A straightforward method for disaccharide characterization by a simplified version of time domain nuclear magnetic resonance

A straightforward method for disaccharide characterization by a simplified version of time domain nuclear magnetic resonance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A straightforward method for disaccharide characterization by a simplified version of time domain nuclear magnetic resonance Afroza Sultana, Ali Asghari, Seddik Khalloufi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4764910/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 24 You are reading this latest preprint version Abstract The necessity of identifying and quantifying sugars in food processing is endless for maintaining food quality attributes such as color, taste, and texture, monitoring regulatory compliance, labeling packages, and maintaining authenticity. Despite available analytical methods for characterizing sugar molecules, the limitations of conventional methods drive researchers to seek more convenient alternatives. This study aimed to characterize common disaccharides such as saccharose, lactose, maltose, and trehalose using a simplified version of time domain nuclear magnetic resonance (TD-NMR), facilitating a quick, cost-effective, and user-friendly approach. In the transverse relaxometry, secondary peak(s) were observed for all the disaccharides with a main peak. Although they have similar molecular formulas and weights, lactose exhibited the longest relaxation time for the secondary peak, followed by trehalose, saccharose, and maltose. This behavior was assumed due to the interaction of sugar molecules with water. The increasing concentration of disaccharide in the solution displayed the leftward shifting of peaks. Maltose showed two secondary peaks, which were not observed in other sugar samples. This TD-NMR showed potential to distinguish disaccharides from unknown powders and solutions by analyzing either the relaxation time of the secondary peak or the ratio of the secondary to the total peak. Moreover, quantification is possible from the standard curves of relaxation time and the combined surface area of the main and secondary peaks with the corresponding sugar concentration. However, it shows challenges in discrimination between α- and β-isomers. disaccharide TD-NMR water mobility T2 relaxometry characterization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Disaccharides, the major source of dietary carbohydrates, play a significant role in all aspects related to the food industry, such as processing, preservation, and quality measures of foods. They are composed of two monomers formed by a glycoside linkage between an anomeric carbon of one monosaccharide and any hydroxyl group of another monosaccharide (Pokrzywnicka & Koncki, 2018 ). The disaccharides that are most commonly used include saccharose, lactose, and maltose. Trehalose, although expensive, is also used as an additive during processing in the food industry. These four disaccharides have similar molecular formula and weight; however, they differ from each other based on their molecular structure and other physical properties (see Table 1 ). Table 1 Comparative properties between disaccharides (adapted from Chen et al., 2022 ; Pokrzywnicka & Koncki, 2018 ) Saccharose β-Lactose Maltose Trehalose Chemical formula C 12 H 22 O 11 C 12 H 22 O 11 C 12 H 22 O 11 C 12 H 22 O 11 Molecular weight (g/mol) 342.3 342.3 342.3 342.3 Structure Associated monomers with linkage Glucose + Fructose (C1–C2 bond) Galactose + Glucose (C1–C4 bond) Glucose + Glucose (C1–C4 bond) Glucose + Glucose (C1–C1 bond) Major source Sugarcane and sugar beet Milk and dairy Germinating seeds and hydrolysis of starch Organisms (bacteria, insects, fungi) and commercially produced from starch by enzyme catalysis Alternative name(s) Sucrose, table sugar Milk sugar Malt sugar - Solubility (g/g water at 23°C) 2.0 0.20 1.08 0.41 Reducing properties Non-reducing Reducing Reducing Non-reducing Some disaccharides are considered free sugars, which are intentionally added to foods and drinks during manufacturing, and these sugars cause non-communicable diseases (Bridge et al., 2024 ). Free sugar such as saccharose (also called sucrose or table sugar) is one of the concerning factors reported by the WHO (World Health Organization, 2015 ), overconsumption of which leads to several health issues such as obesity, fatty liver, and tooth decay (Qi & Tester, 2020 ). Therefore, many studies recommend reducing its intake to avoid these health concerns (Herforth et al., 2019 ; Muka et al., 2015 ). Additionally, many people are suffering from some disorders, such as lactose intolerance and galatosemia, to tolerate lactose and consequently suffer from abdominal pain, diarrhea, or other digestion-related problems (Yang et al., 2021 ). Lactose is the disaccharide of galactose and glucose monomers, which is found in mammalian milk and other dairy products (Conzuelo et al., 2010 ; da Costa et al., 2016 ; Yang et al., 2021 ). Maltose is another important and commonly used disaccharide produced by the hydrolysis of starch and naturally found in germinated seeds (BeMiller, 2019 ), requiring determination in alcoholic drinks, starch hydrolytes, and other cereal grains to understand the advancement of the fermentation process (Pokrzywnicka & Koncki, 2018 ). Although the widespread use of trehalose is limited because of its high cost, some unique properties of trehalose (for example, a very good stabilizer for protein and fat, excellent stability, and high glass-transition temperature) are gaining interest in being used in the food industry (Chen et al., 2022 ; Ohtake & Wang, 2011 ). Therefore, the significance of the identification and quantification of disaccharides in the food industry is endless. For example, labeling specifically in the package of processed products (such as the presence or absence of lactose in a lactose-free diet), checking nutritional analysis (such as the quantity of fructose in beverages), maintaining regulatory compliances (such as sugar in infant formula), and controlling the quality of food products (such as detection of adulterated sugar in honey) are some of them (Akyüz et al., 2021 ; Başar & Özdemir, 2018 ; Walker et al., 2014 ; Walker & Goran, 2015 ). Moreover, the added sugars influence different quality attributes during the processing of food products, such as color (such as caramel), texture (such as jam or jelly), and taste (such as sweetness) (Clemens et al., 2016 ). Several analytical methods are available to determine sugar samples, including high-performance liquid chromatography, gas chromatography-mass spectroscopy, paper chromatography, ion exclusion chromatography, near-infrared, medium-infrared, and Raman spectroscopy (Akyüz et al., 2021 ; Pokrzywnicka & Koncki, 2018 ). However, the challenges encountered in conventional techniques for determining sugar molecules, such as high cost, low selectivity, and long analyzing times, drive the investigation of advancements in alternative techniques to overcome these constraints (Akyüz et al., 2021 ). Nuclear magnetic resonance (NMR) is recognized as a powerful tool for characterizing sugar molecules (Bewley & Shahzad-ul‐Hussan, 2013 ; Buda et al., 2016 ; Schievano et al., 2017 ). Although high-frequency NMR dominates the market due to its strong magnetic field (1.5T), it requires more maintenance and installation, along with high energy consumption and the need for high-volume operation (Niumag Corporation, 2020 ). In contrast, low-frequency or time-domain NMR (TD-NMR) offers numerous advantages, such as shorter analyzing times (within a few seconds), lower setup and operation costs, reduced space requirements due to the presence of a permanent magnet, non-invasiveness, and less impact on the surrounding environment (Niumag Corporation, 2020 ). Therefore, this study aimed to investigate the applicability of a simplified version of 1 H TD-NMR to characterize the most commonly used disaccharides (such as saccharose, lactose, maltose, and trehalose) in binary solution (single sugar in water) by analyzing spin-spin (T 2 ) relaxation spectra. The study considers those disaccharides due to their same molecular formula and weight to explore the feasibility of this type of NMR. This investigation focused on characterizing the disaccharides with minimal time and analysis requirements, selecting a single sugar in solution to compensate for any possible effect that might occur in a complex system (the presence of multiple sugars), and thus aiming for a comprehensive understanding. 2. Materials and Methods 2.1. Preparation of disaccharide solutions Saccharose anhydrous (Redpath Sugar Ltd., Canada), maltose monohydrate (Ward's Science, United States), trehalose dihydrate (VWR, United States), α-lactose monohydrate (Sigma-Aldrich, United States), and β-lactose anhydrous (Sigma-Aldrich, United States) powders were of analytical grade and used for the preparation of disaccharide solutions. The quantity of added water to prepare the solution was adjusted, taking into account the presence of sugar hydrates. The solutions were prepared using deionized water with different concentrations by considering their solubility, as shown in Table 2 . The prepared sugar solutions were shaken properly and kept in airtight test tubes at 32°C for a minimum of one hour to reduce temperature fluctuations during relaxation time. Finally, the samples were ready to obtain a relaxation curve by scanning them through TD-NMR. Table 2 Disaccharide to water ratio for the preparation of sugar solutions Name of disaccharide Disaccharide content in sugar solution (g/100 g pure water) Saccharose 0, 5, 10, 15, 20, 30, 40, 50, 60 and 70 Maltose Trehalose 0, 5, 10, 15, 20, 30, 40, and 50 α-Lactose 0, 5, 10, 15, 20, and 25 β-Lactose 2.2. Spin-spin relaxometry by TD-NMR TD-NMR (PQ001-20-025V Pulsed NMR Analyzer manufactured by Niumag Corporation, China, was used to measure the spin-spin (transverse) relaxation time (T 2 ) distribution curve. Experiments were performed at a magnetic field intensity of 0.5 ± 0.08 Tesla (designed with the attached permanent magnet) and a constant temperature of 32°C. Experimental parameters were determined through initial Free Induction Decay (FID) tests, setting the frequency bandwidth (SW) (100 Hz), analog gain (RG1) (20 db), digital gain (DRG1) (3), preamplifier gain (PRG) (0), and radio frequency delay (RFD) time (0.25 ms). The Carr-Purcell-Meiboom-Gill (CPMG) sequence was processed at the following conditions: Simultaneous Iterative Reconstruction Technique (SIRT) within a time domain of 1 to 4000 ms (Carr & Purcell, 1954 ; Meiboom & Gill, 1958 ; Trampert & Leveque, 1990 ), signal-to-noise ratio (SNR) > 4000, intervals between observations 240 seconds. The artifact peaks were eliminated, and the final signals were normalization based on the scan number and sample mass. The triplicate measurement of each sample with five observations was conducted in this study. The data presented here was the mean ± standard deviation of the total 15 observations of each sample. 3. Results and Discussions 3.1. T 2 relaxation curve of disaccharides A T 2 distribution curve of saccharose, maltose, trehalose, and β-lactose is presented in Fig. 1 . The maximum concentrations of trehalose and lactose (50 g and 25 g/ 100 g of water respectively) were chosen to avoid saturation zone of each disaccharide. Two distinguishable peaks (proton population) were observed in this Fig., termed main and secondary at the relaxation times T 2Main and T 2Secondary , respectively. However, pure water showed only one main peak, with T 2Main ranging from 2630 to 2700 ms and no secondary peak, indicating more mobile behavior compared to bound water (typically found at short relaxation time) (Cheng et al., 2019 ; Li et al., 2014 ). When the quantity of disaccharides increased, the main peak appeared in a shorter relaxation time than the solution with less sugar content. It was clearly seen that the higher content of disaccharide in solution had an inverse relationship with T 2 relaxation time, as expected. For example, 5 g/100 g water solution of disaccharides exhibited the shoulder of the main peak at around 2100–2200 ms, whereas the highest added content of saccharose (70 g/100 g water), maltose (70 g/100 g water), trehalose (50 g/100 g water), and β-lactose (25 g/100 g water) showed at around 540, 255, 755, and 1245 ms, respectively. The higher the concentration, the more sugar molecules are able to associate in the water and interact with each other. This interaction, particularly at higher concentrations, is more likely between solid-solid (sugar-sugar) than solid-liquid (Ilhan et al., 2020 ). Additionally, the hydrogen bond formed between the hydrogen atom of the hydroxyl group in sugar and water exhibits greater strength compared to the hydrogen bonding occurring between water molecules (Edelman et al., 2015 ), indicating a more bound (less mobile) condition than pure water. However, the peak (longer T 2 ) obtained for a less concentrated solution might be attributed to both solid-solid and solid-liquid interactions (Ilhan et al., 2020 ), behaving like an immobilized condition. On the other hand, the signal intensity, as shown in the Y-axis of Fig. 1 , is proportional to the increased quantity of disaccharides. The signal intensity has been normalized considering the total mass of the solution and presented in an arbitrary unit (a.u.). An exception was found for maltose solution, where two non-equal secondary peaks were noticed; unlike other sugar solutions, the secondary peak of maltose disintegrated into two non-uniform peaks. The secondary peaks were not completely connected and both moved together when the sugar content increased in the solution. Moreover, proton relaxed faster (shorter T 2 ) in maltose solution as compared to other sugar solutions. For example, secondary peak appeared for maltose and ranged from 10–190 ms, particularly for peak 1: 20–190 ms and peak 2: 10–30 ms, whereas saccharose, trehalose, and β-lactose showed secondary peak ranged between 100–260 ms, 130–330 ms, and 300–460 ms, respectively. The main peak also showed similar trends for all the sugars. This behavior of maltose aligns with the study conducted by Pocan et al. ( 2022 ), who observed the behavior of different sugar syrups applied in the preparation of “Turkish delight.” They revealed that corn syrup containing delights had less T 2 due to the presence of a considerable amount of maltose, which is a more humectant (indicating more water-binding capacity). In this study, both α- and β-lactose were considered for analyzing the T 2 relaxation curve, and the relaxation time for the main and secondary peaks was almost similar, as shown in Table 3 . Therefore, further analysis is shown for β-lactose only, and throughout the entire manuscript, any mention of lactose refers to β-lactose. Table 3 T 2 relaxation time for α- and β-lactose Isomer Lactose to water ratio (g/100 g) 5 10 15 20 25 T 2 of main peak (ms) α-Lactose 2741 ± 29 2537 ± 29 2259 ± 55 2061 ± 68 1812 ± 00 β-Lactose 2744 ± 06 2413 ± 34 2135 ± 22 1952 ± 33 1782 ± 36 T 2 of secondary peak (ms) α-Lactose 326 ± 25 394 ± 15 375 ± 12 360 ± 10 327 ± 06 β-Lactose 314 ± 28 369 ± 12 364 ± 08 348 ± 07 328 ± 00 3.2. Behavior of peaks observed in disaccharides solutions The percentage of the total surface area of the main and secondary peaks corresponding to the disaccharide content in the sugar solution is presented in Fig. 2 . In this Fig., S T , S M , S S indicate the total, main, and secondary peak surface areas, respectively. The Fig. illustrates that an increasing amount of disaccharide content generates a secondary peak with a concave upward shape (except maltose), indicating a positive correlation between disaccharide concentration and the increase rate of the secondary peak and an inverse correlation with the main peak. In another way, it can be said that the hydrogen atom in a sugar solution with a low sugar-to-water ratio behaves like the hydrogen atom in pure water. In case of maltose, almost linear-shaped upward secondary peaks (S s1 and S S2 ) relative to the total peak were observed. However, the surface area of S S2 was much smaller than S s1 . This deviation became larger with an increased maltose-to-water ratio, indicating stronger increase of S S1 with maltose concentration. Another noticeable observation is the value of S M /S T , which can provide a preliminary hypothesis related to disaccharide solubility. For example, higher S M /S T value can be linked to higher solubility of saccharose, trehalose, and lactose. However, the behavior of maltose is excluded from this hypothesis. A clear distinction between disaccharides in terms of relaxation times of the main and secondary peaks can be seen in Fig. 3 . At the initial concentration, until a sugar-to-water ratio of 10 g/100 g, the main peak of all the disaccharides appeared at almost the same T 2 , gradually declining with increasing sugar content. However, T 2 for maltose sharply decreased with increasing sugar content, followed by trehalose, saccharose, and lactose, respectively. This increased amount of sugar limits the availability of free water molecules, hence reducing mobility and relaxation time. Similarly, at the initial concentration until a sugar-to-water ratio of 20 g/100 g, the secondary peak showed a larger standard deviation for saccharose, trehalose, and maltose. This is probably due to the more compatible nature of water when the solute is at a low concentration (Aroulmoji et al., 2000 ), indicating increased freedom of protons to exist in the main peak. The T 2 for the secondary peak follows the order: lactose > trehalose > saccharose > maltose. Although these sugars have similar molecular weight and the same number of OH groups, their hydration number, hydrogen bonding between water-sugar molecules, sugar-sugar molecule clustering, and viscosity differ, influencing the interaction of sugar molecules with water and hence the magnetic environment. Studies have revealed that the hydration of disaccharides follows the order of lactose > trehalose > maltose > sucrose (Furuki et al., 2009 ; Lerbret et al., 2005 ; Pocan et al., 2022 ). However, the behavior of maltose is hypothesized to involve intermolecular (sugar-sugar) interactions through hydrogen bonding. A study conducted by Lerbret et al., ( 2005 ) found that the intermolecular hydrogen bonding of maltose is higher than that of trehalose and sucrose across all concentrations ranging from 33–66 wt%. Moreover, the intramolecular bonding (sugar-water) of maltose was found to be less than that of sucrose and higher than that of trehalose, indicating that maltose has more OH groups available to interact with other maltose molecules than water molecules (Lerbret et al., 2005 ). Therefore, in sugar network formation, maltose tends to organize itself into larger clusters compared to sucrose and trehalose (Lerbret et al., 2005 ). Our hypothesis is that the main peak belongs to the signal generated from the proton of hydrogen bonding between sugar and water molecules, while the secondary peak belongs to the hydrogen bonding between sugar molecules. The stronger sugar-sugar bonding of maltose may lead to the generation of secondary peaks with less mobility and reduced relaxation time. Additionally, the formation of larger sugar-sugar bonded clusters of maltose makes the sugar-water solution less homogeneous in terms of the number of peaks compared to trehalose and sucrose (Lerbret et al., 2005 ). Therefore, it is assumed that the lower homogeneity, in terms of the number of peaks, of maltose might influence the synchronization of 1 H in generating two secondary peaks. On the other hand, lactose, with lower solubility compared to other disaccharides studied here, is highly hydrophilic and has a stronger ability to interact with water molecules (Wijayasinghe et al., 2015 ). Consequently, a low concentration of lactose molecules has more available OH in solution and fewer bound inter- and intra-molecular networks, implying greater mobility and higher T 2 compared to others. Regarding the T 2Secondary of trehalose and sucrose, trehalose required more time to relax than sucrose, possibly due to the viscosity of the sugar in the water solution. At higher concentrations, the difference in viscosity between trehalose and sucrose was significantly greater compared to lower concentrations, with trehalose exhibiting markedly higher viscosity levels than sucrose (Galmarini et al., 2011 ). Higher viscosity values of sugar molecules tend to accumulate more sugar molecules in solution, resulting in a less mobile situation and reduced T 2 values (Aroulmoji et al., 2012 ; Lai & Schmidt, 1993 ). Another study found that trehalose has a stronger and more rigid structure with water than a sucrose-water mixture (Magazu et al., 2003 ), supporting the findings that sucrose requires more T 2 to display the main peak (due to bonding between sugar and water) and less T 2 for the secondary peak (due to bonding between sugar molecules) compared to trehalose. A linear regression equation was observed when plotting disaccharide content in water as the independent variable and the ratio of relaxation time between the main peak of the disaccharide and water as the dependent variable, as shown in Fig. 4 . The values of the dependent variables were considered on a logarithmic basis for obtaining better correlation. Each sugar displayed a high regression coefficient (R 2 = 0.99), indicating excellent fittings and a strong dependency of the dataset of the main peak on the concentration of disaccharide. 3.3. Analysis of peak area and width corresponding to disaccharide content in the solution Figure 5 illustrates the impact of disaccharide content on the total surface area of disaccharide (S T ) compared to pure water (S 0 ), where S T = S Main + S Secondary . The ratio of S T /S 0 represents the change of the total surface of the solution while compensating for the effect of signal amplification coefficients used by the user of NMR. Both normalized and non-normalized surface area shown in the Fig. have a good fit with R 2 ≥ 0.98, indicating a strong correlation with the concentration of disaccharides. These findings also represent the increment rate of hydrogen atoms in the solution with the increasing amount of disaccharide content relative to the initial hydrogen in water and can be expressed as follows: $$\:\frac{{S}_{T}}{{S}_{0}}=\frac{{{H}_{Water}+\:H}_{Sugar}}{{H}_{Water}}$$ The main peaks observed in the disaccharide solution were further analyzed compared to pure water considering the peak width (∆T) (the gap of T 2 between onset and endpoint) and presented in Fig. 6 . Each case explained that with the increase in the concentration of disaccharides, the main peak width decreased compared to the main peak observed for pure water. However, at the initial condition (5 g sugar/100 g water), all the sugars showed similar behavior to water, indicating that at very low concentrations, there is availability of binding positions of water molecules with sugar molecules. From Fig. 6 , it is noticeable that the deviation of ∆T for the secondary peak was higher until the sugar content reached 20 g/100 g water. This deviation in initial cases might be due to the greater freedom of forming hydrogen bonding between sugar and water molecules due to sufficient OH groups in the solution. The rationale behind the varied behavior of different disaccharides in water solutions has been previously explained. However, NMR users may also utilize this type of analysis to identify disaccharides in unknown solutions. 3.4. Correlation of peak characteristics and hydrogen atom in the solution Figure 7 illustrates a linear relationship between hydrogen content and signal intensity as observed in TD-NMR. The intercept values (ranging from 140–356 a.u.) of the linear regression equations for all disaccharides are negligible when compared to the substantial slopes (ranging from 2013–2570 a.u.), confirming that the signal intensity primarily originates from 1 H. According to our hypothesis, the generation of secondary peaks results from sugar-sugar bonding rather than sugar-water interactions. Therefore, the distraction of the main peak signal from the total signal and the subsequent appearance of secondary peak(s) are attributed to the hydrogen bonding among disaccharide molecules and their interactions with water. Notably, saccharose and trehalose exhibit a similar diversion point of the main peak at a sugar content of 15 g/100 g, whereas lactose diverges earlier at 10 g/100 g. In Fig. 7 , the fastest diversion was observed in maltose solution, where 5 g/100 g can be considered a critical concentration (the onset of diversion). The main peaks of saccharose, trehalose, and lactose exhibited convex upward shapes, while secondary peaks displayed concave upward growth with increasing concentration. This finding is in good agreement with numerous other studies, which have confirmed that the addition of sugar significantly retards water dynamics (mobility) (Heyden et al., 2008 ; Lerbret et al., 2011 ; Paolantoni et al., 2009 ; Paolantoni et al., 2007 ). This slowing-down process (reducing water mobility) depends on the total number of hydrogen bonds formed with water (Magazu et al., 2001 ) and restricts the flexibility or mobility of water molecules. Furthermore, another influential parameter for slowing down water mobility is the percolation of the hydrogen bonding network, which increases sharply with the threshold concentration of sugar in the solution (Bordat et al., 2004 ). At this threshold concentration, the mean cluster size of water-water hydrogen bonding prominently begins to decrease, and sugar starts to influence water molecules (Bordat et al., 2004 ). In this Fig., the diversion of the main peak from the total peak signal and the appearance of secondary peak(s) with increasing sugar concentration indicate the beginning of interaction between sugar molecules in water, leading to a decrease in water-water interaction. Lee et al. ( 2005 ) mentioned that sugar-water bonding is more stable than water-water bonding, which aligns with our findings, and thus the increase of secondary peaks with concentration. Notably, the increase of the secondary peak was more pronounced for trehalose compared to sucrose. The formation of larger cluster sizes for trehalose may drive this behavior, as the association of sugar molecules generates stronger bonding, while sucrose creates smaller cluster sizes (Lerbret et al., 2011 ; Lerbret et al., 2005 ). On the other hand, maltose exhibited different behavior, producing two secondary peaks with almost linear behavior. As described earlier, the strong intermolecular bonding between maltose molecules might divert the primary peak faster than other disaccharides. However, the comprehension of maltose displaying a completely different trend with two secondary peaks is still unclear, requiring further investigation for a comprehensive understanding. 3.5. Applicability of TD-NMR in characterizing disaccharides This study aimed to address a general question: "Can we use TD-NMR to characterize disaccharides in a simple and rapid way from unknown samples in both solid (dried powder) and liquid (solution) form, with known or unknown concentrations?" The answer to this question is summarized in Fig. 8 , which outlines the characterization of disaccharides. Firstly, all samples (relevant disaccharides in this study) require the preparation of a solution of 20 g/100 g pure water to compensate for their wide range of T 2 deviations below this concentration. Users can identify the sugar samples based on their secondary peak. Analyzing either the observation of relaxation time or calculating the ratio of the surface area of the secondary peak to the total peak (S S /S T ) facilitates the characterization of the sugar sample in solution. Maltose is easily characterized by observing its two secondary peaks. If S S /S T is less than 2.5%, it is saccharose; if it falls between 2.5 to 4.5, then it is trehalose; and if it exceeds 4.5, then it is lactose. In terms of observing T 2 relaxation time, saccharose, trehalose, and lactose can be identified as 290 ms, respectively. Using this method, TD-NMR users can compile a list of S S /S T and T 2 of different sugar samples for easier and more rapid identification in a cost-effective manner. 4. Conclusion This study investigated the T 2 relaxation distribution curve of saccharose, maltose, trehalose, and lactose in solutions, aiming to assess the potential of a simplified version of TD-NMR, which encompasses fundamental options, in characterizing disaccharides. Saccharose, trehalose, and lactose exhibit two distinguishable peaks, considered the main and secondary peaks, while pure water displays no secondary peaks. In contrast, maltose shows two secondary peaks along with the main peak. The findings confirm that as the disaccharide content increases, the relaxation time decreases, indicating reduced mobility due to increased interactions between sugar molecules and water. It is hypothesized that the main peak originates from proton bonding in water-water or sugar-water interactions, while the secondary peak arises from sugar-sugar interactions. The relaxation time for the secondary peak of saccharose, trehalose, β-lactose, and maltose ranged from 100–260 ms, 130–330 ms, 300–460 ms, and 10–190 ms, respectively. However, factors such as hydrogen bonding between sugar molecules, viscosity, hydration number, and solubility may influence relaxation time. The unique behavior of maltose compared to other disaccharides is hypothesized to be due to stronger intermolecular bonding. Further investigation is needed to determine the exact reasons for the differing trends observed in maltose, with the possibility that spin-lattice (T 1 ) relaxation analysis could provide greater insights. However, the standard curves of T 2 , or the surface area of the main combined with secondary peaks, as a function of sugar concentration obtained from the analysis of the peak are applicable for the quantification of disaccharides in binary mixtures (water and one disaccharide). Overall, this study enhances our comprehension of the feasibility and reliability of TD-NMR as an effective tool for characterizing disaccharide samples in both unknown powder and homogeneous sugar solutions, primarily by analyzing relaxation time or the surface area of the secondary peak. However, the machine faces challenges in discriminating between α- and β-isomers. Finally, further exploration in characterizing monosaccharides will improve the comprehensibility to assess the applicability of this simplified version of TD-NMR. Declarations The authors declare no conflict of interest. Funding statement Natural Sciences and Engineering Research Council of Canada Author Contribution Afroza Sultana: Writing - original draft preparation; Formal analysis and investigation; Ali Asghari: Conceptualization, Methodology, Formal analysis and investigation; Writing - review and editing; Seddik Khalloufi: Conceptualization, Methodology, Formal analysis and investigation; Writing - review and editing; Supervision, Validation, Visualization Data availability Data will be available upon request. References Akyüz E, Başkan KS, Tütem E, Apak R (2021) High performance liquid chromatographic method with post-column detection for quantification of reducing sugars in foods. J Chromatogr A 1660:462664 Aroulmoji V, Mathlouthi M, Birch G (2000) Hydration properties of Na, K, Mg gluconates and gluconate/sucrose mixtures and their possible taste effect. 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J Chem Phys, 122 (20) Lerbret A, Affouard F, Bordat P, Hedoux A, Guinet Y, Descamps M (2011) Slowing down of water dynamics in disaccharide aqueous solutions. J Non-cryst Solids 357(2):695–699 Lerbret A, Bordat P, Affouard F, Descamps M, Migliardo F (2005) How homogeneous are the trehalose, maltose, and sucrose water solutions? An insight from molecular dynamics simulations. J Phys Chem B 109(21):11046–11057 Li W, Wang P, Xu X, Xing T, Zhou G (2014) Use of low-field nuclear magnetic resonance to characterize water properties in frozen chicken breasts thawed under high pressure. Eur Food Res Technol 239:183–188 Magazu S, Migliardo F, Mondelli C, Romeo G (2003) Temperature dependence of mean square displacement by IN13: a comparison between trehalose and sucrose water mixtures. Chem Phys 292(2–3):247–251 Magazu S, Villari V, Migliardo P, Maisano G, Telling M (2001) Diffusive Dynamics of Water in the Presence of Homologous Disaccharides: A Comparative Study by Quasi Elastic Neutron Scattering. IV. J Phys Chem B 105(9):1851–1855 Meiboom S, Gill D (1958) Modified spin-echo method for measuring nuclear relaxation times. Rev Sci Instrum 29(8):688–691 Muka T, Imo D, Jaspers L, Colpani V, Chaker L, van der Lee SJ, Mendis S, Chowdhury R, Bramer WM, Falla A (2015) The global impact of non-communicable diseases on healthcare spending and national income: a systematic review. Eur J Epidemiol 30:251–277 Niumag Corporation (2020) https://www.nmranalyzer.com/why-choose-lf-nmr-low-field-nmr.html Ohtake S, Wang YJ (2011) Trehalose: current use and future applications. J Pharm Sci 100(6):2020–2053 Paolantoni M, Comez L, Gallina M, Sassi P, Scarponi F, Fioretto D, Morresi A (2009) Light scattering spectra of water in trehalose aqueous solutions: Evidence for two different solvent relaxation processes. J Phys Chem B 113(22):7874–7878 Paolantoni M, Sassi P, Morresi A, Santini S (2007) Hydrogen bond dynamics and water structure in glucose-water solutions by depolarized Rayleigh scattering and low-frequency Raman spectroscopy. J Chem Phys, 127 (2) Pocan P, Grunin L, Oztop MH (2022) Effect of Different Syrup Types on Turkish Delights (Lokum): A TD-NMR Relaxometry Study. ACS Food Sci Technol 2(12):1819–1831 Pokrzywnicka M, Koncki R (2018) Disaccharides determination: A review of analytical methods. Crit Rev Anal Chem 48(3):186–213 Qi X, Tester RF (2020) Lactose, maltose, and sucrose in health and disease. Mol Nutr Food Res 64(8):1901082 Schievano E, Tonoli M, Rastrelli F (2017) NMR quantification of carbohydrates in complex mixtures. A challenge on honey. Anal Chem 89(24):13405–13414 Trampert J, Leveque JJ (1990) Simultaneous iterative reconstruction technique: Physical interpretation based on the generalized least squares solution. J Geophys Research: Solid Earth 95(B8):12553–12559 Walker RW, Dumke KA, Goran MI (2014) Fructose content in popular beverages made with and without high-fructose corn syrup. Nutrition 30(7–8):928–935 Walker RW, Goran MI (2015) Laboratory determined sugar content and composition of commercial infant formulas, baby foods and common grocery items targeted to children. Nutrients 7(7):5850–5867 Wijayasinghe R, Vasiljevic T, Chandrapala J (2015) Water-lactose behavior as a function of concentration and presence of lactic acid in lactose model systems. J Dairy Sci 98(12):8505–8514 World Health Organization (2015) Guideline: sugars intake for adults and children. World Health Organization Yang J, Rainville P, Liu K, Pointer B (2021) Determination of lactose in low-lactose and lactose-free dairy products using LC-MS. J Food Compos Anal 100:103824 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 06 Aug, 2024 Reviews received at journal 05 Aug, 2024 Reviews received at journal 04 Aug, 2024 Reviews received at journal 01 Aug, 2024 Reviews received at journal 01 Aug, 2024 Reviews received at journal 31 Jul, 2024 Reviews received at journal 30 Jul, 2024 Reviews received at journal 30 Jul, 2024 Reviews received at journal 29 Jul, 2024 Reviewers agreed at journal 28 Jul, 2024 Reviewers agreed at journal 27 Jul, 2024 Reviewers agreed at journal 27 Jul, 2024 Reviewers agreed at journal 24 Jul, 2024 Reviewers agreed at journal 24 Jul, 2024 Reviewers agreed at journal 24 Jul, 2024 Reviewers agreed at journal 23 Jul, 2024 Reviewers agreed at journal 23 Jul, 2024 Reviewers agreed at journal 22 Jul, 2024 Reviewers agreed at journal 22 Jul, 2024 Reviewers agreed at journal 22 Jul, 2024 Reviewers invited by journal 22 Jul, 2024 Editor assigned by journal 18 Jul, 2024 Submission checks completed at journal 18 Jul, 2024 First submitted to journal 18 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4764910","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":336751886,"identity":"ecdb006e-71d1-4d81-90c8-89dc70626513","order_by":0,"name":"Afroza Sultana","email":"","orcid":"","institution":"Laval University","correspondingAuthor":false,"prefix":"","firstName":"Afroza","middleName":"","lastName":"Sultana","suffix":""},{"id":336751887,"identity":"2d031052-36a5-46a5-ab9d-994f9a1ed0c5","order_by":1,"name":"Ali Asghari","email":"","orcid":"","institution":"Laval University","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Asghari","suffix":""},{"id":336751888,"identity":"a1f5769b-762c-4ed0-a3ad-9c2f41b3ea26","order_by":2,"name":"Seddik Khalloufi","email":"data:image/png;base64,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","orcid":"","institution":"Laval University","correspondingAuthor":true,"prefix":"","firstName":"Seddik","middleName":"","lastName":"Khalloufi","suffix":""}],"badges":[],"createdAt":"2024-07-18 20:07:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4764910/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4764910/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62397776,"identity":"b59ac874-c44c-4438-a9e7-0d29f850d0d1","added_by":"auto","created_at":"2024-08-13 17:46:08","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":166893,"visible":true,"origin":"","legend":"\u003cp\u003eNMR relaxation curve for disaccharide solution (g disaccharide per 100 g deionized water)\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4764910/v1/336b05c197ffc863772623d9.jpg"},{"id":62397778,"identity":"9fb69346-163a-467e-95dc-c67585a40248","added_by":"auto","created_at":"2024-08-13 17:46:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96074,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent surface area ratios as a function sugar concentration (S\u003csub\u003eM\u003c/sub\u003e: Surface area of main peak; S\u003csub\u003eS\u003c/sub\u003e: Surface area of secondary peak; S\u003csub\u003eT\u003c/sub\u003e: Total surface area of peaks)\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4764910/v1/bf8fe9b7fa7032e20d4b6547.jpg"},{"id":62397303,"identity":"7da7f424-89b4-4cb7-82b9-26a70d498c6a","added_by":"auto","created_at":"2024-08-13 17:38:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":48851,"visible":true,"origin":"","legend":"\u003cp\u003eVariation of relaxation time as a function of sugar concentration\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4764910/v1/a6c1cf4a3c42a11ce1aaacde.jpg"},{"id":62397307,"identity":"3db92510-4e61-42de-a6cd-b331e126fa51","added_by":"auto","created_at":"2024-08-13 17:38:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":78623,"visible":true,"origin":"","legend":"\u003cp\u003eLinear correlation between relaxation time of the main peak and sugar concentration\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4764910/v1/140fef054d67579547fc979c.jpg"},{"id":62397310,"identity":"174e769a-32c8-49cf-80dc-4af4e4f0a2f2","added_by":"auto","created_at":"2024-08-13 17:38:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":102162,"visible":true,"origin":"","legend":"\u003cp\u003eLinear correlation between total surface area and sugar concentration.\u003csub\u003e \u003c/sub\u003eS\u003csub\u003eT\u003c/sub\u003e: total surface area of the solution, and S\u003csub\u003e0\u003c/sub\u003e: the surface area of water\u003cstrong\u003e\u003cbr\u003e\n\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4764910/v1/7edc13e8fbd6925d312dc6e8.jpg"},{"id":62397305,"identity":"08c8e6ba-4282-4ca6-9bba-f89a03dd81e4","added_by":"auto","created_at":"2024-08-13 17:38:08","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":99766,"visible":true,"origin":"","legend":"\u003cp\u003eThe width (∆T) (the gap of T\u003csub\u003e2\u003c/sub\u003e between onset and endpoint) as a function of sugar concentrations\u003cstrong\u003e\u003cbr\u003e\n\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4764910/v1/e5d5f3a29e221ea0f8405b51.jpg"},{"id":62397308,"identity":"41dd227b-2165-471b-892e-45092ce73133","added_by":"auto","created_at":"2024-08-13 17:38:08","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":114391,"visible":true,"origin":"","legend":"\u003cp\u003eSurface areas of different peaks as a function hydrogen content in binary mixtures of sugars (water and one disaccharide)\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4764910/v1/275ba76d1a3cf328d8bbf66b.jpg"},{"id":62397777,"identity":"c6613182-9c1c-4ea2-976d-ce0d935a385b","added_by":"auto","created_at":"2024-08-13 17:46:08","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":133821,"visible":true,"origin":"","legend":"\u003cp\u003eApplicability of TD-NMR to characterize disaccharides by analyzing secondary peak of binary mixtures (water and one disaccharide)\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4764910/v1/3c7016eba804f5d662be2657.jpg"},{"id":62398007,"identity":"42d46403-64f4-4375-a527-c9a39acff701","added_by":"auto","created_at":"2024-08-13 17:54:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1466626,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4764910/v1/638d7141-c19f-4bc9-96ba-a41a238e5b22.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A straightforward method for disaccharide characterization by a simplified version of time domain nuclear magnetic resonance","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDisaccharides, the major source of dietary carbohydrates, play a significant role in all aspects related to the food industry, such as processing, preservation, and quality measures of foods. They are composed of two monomers formed by a glycoside linkage between an anomeric carbon of one monosaccharide and any hydroxyl group of another monosaccharide (Pokrzywnicka \u0026amp; Koncki, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). The disaccharides that are most commonly used include saccharose, lactose, and maltose. Trehalose, although expensive, is also used as an additive during processing in the food industry. These four disaccharides have similar molecular formula and weight; however, they differ from each other based on their molecular structure and other physical properties (see Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComparative properties between disaccharides\u003c/p\u003e\n \u003cdiv class=\"Credit\"\u003e\n \u003cp\u003e(adapted from Chen et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pokrzywnicka \u0026amp; Koncki, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSaccharose\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eβ-Lactose\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMaltose\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTrehalose\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChemical formula\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMolecular weight (g/mol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e342.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e342.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e342.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e342.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStructure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n 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\"\u003e\u003c/p\u003e\n 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAssociated monomers with linkage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlucose + Fructose (C1–C2 bond)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGalactose + Glucose (C1–C4 bond)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlucose + Glucose (C1–C4 bond)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlucose + Glucose (C1–C1 bond)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMajor source\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSugarcane and sugar beet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMilk and dairy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGerminating seeds and hydrolysis of starch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOrganisms (bacteria, insects, fungi) and commercially produced from starch by enzyme catalysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlternative name(s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSucrose, table sugar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMilk sugar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMalt sugar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSolubility (g/g water at 23°C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eReducing properties\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNon-reducing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eReducing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eReducing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNon-reducing\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eSome disaccharides are considered free sugars, which are intentionally added to foods and drinks during manufacturing, and these sugars cause non-communicable diseases (Bridge et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). Free sugar such as saccharose (also called sucrose or table sugar) is one of the concerning factors reported by the WHO (World Health Organization, \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e), overconsumption of which leads to several health issues such as obesity, fatty liver, and tooth decay (Qi \u0026amp; Tester, \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, many studies recommend reducing its intake to avoid these health concerns (Herforth et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Muka et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Additionally, many people are suffering from some disorders, such as lactose intolerance and galatosemia, to tolerate lactose and consequently suffer from abdominal pain, diarrhea, or other digestion-related problems (Yang et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Lactose is the disaccharide of galactose and glucose monomers, which is found in mammalian milk and other dairy products (Conzuelo et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; da Costa et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yang et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Maltose is another important and commonly used disaccharide produced by the hydrolysis of starch and naturally found in germinated seeds (BeMiller, \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e), requiring determination in alcoholic drinks, starch hydrolytes,\u003c/p\u003e\n\u003cp\u003eand other cereal grains to understand the advancement of the fermentation process (Pokrzywnicka \u0026amp; Koncki, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Although the widespread use of trehalose is limited because of its high cost, some unique properties of trehalose (for example, a very good stabilizer for protein and fat, excellent stability, and high glass-transition temperature) are gaining interest in being used in the food industry (Chen et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ohtake \u0026amp; Wang, \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). Therefore, the significance of the identification and quantification of disaccharides in the food industry is endless. For example, labeling specifically in the package of processed products (such as the presence or absence of lactose in a lactose-free diet), checking nutritional analysis (such as the quantity of fructose in beverages), maintaining regulatory compliances (such as sugar in infant formula), and controlling the quality of food products (such as detection of adulterated sugar in honey) are some of them (Akyüz et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Başar \u0026amp; Özdemir, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Walker et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e; Walker \u0026amp; Goran, \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Moreover, the added sugars influence different quality attributes during the processing of food products, such as color (such as caramel), texture (such as jam or jelly), and taste (such as sweetness) (Clemens et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eSeveral analytical methods are available to determine sugar samples, including high-performance liquid chromatography, gas chromatography-mass spectroscopy, paper chromatography, ion exclusion chromatography, near-infrared, medium-infrared, and Raman spectroscopy (Akyüz et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Pokrzywnicka \u0026amp; Koncki, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, the challenges encountered in conventional techniques for determining sugar molecules, such as high cost, low selectivity, and long analyzing times, drive the investigation of advancements in alternative techniques to overcome these constraints (Akyüz et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Nuclear magnetic resonance (NMR) is recognized as a powerful tool for characterizing sugar molecules (Bewley \u0026amp; Shahzad-ul‐Hussan, \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e; Buda et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Schievano et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). Although high-frequency NMR dominates the market due to its strong magnetic field (1.5T), it requires more maintenance and installation, along with high energy consumption and the need for high-volume operation (Niumag Corporation, \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). In contrast, low-frequency or time-domain NMR (TD-NMR) offers numerous advantages, such as shorter analyzing times (within a few seconds), lower setup and operation costs, reduced space requirements due to the presence of a permanent magnet, non-invasiveness, and less impact on the surrounding environment (Niumag Corporation, \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eTherefore, this study aimed to investigate the applicability of a simplified version of \u003csup\u003e1\u003c/sup\u003eH TD-NMR to characterize the most commonly used disaccharides (such as saccharose, lactose, maltose, and trehalose) in binary solution (single sugar in water) by analyzing spin-spin (T\u003csub\u003e2\u003c/sub\u003e) relaxation spectra. The study considers those disaccharides due to their same molecular formula and weight to explore the feasibility of this type of NMR. This investigation focused on characterizing the disaccharides with minimal time and analysis requirements, selecting a single sugar in solution to compensate for any possible effect that might occur in a complex system (the presence of multiple sugars), and thus aiming for a comprehensive understanding.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Preparation of disaccharide solutions\u003c/h2\u003e \u003cp\u003eSaccharose anhydrous (Redpath Sugar Ltd., Canada), maltose monohydrate (Ward's Science, United States), trehalose dihydrate (VWR, United States), α-lactose monohydrate (Sigma-Aldrich, United States), and β-lactose anhydrous (Sigma-Aldrich, United States) powders were of analytical grade and used for the preparation of disaccharide solutions. The quantity of added water to prepare the solution was adjusted, taking into account the presence of sugar hydrates. The solutions were prepared using deionized water with different concentrations by considering their solubility, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The prepared sugar solutions were shaken properly and kept in airtight test tubes at 32\u0026deg;C for a minimum of one hour to reduce temperature fluctuations during relaxation time. Finally, the samples were ready to obtain a relaxation curve by scanning them through TD-NMR.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDisaccharide to water ratio for the preparation of sugar solutions\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName of disaccharide\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDisaccharide content in sugar solution (g/100 g pure water)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSaccharose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0, 5, 10, 15, 20, 30, 40, 50, 60 and 70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaltose\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrehalose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0, 5, 10, 15, 20, 30, 40, and 50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eα-Lactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0, 5, 10, 15, 20, and 25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-Lactose\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Spin-spin relaxometry by TD-NMR\u003c/h2\u003e \u003cp\u003eTD-NMR (PQ001-20-025V Pulsed NMR Analyzer manufactured by Niumag Corporation, China, was used to measure the spin-spin (transverse) relaxation time (T\u003csub\u003e2\u003c/sub\u003e) distribution curve. Experiments were performed at a magnetic field intensity of 0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 Tesla (designed with the attached permanent magnet) and a constant temperature of 32\u0026deg;C. Experimental parameters were determined through initial Free Induction Decay (FID) tests, setting the frequency bandwidth (SW) (100 Hz), analog gain (RG1) (20 db), digital gain (DRG1) (3), preamplifier gain (PRG) (0), and radio frequency delay (RFD) time (0.25 ms). The Carr-Purcell-Meiboom-Gill (CPMG) sequence was processed at the following conditions: Simultaneous Iterative Reconstruction Technique (SIRT) within a time domain of 1 to 4000 ms (Carr \u0026amp; Purcell, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1954\u003c/span\u003e; Meiboom \u0026amp; Gill, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1958\u003c/span\u003e; Trampert \u0026amp; Leveque, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), signal-to-noise ratio (SNR)\u0026thinsp;\u0026gt;\u0026thinsp;4000, intervals between observations 240 seconds. The artifact peaks were eliminated, and the final signals were normalization based on the scan number and sample mass. The triplicate measurement of each sample with five observations was conducted in this study. The data presented here was the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of the total 15 observations of each sample.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussions","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.1. T\u003csub\u003e2\u003c/sub\u003e relaxation curve of disaccharides\u003c/h2\u003e \u003cp\u003eA T\u003csub\u003e2\u003c/sub\u003e distribution curve of saccharose, maltose, trehalose, and β-lactose is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The maximum concentrations of trehalose and lactose (50 g and 25 g/ 100 g of water respectively) were chosen to avoid saturation zone of each disaccharide. Two distinguishable peaks (proton population) were observed in this Fig., termed main and secondary at the relaxation times T\u003csub\u003e2Main\u003c/sub\u003e and T\u003csub\u003e2Secondary\u003c/sub\u003e, respectively. However, pure water showed only one main peak, with T\u003csub\u003e2Main\u003c/sub\u003e ranging from 2630 to 2700 ms and no secondary peak, indicating more mobile behavior compared to bound water (typically found at short relaxation time) (Cheng et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen the quantity of disaccharides increased, the main peak appeared in a shorter relaxation time than the solution with less sugar content. It was clearly seen that the higher content of disaccharide in solution had an inverse relationship with T\u003csub\u003e2\u003c/sub\u003e relaxation time, as expected. For example, 5 g/100 g water solution of disaccharides exhibited the shoulder of the main peak at around 2100\u0026ndash;2200 ms, whereas the highest added content of saccharose (70 g/100 g water), maltose (70 g/100 g water), trehalose (50 g/100 g water), and β-lactose (25 g/100 g water) showed at around 540, 255, 755, and 1245 ms, respectively. The higher the concentration, the more sugar molecules are able to associate in the water and interact with each other. This interaction, particularly at higher concentrations, is more likely between solid-solid (sugar-sugar) than solid-liquid (Ilhan et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, the hydrogen bond formed between the hydrogen atom of the hydroxyl group in sugar and water exhibits greater strength compared to the hydrogen bonding occurring between water molecules (Edelman et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), indicating a more bound (less mobile) condition than pure water. However, the peak (longer T\u003csub\u003e2\u003c/sub\u003e) obtained for a less concentrated solution might be attributed to both solid-solid and solid-liquid interactions (Ilhan et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), behaving like an immobilized condition. On the other hand, the signal intensity, as shown in the Y-axis of Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, is proportional to the increased quantity of disaccharides. The signal intensity has been normalized considering the total mass of the solution and presented in an arbitrary unit (a.u.).\u003c/p\u003e \u003cp\u003eAn exception was found for maltose solution, where two non-equal secondary peaks were noticed; unlike other sugar solutions, the secondary peak of maltose disintegrated into two non-uniform peaks. The secondary peaks were not completely connected and both moved together when the sugar content increased in the solution. Moreover, proton relaxed faster (shorter T\u003csub\u003e2\u003c/sub\u003e) in maltose solution as compared to other sugar solutions. For example, secondary peak appeared for maltose and ranged from 10\u0026ndash;190 ms, particularly for peak 1: 20\u0026ndash;190 ms and peak 2: 10\u0026ndash;30 ms, whereas saccharose, trehalose, and β-lactose showed secondary peak ranged between 100\u0026ndash;260 ms, 130\u0026ndash;330 ms, and 300\u0026ndash;460 ms, respectively. The main peak also showed similar trends for all the sugars. This behavior of maltose aligns with the study conducted by Pocan et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), who observed the behavior of different sugar syrups applied in the preparation of \u0026ldquo;Turkish delight.\u0026rdquo; They revealed that corn syrup containing delights had less T\u003csub\u003e2\u003c/sub\u003e due to the presence of a considerable amount of maltose, which is a more humectant (indicating more water-binding capacity).\u003c/p\u003e \u003cp\u003eIn this study, both α- and β-lactose were considered for analyzing the T\u003csub\u003e2\u003c/sub\u003e relaxation curve, and the relaxation time for the main and secondary peaks was almost similar, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Therefore, further analysis is shown for β-lactose only, and throughout the entire manuscript, any mention of lactose refers to β-lactose.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eT\u003csub\u003e2\u003c/sub\u003e relaxation time for α- and β-lactose\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIsomer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eLactose to water ratio (g/100 g)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eT\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eof\u003c/b\u003e \u003cb\u003emain peak (ms)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eα-Lactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2741\u0026thinsp;\u0026plusmn;\u0026thinsp;29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2537\u0026thinsp;\u0026plusmn;\u0026thinsp;29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2259\u0026thinsp;\u0026plusmn;\u0026thinsp;55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2061\u0026thinsp;\u0026plusmn;\u0026thinsp;68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1812\u0026thinsp;\u0026plusmn;\u0026thinsp;00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eβ-Lactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2744\u0026thinsp;\u0026plusmn;\u0026thinsp;06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2413\u0026thinsp;\u0026plusmn;\u0026thinsp;34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2135\u0026thinsp;\u0026plusmn;\u0026thinsp;22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1952\u0026thinsp;\u0026plusmn;\u0026thinsp;33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1782\u0026thinsp;\u0026plusmn;\u0026thinsp;36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eT\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eof\u003c/b\u003e \u003cb\u003esecondary peak (ms)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eα-Lactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e326\u0026thinsp;\u0026plusmn;\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e394\u0026thinsp;\u0026plusmn;\u0026thinsp;15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e375\u0026thinsp;\u0026plusmn;\u0026thinsp;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e360\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e327\u0026thinsp;\u0026plusmn;\u0026thinsp;06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eβ-Lactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e314\u0026thinsp;\u0026plusmn;\u0026thinsp;28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e369\u0026thinsp;\u0026plusmn;\u0026thinsp;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e364\u0026thinsp;\u0026plusmn;\u0026thinsp;08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e348\u0026thinsp;\u0026plusmn;\u0026thinsp;07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e328\u0026thinsp;\u0026plusmn;\u0026thinsp;00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Behavior of peaks observed in disaccharides solutions\u003c/h2\u003e \u003cp\u003eThe percentage of the total surface area of the main and secondary peaks corresponding to the disaccharide content in the sugar solution is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. In this Fig., S\u003csub\u003eT\u003c/sub\u003e, S\u003csub\u003eM\u003c/sub\u003e, S\u003csub\u003eS\u003c/sub\u003e indicate the total, main, and secondary peak surface areas, respectively. The Fig. illustrates that an increasing amount of disaccharide content generates a secondary peak with a concave upward shape (except maltose), indicating a positive correlation between disaccharide concentration and the increase rate of the secondary peak and an inverse correlation with the main peak. In another way, it can be said that the hydrogen atom in a sugar solution with a low sugar-to-water ratio behaves like the hydrogen atom in pure water. In case of maltose, almost linear-shaped upward secondary peaks (S\u003csub\u003es1\u003c/sub\u003e and S\u003csub\u003eS2\u003c/sub\u003e) relative to the total peak were observed. However, the surface area of S\u003csub\u003eS2\u003c/sub\u003e was much smaller than S\u003csub\u003es1\u003c/sub\u003e. This deviation became larger with an increased maltose-to-water ratio, indicating stronger increase of S\u003csub\u003eS1\u003c/sub\u003e with maltose concentration. Another noticeable observation is the value of S\u003csub\u003eM\u003c/sub\u003e/S\u003csub\u003eT\u003c/sub\u003e, which can provide a preliminary hypothesis related to disaccharide solubility. For example, higher S\u003csub\u003eM\u003c/sub\u003e/S\u003csub\u003eT\u003c/sub\u003e value can be linked to higher solubility of saccharose, trehalose, and lactose. However, the behavior of maltose is excluded from this hypothesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA clear distinction between disaccharides in terms of relaxation times of the main and secondary peaks can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. At the initial concentration, until a sugar-to-water ratio of 10 g/100 g, the main peak of all the disaccharides appeared at almost the same T\u003csub\u003e2\u003c/sub\u003e, gradually declining with increasing sugar content. However, T\u003csub\u003e2\u003c/sub\u003e for maltose sharply decreased with increasing sugar content, followed by trehalose, saccharose, and lactose, respectively. This increased amount of sugar limits the availability of free water molecules, hence reducing mobility and relaxation time. Similarly, at the initial concentration until a sugar-to-water ratio of 20 g/100 g, the secondary peak showed a larger standard deviation for saccharose, trehalose, and maltose. This is probably due to the more compatible nature of water when the solute is at a low concentration (Aroulmoji et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), indicating increased freedom of protons to exist in the main peak. The T\u003csub\u003e2\u003c/sub\u003e for the secondary peak\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003efollows the order: lactose\u0026thinsp;\u0026gt;\u0026thinsp;trehalose\u0026thinsp;\u0026gt;\u0026thinsp;saccharose\u0026thinsp;\u0026gt;\u0026thinsp;maltose. Although these sugars have similar molecular weight and the same number of OH groups, their hydration number, hydrogen bonding between water-sugar molecules, sugar-sugar molecule clustering, and viscosity differ, influencing the interaction of sugar molecules with water and hence the magnetic environment. Studies have revealed that the hydration of disaccharides follows the order of lactose\u0026thinsp;\u0026gt;\u0026thinsp;trehalose\u0026thinsp;\u0026gt;\u0026thinsp;maltose\u0026thinsp;\u0026gt;\u0026thinsp;sucrose (Furuki et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Lerbret et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Pocan et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, the behavior of maltose is hypothesized to involve intermolecular (sugar-sugar) interactions through hydrogen bonding. A study conducted by Lerbret et al., (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) found that the intermolecular hydrogen bonding of maltose is higher than that of trehalose and sucrose across all concentrations ranging from 33\u0026ndash;66 wt%. Moreover, the intramolecular bonding (sugar-water) of maltose was found to be less than that of sucrose and higher than that of trehalose, indicating that maltose has more OH groups available to interact with other maltose molecules than water molecules (Lerbret et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Therefore, in sugar network formation, maltose tends to organize itself into larger clusters compared to sucrose and trehalose (Lerbret et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur hypothesis is that the main peak belongs to the signal generated from the proton of hydrogen bonding between sugar and water molecules, while the secondary peak belongs to the hydrogen bonding between sugar molecules. The stronger sugar-sugar bonding of maltose may lead to the generation of secondary peaks with less mobility and reduced relaxation time. Additionally, the formation of larger sugar-sugar bonded clusters of maltose makes the sugar-water solution less homogeneous in terms of the number of peaks compared to trehalose and sucrose (Lerbret et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Therefore, it is assumed that the lower homogeneity, in terms of the number of peaks, of maltose might influence the synchronization of \u003csup\u003e1\u003c/sup\u003eH in generating two secondary peaks. On the other hand, lactose, with lower solubility compared to other disaccharides studied here, is highly hydrophilic and has a stronger ability to interact with water molecules (Wijayasinghe et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Consequently, a low concentration of lactose molecules has more available OH in solution and fewer bound inter- and intra-molecular networks, implying greater mobility and higher T\u003csub\u003e2\u003c/sub\u003e compared to others. Regarding the T\u003csub\u003e2Secondary\u003c/sub\u003e of trehalose and sucrose, trehalose required more time to relax than sucrose, possibly due to the viscosity of the sugar in the water solution. At higher concentrations, the difference in viscosity between trehalose and sucrose was significantly greater compared to lower concentrations, with trehalose exhibiting markedly higher viscosity levels than sucrose (Galmarini et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Higher viscosity values of sugar molecules tend to accumulate more sugar molecules in solution, resulting in a less mobile situation and reduced T\u003csub\u003e2\u003c/sub\u003e values (Aroulmoji et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Lai \u0026amp; Schmidt, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Another study found that trehalose has a stronger and more rigid structure with water than a sucrose-water mixture (Magazu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), supporting the findings that sucrose requires more T\u003csub\u003e2\u003c/sub\u003e to display the main peak (due to bonding between sugar and water) and less T\u003csub\u003e2\u003c/sub\u003e for the secondary peak (due to bonding between sugar molecules) compared to trehalose.\u003c/p\u003e \u003cp\u003eA linear regression equation was observed when plotting disaccharide content in water as the independent variable and the ratio of relaxation time between the main peak of the disaccharide and water as the dependent variable, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The values of the dependent variables were considered on a logarithmic basis for obtaining better correlation. Each sugar displayed a high regression coefficient (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.99), indicating excellent fittings and a strong dependency of the dataset of the main peak on the concentration of disaccharide.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Analysis of peak area and width corresponding to disaccharide content in the solution\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e illustrates the impact of disaccharide content on the total surface area of disaccharide (S\u003csub\u003eT\u003c/sub\u003e) compared to pure water (S\u003csub\u003e0\u003c/sub\u003e), where S\u003csub\u003eT\u003c/sub\u003e = S\u003csub\u003eMain\u003c/sub\u003e + S\u003csub\u003eSecondary\u003c/sub\u003e. The ratio of S\u003csub\u003eT\u003c/sub\u003e/S\u003csub\u003e0\u003c/sub\u003e represents the change of the total surface of the solution while compensating for the effect of signal amplification coefficients used by the user of NMR. Both normalized and non-normalized surface area shown in the Fig. have a good fit with R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.98, indicating a strong correlation with the concentration of disaccharides. These findings also represent the increment rate of hydrogen atoms in the solution with the increasing amount of disaccharide content relative to the initial hydrogen in water and can be expressed as follows:\u003c/p\u003e \u003cp\u003e \u003cdiv id=\"Equa\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\frac{{S}_{T}}{{S}_{0}}=\\frac{{{H}_{Water}+\\:H}_{Sugar}}{{H}_{Water}}$$\u003c/div\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe main peaks observed in the disaccharide solution were further analyzed compared to pure water considering the peak width (∆T) (the gap of T\u003csub\u003e2\u003c/sub\u003e between onset and endpoint) and presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Each case explained that with the increase in the concentration of disaccharides, the main peak width decreased compared to the main peak observed for pure water. However, at the initial condition (5 g sugar/100 g water), all the sugars showed similar behavior to water, indicating that at very low concentrations, there is availability of binding positions of water molecules with sugar molecules. From Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, it is noticeable that the deviation of ∆T for the secondary peak was higher until the sugar content reached 20 g/100 g water. This deviation in initial cases might be due to the greater freedom of forming hydrogen bonding between sugar and water molecules due to sufficient\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOH groups in the solution. The rationale behind the varied behavior of different disaccharides in water solutions has been previously explained. However, NMR users may also utilize this type of analysis to identify disaccharides in unknown solutions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Correlation of peak characteristics and hydrogen atom in the solution\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e illustrates a linear relationship between hydrogen content and signal intensity as observed in TD-NMR. The intercept values (ranging from 140\u0026ndash;356 a.u.) of the linear regression equations for all disaccharides are negligible when compared to the substantial slopes (ranging from 2013\u0026ndash;2570 a.u.), confirming that the signal intensity primarily originates from \u003csup\u003e1\u003c/sup\u003eH. According to our hypothesis, the generation of secondary peaks results from sugar-sugar bonding rather than sugar-water interactions. Therefore, the distraction of the main peak signal from the total signal and the subsequent appearance of secondary peak(s) are attributed to the hydrogen bonding among disaccharide molecules and their interactions with water. Notably, saccharose and trehalose exhibit a similar diversion point of the main peak at a sugar content of 15 g/100 g, whereas lactose diverges earlier at 10 g/100 g.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the fastest diversion was observed in maltose solution, where 5 g/100 g can be considered a critical concentration (the onset of diversion). The main peaks of saccharose, trehalose, and lactose exhibited convex upward shapes, while secondary peaks displayed concave upward growth with increasing concentration. This finding is in good agreement with numerous other studies, which have confirmed that the addition of sugar significantly retards water dynamics (mobility) (Heyden et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Lerbret et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Paolantoni et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Paolantoni et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). This slowing-down process (reducing water mobility) depends on the total number of hydrogen bonds formed with water (Magazu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) and restricts the flexibility or mobility of water molecules.\u003c/p\u003e \u003cp\u003eFurthermore, another influential parameter for slowing down water mobility is the percolation of the hydrogen bonding network, which increases sharply with the threshold concentration of sugar in the solution (Bordat et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). At this threshold concentration, the mean cluster size of water-water hydrogen bonding prominently begins to decrease, and sugar starts to influence water molecules (Bordat et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In this Fig., the diversion of the main peak from the total peak signal and the appearance of secondary peak(s) with increasing sugar concentration indicate the beginning of interaction between sugar molecules in water, leading to a decrease in water-water interaction. Lee et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) mentioned that sugar-water bonding is more stable than water-water bonding, which aligns with our findings, and thus the increase of secondary peaks with concentration. Notably, the increase of the secondary peak was more pronounced for trehalose compared to sucrose. The formation of larger cluster sizes for trehalose may drive this behavior, as the association of sugar molecules generates stronger bonding, while sucrose creates smaller cluster sizes (Lerbret et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Lerbret et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). On the other hand, maltose exhibited different behavior, producing two secondary peaks with almost linear behavior. As described earlier, the strong intermolecular bonding between maltose molecules might divert the primary peak faster than other disaccharides. However, the comprehension of maltose displaying a completely different trend with two secondary peaks is still unclear, requiring further investigation for a comprehensive understanding.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Applicability of TD-NMR in characterizing disaccharides\u003c/h2\u003e \u003cp\u003eThis study aimed to address a general question: \"Can we use TD-NMR to characterize disaccharides in a simple and rapid way from unknown samples in both solid (dried powder) and liquid (solution) form, with known or unknown concentrations?\" The answer to this question is summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, which outlines the characterization of disaccharides. Firstly, all samples (relevant disaccharides in this study) require the preparation of a solution of 20 g/100 g pure water to compensate for their wide range of T\u003csub\u003e2\u003c/sub\u003e deviations below this concentration. Users can identify the sugar samples based on their secondary peak. Analyzing either the observation of relaxation time or calculating the ratio of the surface area of the secondary peak to the total peak (S\u003csub\u003eS\u003c/sub\u003e/S\u003csub\u003eT\u003c/sub\u003e) facilitates the characterization of the sugar sample in solution. Maltose is easily characterized by observing its two secondary peaks. If S\u003csub\u003eS\u003c/sub\u003e/S\u003csub\u003eT\u003c/sub\u003e is less than 2.5%, it is saccharose; if it falls between 2.5 to 4.5, then it is trehalose; and if it exceeds 4.5, then it is lactose. In terms of observing T\u003csub\u003e2\u003c/sub\u003e relaxation time, saccharose, trehalose, and lactose can be identified as \u0026lt;\u0026thinsp;210 ms, 210\u0026ndash;290 ms, and \u0026gt;\u0026thinsp;290 ms, respectively. Using this method, TD-NMR users can compile a list of S\u003csub\u003eS\u003c/sub\u003e/S\u003csub\u003eT\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e of different sugar samples for easier and more rapid identification in a cost-effective manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study investigated the T\u003csub\u003e2\u003c/sub\u003e relaxation distribution curve of saccharose, maltose, trehalose, and lactose in solutions, aiming to assess the potential of a simplified version of TD-NMR, which encompasses fundamental options, in characterizing disaccharides. Saccharose, trehalose, and lactose exhibit two distinguishable peaks, considered the main and secondary peaks, while pure water displays no secondary peaks. In contrast, maltose shows two secondary peaks along with the main peak. The findings confirm that as the disaccharide content increases, the relaxation time decreases, indicating reduced mobility due to increased interactions between sugar molecules and water. It is hypothesized that the main peak originates from proton bonding in water-water or sugar-water interactions, while the secondary peak arises from sugar-sugar interactions. The relaxation time for the secondary peak of saccharose, trehalose, β-lactose, and maltose ranged from 100\u0026ndash;260 ms, 130\u0026ndash;330 ms, 300\u0026ndash;460 ms, and 10\u0026ndash;190 ms, respectively. However, factors such as hydrogen bonding between sugar molecules, viscosity, hydration number, and solubility may influence relaxation time. The unique behavior of maltose compared to other disaccharides is hypothesized to be due to stronger intermolecular bonding. Further investigation is needed to determine the exact reasons for the differing trends observed in maltose, with the possibility that spin-lattice (T\u003csub\u003e1\u003c/sub\u003e) relaxation analysis could provide greater insights. However, the standard curves of T\u003csub\u003e2\u003c/sub\u003e, or the surface area of the main combined with secondary peaks, as a function of sugar concentration obtained from the analysis of the peak are applicable for the quantification of disaccharides in binary mixtures (water and one disaccharide). Overall, this study enhances our comprehension of the feasibility and reliability of TD-NMR as an effective tool for characterizing disaccharide samples in both unknown powder and homogeneous sugar solutions, primarily by analyzing relaxation time or the surface area of the secondary peak. However, the machine faces challenges in discriminating between α- and β-isomers. Finally, further exploration in characterizing monosaccharides will improve the comprehensibility to assess the applicability of this simplified version of TD-NMR.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003ch2\u003eFunding statement\u003c/h2\u003e \u003cp\u003eNatural Sciences and Engineering Research Council of Canada\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAfroza Sultana: Writing - original draft preparation; Formal analysis and investigation; Ali Asghari: Conceptualization, Methodology, Formal analysis and investigation; Writing - review and editing; Seddik Khalloufi: Conceptualization, Methodology, Formal analysis and investigation; Writing - review and editing; Supervision, Validation, Visualization\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eData will be available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAky\u0026uuml;z E, Başkan KS, T\u0026uuml;tem E, Apak R (2021) High performance liquid chromatographic method with post-column detection for quantification of reducing sugars in foods. 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J Food Compos Anal 100:103824\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-analytical-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Analytical Methods](https://www.springer.com/journal/12161)","snPcode":"12161","submissionUrl":"https://submission.nature.com/new-submission/12161/3","title":"Food Analytical Methods","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"disaccharide, TD-NMR, water mobility, T2 relaxometry, characterization","lastPublishedDoi":"10.21203/rs.3.rs-4764910/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4764910/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe necessity of identifying and quantifying sugars in food processing is endless for maintaining food quality attributes such as color, taste, and texture, monitoring regulatory compliance, labeling packages, and maintaining authenticity. Despite available analytical methods for characterizing sugar molecules, the limitations of conventional methods drive researchers to seek more convenient alternatives. This study aimed to characterize common disaccharides such as saccharose, lactose, maltose, and trehalose using a simplified version of time domain nuclear magnetic resonance (TD-NMR), facilitating a quick, cost-effective, and user-friendly approach. In the transverse relaxometry, secondary peak(s) were observed for all the disaccharides with a main peak. Although they have similar molecular formulas and weights, lactose exhibited the longest relaxation time for the secondary peak, followed by trehalose, saccharose, and maltose. This behavior was assumed due to the interaction of sugar molecules with water. The increasing concentration of disaccharide in the solution displayed the leftward shifting of peaks. Maltose showed two secondary peaks, which were not observed in other sugar samples. This TD-NMR showed potential to distinguish disaccharides from unknown powders and solutions by analyzing either the relaxation time of the secondary peak or the ratio of the secondary to the total peak. Moreover, quantification is possible from the standard curves of relaxation time and the combined surface area of the main and secondary peaks with the corresponding sugar concentration. However, it shows challenges in discrimination between α- and β-isomers.\u003c/p\u003e","manuscriptTitle":"A straightforward method for disaccharide characterization by a simplified version of time domain nuclear magnetic resonance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-13 17:38:03","doi":"10.21203/rs.3.rs-4764910/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-06T14:17:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-06T01:49:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-04T13:08:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-01T21:45:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-01T09:37:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-01T03:17:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-30T20:01:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-30T13:26:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-30T01:39:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"95474158795024191386059869030746737696","date":"2024-07-28T09:39:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272819913847755982502815649500397226118","date":"2024-07-27T15:25:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262860428777627961353260788043387000966","date":"2024-07-27T14:35:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197295599419001772344276210512043877304","date":"2024-07-24T21:32:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"337511752346076684274229812259753587462","date":"2024-07-24T10:01:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207555316436655822860777212969771809594","date":"2024-07-24T09:03:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66101609799226377780245286658682172525","date":"2024-07-23T16:26:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"182813396520054954120832904563156294495","date":"2024-07-23T15:40:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8372572245654633894356359003884681515","date":"2024-07-22T12:37:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"38315693551327588000158791313349383069","date":"2024-07-22T11:12:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87136879210154815159285261130990527490","date":"2024-07-22T11:06:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-22T06:36:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-18T23:04:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-18T23:03:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food Analytical Methods","date":"2024-07-18T20:06:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-analytical-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Analytical Methods](https://www.springer.com/journal/12161)","snPcode":"12161","submissionUrl":"https://submission.nature.com/new-submission/12161/3","title":"Food Analytical Methods","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b6ba5592-cb8b-4888-b53e-387db59cd7d9","owner":[],"postedDate":"August 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-09-23T20:08:33+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-13 17:38:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4764910","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4764910","identity":"rs-4764910","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}