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Spin-spin relaxation time attributed to the liquid phase versus the reciprocal of temperature (K À1 ) in a sucrose-water solution. Effect of sucrose concentration and of the addition of protein.
Source publication
This work aimed at characterizing both the spin–spin (T2) and spin–lattice (T1) relaxations of water in frozen samples. Pure water and aqueous solutions (sucrose and/or casein) were studied, temperatures ranging from −13 to 20 °C. Three relaxation components could be distinguished after signal fitting. For example, the shorter spin–spin relaxation...
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Citations
... Earlier studies have leveraged nuclear magnetic resonance (NMR) to discern the amount of unfrozen water in frozen materials. [13][14][15][16] For example, Watanabe and Mizoguchi employed the T 2 relaxation method to gauge unfrozen water quantities in frozen porous glass powders, fujinomori soil, and bentonite. 17 Similarly, Lucas et al. assessed the ice content in frozen sucrose-protein solutions using a congruent approach. ...
Freezing is essential for the stability of biological drug substances and products, particularly in frozen solution formulations and during the primary drying of lyophilized preparations. However, the unfrozen segment within the frozen matrix can alter solute concentration, ionic strength, and stabilizer crystallization, posing risks of increased biophysical instability and faster chemical degradation. While quantifying the unfrozen water content is crucial for designing stable biopharmaceuticals, there is a lack of analytical techniques for in situ quantitative measurements. In this study, we introduce a 1H magic angle spinning NMR technique to identify the freezing point (T_ice) and quantify mobile water content in frozen biologics. Our quantitative results demonstrate that water freezing is influenced by buffer salt properties and formulation composition, including the presence of sugar cryoprotectants and protein concentration. Additionally, the 1H chemical shift can probe pH in the unfrozen phase, potentially predicting the microenvironmental acidity in the frozen state. Our proposed methodology provides fresh insights into the analysis of freeze-concentrated solutions, enhancing our understanding of the stability of frozen and lyophilized biopharmaceuticals.
... It is therefore a good method of analyzing biopolymer gels. The LF NMR method enables the analysis of the molecular dynamics of water, which is a solvent, i.e., the liquid phase of a biopolymer gel [28,29]. The processes of proton relaxation in such systems are, compared to the state of liquid water, slowed down due to the presence of biomolecules forming a spatial lattice. ...
Many different biopolymers are used to stabilize emulsions, of which starch is of particular concern. To improve the characteristics and technical utility of native starch, various types of changes can be made. This article is a report describing the molecular dynamics of water by the low-field nuclear magnetic resonance (LF NMR) of chemically (E 1412 and E 1420) and physically modified starch (LU 1432) gels and the effect of their use on the stability of oil/water emulsions obtained using bovine and porcine fats. The analysis of changes in spin–spin and spin–lattice relaxation times over time showed that the presence of the type of starch modification significantly affects the values of T1 and T2 relaxation times, as well as the correlation times. Research on time-related changes in water binding in oil-in-water emulsions showed that potato starch modified by chemical methods can be used as an emulsifier. Compared to physically modified starch, chemically modified starches have a much better water-binding capacity.
... The NMR technique has been shown to be an appropriate method to calculate the amount of unfrozen water in a food sample. Lucas et al. (2004) [38] examined liquid from the solid water in aqueous sucrose solutions (sucrose and/or casein). They considered spin-spin relaxation measurements that were usually used and also spin-lattice ones. ...
... The NMR technique has been shown to be an appropriate method to calculate the amount of unfrozen water in a food sample. Lucas et al. (2004) [38] examined liquid from the solid water in aqueous sucrose solutions (sucrose and/or casein). They considered spin-spin relaxation measurements that were usually used and also spin-lattice ones. ...
Methods of testing and describing the recrystallization process in ice cream systems were characterized. The scope of this study included a description of the recrystallization process and a description and comparison of the following methods: microscopy and image analysis, focused beam reflectance measurement (FBRM), oscillation thermo-rheometry (OTR), nuclear magnetic resonance (NMR), splat-cooling assay, and X-ray microtomography (micro-CT). All the methods presented were suitable for characterization of the recrystallization process, although they provide different types of information, and they should be individually matched to the characteristics of the tested product.
... The relaxation times and relative signal amplitudes were in agreement with the T 2 distributions determined from the CPMG signal, complementing the two relaxation times extracted from the FID signal. The first relaxation time T 2 (1) was very short (around 14-15 ms) and was typical of the relaxation time of protons involved in a crystal network, as already observed in ice (Lucas et al., 2004). Its relative amplitude increased with TBAB content, with values very close to the TBAB concentration of the mixtures. ...
Semiclatrate hydrate crystals of tetra-n-butyl ammonium bromide (TBAB) form at atmospheric pressure and at positive temperature. In suspension with water, they can be used as a heat transport medium or for gas separation. In terms of thermal energy storage and secondary refrigeration applications, TBAB hydrate slurries may be characterized by their flow properties under specific thermophysical conditions. The study presented here investigated one of these important properties, i.e. the crystal content, also called the solid fraction, that is usually estimated using the TBAB:H2O phase diagram. Our investigations revealed that Time-Domain Nuclear Magnetic Resonance (TD-NMR) relaxometry was remarkably sensitive to the solid content of TBAB:H2O hydrate slurries. Non-invasive and non-destructive measurements performed in real time demonstrated the power of NMR relaxometry to identify and quantify TBAB hydrates. Most importantly, NMR both measured and quantified type A and B hydrates, which no other technique can achieve without destroying the crystals or can only perform theoretically on the basis of a phase diagram, and thus without considering the possible conversion of type A to type B hydrates.
... Besides DSC, additional analytical techniques such as microscopy (13), spectrometry (14)(15)(16)(17)(18) and certain types of rheology have been used to study the frozen state. While the spectroscopic approaches are useful, they apply to very local movements and changes at the molecular level. ...
Purpose:
A novel application of oscillatory shear rheology was used to directly monitor global phase behavior of protein formulations in the frozen state and study its correlation with physical instability of frozen protein formulations.
Methods:
Oscillatory rheology was used to measure changes in rheological parameters and to identify mechanical softening temperature (Ts*) and related properties of an IgG2 mAb formulation. Rheological measurements were compared to DSC/MDSC. Physical stability of IgG2 formulations was monitored by SE-HPLC.
Results:
Rheological parameters and Ts* of an IgG2 formulation were sensitive to physical/morphological phase changes during freezing and thawing. Ts* of the frozen formulation was a function of concentration of protein and excipient. Complex modulus, G*, and phase angle, δ, for IgG2 at 70 mg/mL in a sucrose-containing formulation showed the system was not completely frozen at -10°C, which correlated to stability data consistent with ice-induced protein aggregation.
Conclusions:
We report the first application of oscillatory shear rheology to study phase behavior of IgG2 in a sucrose-containing formulation and its correspondence with physical stability not explained by glass transition (Tg'). We provide a mechanism and data suggesting that protein instability occurs at the ice/water interface.
... Nevertheless, benchtop NMR relaxometry has successfully been applied to study crystallisation of fat and water in ice cream (models) during freezing and frozen storage. [247][248][249] Time-Domain NMR Applied to Food Products 7.6.3. Horticultural products Fruits, vegetables, tubers and beans are challenging food stuffs with respect to maintaining quality during the chain between field (or greenhouse) and consumer or processing plant. ...
Time-domain NMR is being used throughout all areas of food science and technology. A wide range of one- and two-dimensional relaxometric and diffusometric applications have been implemented on cost-effective, robust and easy-to-use benchtop NMR equipment. Time-domain NMR applications do not only cover research and development but also quality and process control in the food supply chain. Here the opportunity to further downsize and tailor equipment has allowed for “mobile” sensor applications as well as online quality inspection. The structural and compositional information produced by time-domain NMR experiments requires adequate data-analysis techniques. Here one can distinguish model-driven approaches for hypothesis testing, as well as explorative multi-variate approaches for hypothesis generation. Developments in hardware and software will further enhance measurement speed and reveal more detailed structural features in complex food systems.
... The simultaneous acquisition of the solid and liquid-like relaxation decay signal has also been used to investigate the ice phase during freezing. For example, two components were identified in a binary sucrose solution for temperatures above -2°C [17]. ...
... Using NMR CPMG T 2 dispersion [28][29][30][31] and quantitative analysis of the T 1 magnetic relaxation dispersion (MRD) [27,[32][33][34][35][36], it is now clear that the water relaxation can be explained by taking into account only two relaxation mechanisms, the protein-exchanging protons relaxation and the relaxation from the internal water molecule. Moreover, analysis of 1 H, 2 H and 17 O MRD has demonstrated that the internal molecules involved in this mechanism are only marginally less mobile than the bulk water and should not be considered as "bound", as they were for many years [33], and the number of water molecules is very small. For example, Venu et al [27] shown that a considerable difference in the MRD can be observed between a solution of pancreatic trypsin inhibitor and a mutant protein (G36S) lacking only one of the four internal water molecules. ...
Since the publication of the ISO method for measurement of solid fat content using NMR relaxation at low field, the application of this technique to the characterisation of food composition and food structure on several length scales has expanded considerably. Improvements in the electronic and computational specifications of NMR spectrometers and the use of more sophisticated signal processing methods have led to several new applications, and NMR, previously recognized as a powerful technique to provide information regarding composition, is now widely used for microstructural investigations. Moreover, MRI applications have in recent years been extended from the medical field to food science and this has opened up new opportunities for the understanding of food. We examine here recent developments in these techniques, including NMR relaxation, multidimensional NMR relaxation, diffusion NMR and MRI.
... Adjustment of the relaxation signal of the sugar-water solution was performed by a biexponential model: T 2 (1) ) 38.7 ms and T 2 (2) ) 294.3 ms ( Table 2). This biexponential model was in agreement with previous results obtained on sucrose-water systems (25), where two components were attributed, one for nonexchangeable protons of sucrose and one for water protons in exchange with hydroxyl groups of sucrose. Moreover, because of their long T 2 , some nonexchangeable protons were included in the second component. ...
Proton mobility was studied in molecular fractions of some model systems and of cake using a 1H nuclear magnetic resonance (NMR) relaxation technique. For cake, five spin-spin relaxation times (T2) were obtained from transverse relaxation curves: T2 (1) approximately 20 micros, T2 (2) approximately 0.2 ms, T2 (3) approximately 3 ms, T2 (4) approximately 50 ms, and T2 (2) approximately 165 ms. The faster component was attributed to the solid phase, components 2 and 3 were associated with the aqueous phase, and the two slowest components were linked to the lipid phase. After cooking, the crust contained more fat but less water than the center part of the cake. The amount of gelatinized starch was lower in the crust, and water was more mobile due to less interaction with macromolecules. This preliminary study revealed different effects of storage on the center and crust.
... where T 2 is the proton relaxation time observed, T 2f and T 2b are the transverse relaxation times of the bulk water protons and the exchangeable protein protons, respectively, P a and P b are water protons and the exchangeable protein protons, respectively, and k b is the chemical exchange rate. This model has been used to explain the water relaxation in numerous protein and carbohydrate systems (27,28,(32)(33)(34)(35)(36). ...
The changes in water proton transverse relaxation behavior induced by aggregation of whey proteins are explained in terms of the simple molecular processes of diffusion and chemical exchange. The water self-diffusion coefficient was measured in whey protein solutions and gels by the pulsed field gradient NMR method. As expected, water self-diffusion was reduced with increased protein concentrations. Whatever the concentration, the water molecules were free to diffuse over distances varying from 15 to 47 mum. Water diffusion was constant over these distances, demonstrating that no restrictions were found to explain the water hindrance. The modification in protein structure by gelation induced a decrease in water diffusion. The effects of protein concentration on water diffusion are discussed and modeled. Two approaches were compared, the obstruction effect induced by a spherical particle and the cell model, which considered two water compartments with specific self-diffusion coefficients.
