also be employed using the same probe, thus illustrating how a single instrument can carry out a number of different complementary analytical measurements. Benzoic acid and 4-hydroxybenzoic acid were manipulated with a heated Wollaston wire probe and successfully deposited onto the surface of a piece of CE capillary tubing. The deposited samples were then separated with CE. L-TMA and PT-FTIR were also used to characterize these materials. We have also demonstrated how a nanosample of a nonparticulate material can be taken and then deposited onto the surface of an inert matrix. TAN of a nonparticulate material was explored using polyethylene as the analyte and fluorene as the matrix. These examples show that thermal probe techniques provide a versatile "tool box" of modes of analysis with the potential to analyze a wide range of samples in a spatially resolved way.

Effect of variety and environmental factors on gluten proteins: an analytical, spectroscopic, and rheological study.

Four cultivars of winter wheat with contrasting qualities for breadmaking were selected to study the effects of environmental factors on grain protein composition and properties. They were grown in the field and under two controlled regimens designed to mimic typical "hot/dry" and "cold/wet" conditions experienced during grain development in the United Kingdom. The composition of the gluten proteins determined by SDS-PAGE and their size distribution determined by SE-HPLC were consistent with the presence of higher proportions of high M r polymers in the two varieties with good breadmaking performance (Spark and Soissons) with limited environmental effects on these parameters. Gluten protein fractions from three of the cultivars were analyzed by Fourier transform infrared (FTIR) spectroscopy and this, combined with creep measurements using a texture analyzer, showed that a conversion from beta-turns to beta-sheets occurred during extension, irrespective of the growth conditions. However, the breadmaking varieties Soissons and Spark showed greater differences related to environmental conditions than the variety Rialto, which has poorer processing quality.

A study on maize proteins as a potential new tablet excipient.

This investigation has examined the use of zein proteins from maize as the major component in oral controlled-release tablets, such formulations often being required to improve patient compliance. Tablets containing ground zein proteins, calcium hydrogen orthophosphate, polyvinyl pyrrolidone, theophylline and magnesium stearate were produced by wet granulation and compression on a single station tablet press and were compared to directly compressed tablets based on zein proteins, calcium hydrogen orthophosphate and theophylline. Non invasive techniques such as Fourier Transform infrared spectroscopy and Fourier Transform Raman spectroscopy were employed to investigate any changes in the secondary structure of zein proteins during tablet production. Random coils, alpha helices and beta sheets predominated and their relative content remained unaffected during grinding, wet granulation and compression, indicating that formulations based on zeins will be robust, i.e. insensitive to minor changes in the production conditions. Drug release from the tablets was studied using a standard pharmacopoeial dissolution test. Dissolution profiles in water, 0.1M HCl (pH=1) and phosphate buffer (pH=6.8) show that only a limited amount of theophylline was released after 4.5h, suggesting that zein proteins could act as a potential vehicle for oral controlled drug release. Analysis of the theophylline release profiles using the Peppas and Sahlin model reveals that diffusion and polymer relaxation occurred in acidic (pH=1) and buffered (pH=6.8) conditions for wet granulated tablets, whereas diffusion was predominant in directly compressed tablets. In conclusion, the present study has shown that zeins can be successfully used as a pharmaceutical excipient, and in particular as a matrix in monolithic controlled release tablets.

Hydration of gluten: a dielectric, calorimetric, and fourier transform infrared study.

The nature of the hydration of proteins and the subsequent implications for functionality is a matter of importance in both pharmaceutical and food applications. Most published studies rely on the use of one technique and attempt to characterize the system. Few studies have used combinations of techniques. In this paper we report on the use of infrared, dielectric, and calorimetric methods to examine the hydration process of wheat gluten. This has been the subject of considerable study by other techniques and has been well characterized by our group. Results show that in both the infrared and dielectric measurements there is a change in behavior at about 35% water content. This is also the water content below which lowering the temperature of the sample does not result in ice formation. We suggest that at this water content the protein amide groups are fully hydrated, and beyond this point addition of water results in protein dilution rather than further hydration.

Plasticization of a protein-based film by glycerol: a spectroscopic, mechanical, and thermal study.

Kafirin, the seed storage protein of the cereal sorghum, is highly homologous with the maize storage protein zein. The effects of plasticisation of a kafirin film by glycerol in the absence of water were examined by a combination of spectroscopic (NMR and infrared), rheological, and calorimetric methods. The results suggest that at low glycerol levels the glycerol is absorbed onto and possibly into the protein. Increasing the level of glycerol increases the motion of the protein and changes the protein conformation. There are corresponding changes of the mechanical properties of protein films. At 40% (w/w) of glycerol, two glass transition temperatures were observed, one of which corresponded to the glass transition temperature of pure glycerol. This result indicates that at this level of plasticizer there are sufficient glycerol/glycerol interactions occurring to allow a separate glass formation process for glycerol.

Comparison of repetitive sequences derived from high molecular weight subunits of wheat glutenin, an elastomeric plant protein.

A strategy has been developed to create repetitive peptides incorporating substitutions in the PGQGQQGYYPTSLQQ consensus repeat sequence of high molecular weight subunits in order to investigate natural sequence variations in elastomeric proteins of wheat gluten. After introduction of glutamic and aspartic acid residues, the peptide behaved similarly to the unmodified form at low pH, but became readily water soluble at pH > 6. Substitution of Gln for Leu at position 13 resulted in only small changes to the secondary structure of the water-insoluble peptides, as did Tyr8His and Thr11Ala. The effects of proline substitutions depended on their location: Leu13Pro substitution had little effect on solubility and structure, but Gln6Pro substitution resulted in dramatic changes. Peptides with two Gln6Pro substitutions had similar properties to the water-insoluble parental peptide, but those with 6 or 10 substitutions were readily soluble. The results indicated that specific sequences influence noncovalent intermolecular interactions in wheat gluten proteins.

Effects of temperature and water content on the secondary structure of wheat gluten studied by FTIR spectroscopy.

The effect of temperature on gluten conditioned at the following water contents, 0%, 13%, and 47% (wet weight basis), was studied by FTIR spectroscopy over the temperature range of 25-85 degrees C. A detailed discussion of the assignment of the amide I band is given. At 0% hydration no changes in the secondary structure with temperature could be detected; spectra were consistent with a tight disordered structure with many protein-protein interactions. At 13% hydration, distinctive changes occurred in the low-frequency region of the amide I band (1,630-1,613 cm(-1)). This was attributed to changes in the beta-sheet structure. On cooling to 25 degrees C, these changes were mainly reversed. It was noted that most of the changes observed occurred above the glass transition temperature. At 47% hydration, more complex changes took place: as the temperature was raised distinct bands at 1,630 and 1,613 cm(-1) merged. However, this process was partially reversed, with recovery of both bands, on cooling. The significance of these results in relation to other changes in gluten proteins in flour and dough with temperature and water content is discussed.