Separation Of Carbohydrates Research Articles

Separation of carbohydrates using dynamically adsorbed borate stationary phase for hydrophilic interaction liquid chromatography

In this work, a chromatographic method for the separation of carbohydrates was proposed. Tris-(hydroxymethyl)-amine (TRIS) functionalized silica-based hydrophilic interaction liquid chromatography (HILIC) stationary was synthesized. The dynamically absorbed borate layer is generated by using borate buffer as a polar modifier due to the complexation of borate with TRIS ligand in the stationary phase. The chromatographic systems were analyzed by the linear solvation energy relationship model. The calculated system constants revealed the enhancement of anionic exchange by the addition of borate in the mobile phase system. In addition, ligand exchange is critical for the retention and elution order of sugars and sugar alcohols. Carbohydrates displayed prolonged retention with different selectivity profiles relating to their complexation coefficients with borate. Experiment results showed that the effect of borate in this chromatographic system was stable within the range of pH 3–7 and borate concentration of 5–15 mM. This work provides a complementary solution for the separation of carbohydrates. It can also be extended to the separation of glycosides.

Exploring drying-induced separation of main constituents in melamine-blended milk infant formula using DCDR spectroscopy

Drop coating deposition Raman (DCDR) spectroscopy was employed to investigate the melamine compound susceptibility to milk infant formula main constituents as lactose and lipids (specifically 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)). DCDR approach is based on the deposition and drying of a small droplet (a few µl) of liquid sample on a hydrophobic substrate. After complete evaporation, the analyte is preconcentrated into the dried pattern from which the classical Raman spectra are acquired. A simple melamine-blended model reactions were tested (melamine-blended lactose, DOPC, and DOPE). From these, lactose was shown to be the most potent reaction partner for melamine, where a significant difference in dried pattern between pure and blended lactose was noticed, and the relevant Raman spectral changes were observed. Pure and melamine-blended infant formula solutions from two kinds of powdered infant formula purchased in the local market were then studied. For pure milk solutions, the drying process, as an integral part of the DCDR approach, led to the spatial separation of lipids and carbohydrates in the resulting pattern. Acquired Raman spectra revealed that the ring edge of the dried pattern was composed mainly of lipids, while the thin film in the central part contained mainly carbohydrates, especially lactose. For the melamine-blended formula, the DCDR approach identified that melamine was present only in the central part of the dried pattern together with carbohydrates. As a result, it was assumed that melamine has a higher susceptibility for carbohydrates than for lipid molecules, even in milk infant formula.

Detection techniques for carbohydrates in capillary electrophoresis – a comparative study

Capillary electrophoresis methods for the separation of carbohydrates with four different detection techniques, namely direct UV, indirect UV, capacitively coupled conductivity, and laser-induced fluorescence detection, were tested and their performance was evaluated and compared in terms of linearity, limits of detection and quantitation, repeatability, recovery, analysis time, and sample treatment. The test set of analytes comprised sucrose, glucose, and fructose. The effect of using lactose as an internal standard on the individual methods was investigated, too. The results showed that laser-induced fluorescence detection is a technique of choice for applications requiring the detection of very low amounts of reducing carbohydrates. Contactless conductivity detection is favorable when detection sensitivity is not a crucial parameter but fast and reliable analysis is required. When only a UV detector is available as a standard part of capillary electrophoresis instruments, direct UV detection can be used when analysis time is not a critical parameter. For fast analysis with UV detectors, indirect UV detection is the technique of choice. Finally, to verify the applicability of the tested methods, samples of cola beverage, honey, and orange juice were analyzed and the results obtained by all four methods were compared.Graphical abstract

L-Poly(lactic acid) Production by Microwave Irradiation of Lactic Acid Obtained from Lignocellulosic Wastes.

L-polylactic acid (PLA), a semi-crystalline aliphatic polyester, is one of the most manufactured biodegradable plastics worldwide. The objective of the study was to obtain L-polylactic acid (PLA) from lignocellulosic plum biomass. Initially, the biomass was processed via pressurized hot water pretreatment at a temperature of 180 °C for 30 min at 10 MPa for carbohydrate separation. Cellulase and the beta-glucosidase enzymes were then added, and the mixture was fermented with Lacticaseibacillus rhamnosus ATCC 7469. The resulting lactic acid was concentrated and purified after ammonium sulphate and n-butanol extraction. The productivity of L-lactic acid was 2.04 ± 0.18 g/L/h. Then, the PLA was synthesized in two stages. Firstly, lactic acid was subjected to azeotropic dehydration at 140 °C for 24 h in the presence of xylene, using SnCl2 (0.4 wt.%) as a catalyst, resulting in lactide (CPLA). Secondly, microwave-assisted polymerization was carried out at 140 °C for 30 min with 0.4 wt.% SnCl2. The resulting powder was purified with methanol to produce PLA with 92.1% yield. The obtained PLA was confirmed using electrospray ionization mass spectrometry, nuclear magnetic resonance, thermogravimetric analysis, Fourier transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction. Overall, the resulting PLA can successfully replace the traditional synthetic polymers used in the packaging industry.

Combined liquid hot water and sulfonation pretreatment of sugarcane bagasse to maximize fermentable sugars production

Sugarcane bagasse (SCB) is a widely available biomass feedstock currently used for ethanol production in various parts of the world. Here, we carried out a combined liquid hot water and neutral/alkaline sulfonation pretreatments of SCB at relatively low temperatures to maximize enzymatic hydrolysis yields. To describe multi-step pretreatments severities, analytical equations for modified combind severity factors (CSF´m) were introduced. Although the chemical composition of SCB after the liquid hot water pretreatment alone has not been significantly changed under tested conditions, this pretreatment led to an increase in the efficiency of its enzymatic hydrolysis. Furthermore, a combination of liquid hot water pretreatment (at 120 °C for 1 h) and sulfonation of SCB (at 120 °C for 2 h) resulted in excellent enzymatic hydrolysis yields. After alkaline sulfonation, glucose yields reached 99.6 ± 0.21% for autoclaved and alkaline sulfonated bagasse (AASB) and 97.47 ± 0.75% for boiled and alkaline sulfonated bagasse (BASB), whereas xylose yields were 86.9 ± 1.9% and 88.0 ± 1.8% for AASB and BASB, respectively. High enzymatic hydrolysis yields are in line with elevated CSF´m of the latter pretreatments. X-ray diffraction, confocal laser scanning microscopy, and solid-state 13C NMR spectroscopy analyses showed that the structural desorganization of bagasse after liquid hot water pretreatment associated to chemical modifications of lignin and its massive removal promoted by alkaline sulfonation led to efficient separation of carbohydrates and lignin, revealing underlying structural reasons of the observed efficient enzymatic hydrolysis.

Analysis of Sugars in Honey Samples by Capillary Zone Electrophoresis Using Fluorescence Detection

The applicability of capillary electrophoresis (CE) with light-emitting diode-induced fluorescence detection (LEDIF) for the separation of sugars in honey samples was studied. An amount of 25 mM ammonium acetate (pH 4.5) with 0.3% polyethylene oxide (PEO) was found to be optimal for the efficient separation of carbohydrates. 8-aminopyrene-1,3,6-trisulfonic acid (APTS) was used for the labeling of the carbohydrate standards and honey sugars for fluorescence detection. The optimized method was applied in the quantitative analysis of fructose and glucose by direct injection of honey samples. Apart from the labeling reaction, no other sample preparation was performed. The mean values of the fructose/glucose ratio for phacelia honey, acacia honey and honeydew honey were 0.86, 1.61 and 1.42, respectively. The proposed method provides high separation efficiency and sensitive detection within a short analysis time. Apart from the labeling reaction, it enables the injection of honeys without sample pretreatment. This is the first time that fluorescence detection has been applied for the CE analysis of sugars in honeys.

Tailored Solid-State Carbohydrate Nanostructures Based on Star-Shaped Discrete Block Co-Oligomers.

Carbohydrates are key building blocks for advanced functional materials owing to their biological functions and unique material properties. Here, we propose a star-shaped discrete block co-oligomer (BCO) platform to access carbohydrate nanostructures in bulk and thin-film states via the microphase separation of immiscible carbohydrate and hydrophobic blocks (maltooligosaccharides with 1-4 glucose units and solanesol, respectively). BCOs with various star-shaped architectures and saccharide volume fractions were synthesized using a modular approach. In the bulk, the BCOs self-assembled into common lamellar, cylindrical, and spherical carbohydrate microdomains as well as double gyroid, hexagonally perforated lamellar, and Fddd network morphologies with domain spacings of ∼7 nm. In thin films, long-range-ordered periodic carbohydrate microdomains were fabricated via spin coating. Such controlled spatial arrangements of functional carbohydrate moieties on the nanoscale have great application potential in biomedical and nanofabrication fields.

Development and chromatographic exploration of stable-bonded cross-linked amino silica against classical amino phases.

The present work reports on a novel stable-bonded amino silica stationary phase obtained by crosslinking of surface aminopropyl moieties using triglycidyl isocyanurate. The obtained cross-linked amido-amino network silica material exhibited superior hydrolytic stability compared to classical 3-aminopropyl phases and showed, inter alia, excellent separation of nine therapeutically effective sulfonamides in hydrophilic interaction/weak anion exchange chromatography elution mode. Additionally, the separation of carbohydrates was investigated under classical hydrophilic interaction chromatography conditions as well proving the suitability of the novel phase for such applications. For the evaluation of the hydrolytic stability the prepared material, as well as two commercially available benchmark columns and a set of in-house synthesized amino-modified materials, were exposed to harsh aqueous mobile phase conditions for in total of 50h at elevated temperature. In this context, the materials were examined by elemental analysis, (13 C and 29 Si cross-polarization/magic angle spinning) solid-state nuclear magnetic resonance, and a chromatographic test before and subsequent to the exposure to these stress conditions. Lastly, the new stationary phase was classified in comparison to a set of commercially available stationary phases by principal component analysis of resultant retention factors gained from chromatographic standard tests.

A green pretreatment approach of corn stalk wastes for obtaining micro/nano-cellulose fibers, monosaccharides and lignin fractions

Efficient separation of carbohydrates and lignin from lignocellulosic biomass, and further high-value conversion are still the main challenge of biorefinery. Oxygen-enhanced alkali treatment as a green method for separating polysaccharides and lignin from gramineous materials with environmental, economic and low energy consumption. Herein, oxygen-enhanced alkali technology was used to pretreatment corn stalk pith, and then realize high-value transformation. The results showed that oxygen-enhanced alkali treatment combined with acid precipitation, ethanol precipitation, enzymatic hydrolysis or ultrasonic can achieve clean, efficient separation and subsequent high-value transformation of various components of corn pith. The enzymatic efficiencies of solid and liquid polysaccharides were 75.42% and 50.52% respectively, and the structure of micro/nano-cellulose fiber is mostly flaky and granular. In addition, the separated lignin contains many small molecular compounds, which has the potential value of further utilization. In this study, oxygen-enhanced alkali treatment can achieve clean, efficient separation and subsequent high-value transformation of various components in corn stalk or lignocellulosic biomass, which has important practical significance.

Quantification of carbohydrates in human serum using gas chromatography–mass spectrometry with the stable isotope-labeled internal standard method

Comparison of two derivatization approaches (silylation and acylation) for carbohydrate separation based on optimizing reaction conditions by artificial neural networks.

Combining high performance thin layer chromatography with minispectrometer-fiber optic probe-coupled to smartphone for in place analysis: Lactose quantification in several matrices

An in place colorimetric method has been proposed for estimation of the quantity of lactose in several matrix (milk, water effluents and surfaces). Analyzing the amount of this carbohydrate it can be control the product, the cleanliness of the parts of the dairy companies and it can avoid contamination of milk products produced there. This method combines the use of HPTLC for sugars separation with novel analytical devices as minispectrophotometer with fiber optic coupled to a smartphone. In order to measure the lactose a colorimetric reaction has been used. Variable volumes of samples or stock solutions were deposited in nano-silica gel layer, a mobile phase of acetonitrile:water:acetic acid was used for carbohydrate separation and a solution of thymol (0.05 g Thymol in 95 mL of EtOH and 5 mL H2SO4) was used for revealed the carbohydrate spot. Finally, the reflectance of samples and stock solutions were measured. The achieved limits of detection were 0.03 mg mL−1 and 0.003 mg mL−1 for the working concentration range and the analysis at traces level respectively.

Chromatographic separation of mannitol from mixtures of other carbohydrates in aqueous solutions

The isolation of mannitol from natural sources, e.g. from plant extracts or broths, requires considerable time and effort. The separation of mannitol from aqueous solutions containing also glucose, fructose, and sucrose was tested using discontinuous preparative anion- and cation-exchange chromatography. The suitability of the application in the separation of carbohydrates and especially mannitol was tested under various conditions and using three different types of ion-exchangers. The effect of sorbent regeneration and modification on the separation was also examined using different concentrations and volumes of chemical agents. The fractions collected after the discontinuous chromatography were analysed on the content of mannitol by the high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) method. The successful isolation of pure mannitol fraction, using water as a mobile phase and a combination of sodium chloride and hydroxide for sorbent regeneration, was achieved only on anion-exchange chromatography.

Separations of Carbohydrates with Noncovalent Shift Reagents by Frequency-Modulated Ion Mobility-Orbitrap Mass Spectrometry.

An increased focus on characterizing the structural heterogeneity of carbohydrates has been driven by their many significant roles in extant life and potential roles in chemical evolution and the origin of life. In this work, multiplexed drift tube ion mobility-Orbitrap mass spectrometry methods were developed to analyze mixtures of disaccharides modified with noncovalent shift reagents. Since traditional coupling of atmospheric pressure drift tube ion mobility cells with Orbitrap mass analyzers suffers from low duty cycles (<0.1%), a frequency modulation scheme was applied to improve the signal-to-noise ratios (SNR). Several parameters such as the resolution setting and maximum injection time of the Orbitrap analyzer and the magnitude and duration of the frequency sweep were investigated for their impact on the sensitivity gains and resolution of disaccharide-shift reagent adducts. The sweep time and disaccharide concentration had a positive correlation with SNR. The magnitude of the frequency sweep had a negative correlation with SNR. However, increasing the frequency sweep improved the resolution of mixtures of disaccharide analytes. Application of frequency-modulated ion mobility-Orbitrap mass spectrometry to four noncovalently modified glucose dimers allowed for the differentiation of three out of these four analytes.

Towards a more complete glycome: Advances in ion chromatography-mass spectrometry (IC-MS) for improved separation and analysis of carbohydrates

To date, few tools are available for the analysis of the glycome without derivatization, a process which is known to introduce issues such as differential loss of sialic acid and incomplete labeling. We have previously reported the use of ion chromatography-mass spectrometry (IC-MS) to analyze native sialylated and sulfated glycans. Here, we introduce improvements to IC column technology, enabling the separation of neutral glycans while maintaining charge separation capabilities. When implemented in an IC-MS workflow, this enables the structural characterization of a broad array of chemically distinct glycans. With the newly developed IC column and modified IC-MS instrumentation configuration, we qualitatively investigated O-glycome profiles in bovine fetuin and porcine gastric mucins. The improved chromatographic resolution in combination with high-resolution MS data present a powerful tool for glycan structural identification.

Comparative study of sugar extraction procedures forHPLCanalysis and proposal of an ethanolic extraction method for plant‐based high‐protein ingredients

The increasing importance of plant-based proteins in the food sector makes a reliable compositional analysis of plant-based high-protein ingredients a necessity. Specifically, the quantification of short-chain carbohydrates is relevant for multiple areas, including food product development, food labelling and fundamental food chemistry and food technology research. Commonly used extraction procedures for subsequent high-performance liquid chromatographic separation and quantification of short-chain carbohydrates have been discussed controversially regarding a range of complications that can potentially lead to inaccurate sugar determination. The present study compares the sugar levels in wheat flour and wholemeal wheat flour determined with different aqueous and ethanolic extraction procedures. These procedures included measures to prevent enzyme activity and microbial growth, which represent two of the most relevant challenges in sugar extraction from food samples. Differences in sugar levels (sum of sucrose/maltose, glucose and fructose) as high as 1.8% dry matter (wheat flour) were observed between the employed extraction procedures. Ethanolic extraction (80% ethanol in ultrapure water) with the use of the antimicrobial agent sodium azide but without Carrez clarification was identified as most promising for sugar determination in plant-based high-protein ingredients. A screening of high-protein ingredients derived from cereals (wheat gluten), pseudocereals (quinoa, amaranth, buckwheat) and legumes (soy, pea, lupin, lentil, carob, chickpea, faba bean) concerning their levels of sucrose, maltose, glucose and fructose confirmed the applicability of the chosen extraction procedure. © 2021 Society of Chemical Industry.

Structural resolution of disaccharides through halogen anion complexation using negative trapped ion mobility spectrometry

Carbohydrates are an indispensable part of early life evolution. The determination of their structures is a key step to analyze their critical roles in biological systems. A variation of composition, glycosidic linkage, and (or) configuration between carbohydrate isomers induces structure diversity and brings challenges for their structural determination. Ion mobility spectrometry (IMS), an emerging gas-phase ion separation technology, has been considered as a promising tool for performing carbohydrate structure elucidation. In this work, eight disaccharides were analyzed by trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) in the negative ion mode as the complexed form of [M + X]-, where M = disaccharide, and X = Cl, Br, and I. As compared to the positive ion analysis of the selected disaccharide in a sodiated form, a reversal charge state provided the ability to eliminate or even reverse the collision cross section (CCS) difference between disaccharide isomers. By the combination of TIMS analysis and the calculation of density functional theory, the only observed two conformers of ions [lactulose + I]- may result from different adduction sites for an iodide anion. Based on the comparison of different halogen adducts, the [M + I]- ion form exhibited more powerful ability for isomeric disaccharide differentiation with an average resolution (RP-P) of 1.17, which results in a 34.5% improvement as compared to the corresponding chloride adducts. This result indicates that the use of negative charge states, especially the complexation of an iodide anion, could be a supplemental strategy to commonly used positive ion analysis for carbohydrate separation.

Rapid capillary gel electrophoresis analysis of human milk oligosaccharides for food additive manufacturing in-process control

Industrial production of human milk oligosaccharides (HMOs) represents a recently growing interest since they serve as key ingredients in baby formulas and are also utilized as dietary supplements for all age groups. Despite their short oligosaccharide chain lengths, HMO analysis is challenging due to extensive positional and linkage variations. Capillary gel electrophoresis primarily separates analyte molecules based on their hydrodynamic volume to charge ratios, thus, offers excellent resolution for most of such otherwise difficult-to-separate isomers. In this work, two commercially available gel compositions were evaluated on the analysis of a mixture of ten synthetic HMOs. The relevant respective separation matrices were then applied to selected analytical in-process control examples. The conventionally used carbohydrate separation matrix was applied for the in-process analysis of bacteria-mediated production of 3-fucosyllactose, lacto-N-tetraose, and lacto-N-neotetraose. The other example showed the suitability of the method for the in vivo in-process control of a shake flask and fermentation approach of 2′-fucosyllactose production. In this latter instance, borate complexation was utilized to efficiently separate the 2′- and 3-fucosylated lactose positional isomers. In all instances, the analysis of the HMOs of interest required only a couple of minutes with high resolution and excellent migration time and peak area reproducibility (average RSD 0.26% and 3.56%, respectively), features representing high importance in food additive manufacturing in-process control.Graphical abstract

Characterization of custard apple seeds for the separation of prebiotic low molecular weight carbohydrates (LMWC)

The concept of waste to wealth is gaining lot of importance in the recent days. In this context, the present study was undertaken to identify the potential prebiotics present in the custard apple seeds. Among the different components identified, the Low Molecular Weight Carbohydrates (LMWC) were chosen for the study and the extraction was performed using Ethanol at three different concentrations (50%, 75% and 95%) and different combination of temperature, time and speed of rotation. The maximum yield of 9.16% was obtained with 50% Ethanol, 37 0C, 72h and 60 rpm. The probiotic organism Lactobacillus bulgaricus displayed a positive growth of 7.54±0.04 log cfu/ml on LMWC over a period of 24 hours which confirmed the prebiotic functionality of LMWC. Scanning Electron Microscopy and FTIR analysis were done to confirm the morphological characteristics. Therefore, all the results obtained showed that LMWC can be used as potential prebiotics in the food industry and the commercial utilization of custard apple seeds.

Effective separation of carbohydrate isomers using metal cation and halogen anion complexes in trapped ion mobility spectrometry

Comprehensive analysis of carbohydrates, one of the critical steps towards the fundamental understanding of biological systems, has been hindered by the identifications of their complex isomeric structures differing in monosaccharide constituents, linkage positions, and configurations. While ion mobility spectrometry-mass spectrometry (IMS-MS) based methods have shown their utilities in performing glycomics measurements, there is still a lack of analytical methods allowing effective isomeric carbohydrate separation. Here, we systematically investigated the effect of alkaline earth cations (Ca2+ and Ba2+) and halogen anions (Cl−, Br− and I−) addition on the separation of 21 pairs of isomeric oligosaccharides using trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). [M + Ca + I]+ complex (M is an oligosaccharide) has shown experimentally to be the optimum complex for the separation of all 21 pairs of isomeric oligosaccharides used in this study with a calculated average resolution (r) ~ 1.82 and resolving power (R) ~ 100–150. The separation capability of this complex was further demonstrated by using mixtures of 15 oligosaccharides. The optimum [M + Ca + I]+ complex can be potentially used as an effective complex for enhancing the IMS separation resolution of isomeric carbohydrates.

On-line two-dimensional liquid chromatography hyphenated to mass spectrometry and ion mobility-mass spectrometry for the separation of carbohydrates from lignocellulosic biomass

Lignocellulosic biomass is a promising resource of renewable energy. Its transformation to ethanol requires efficient pretreatment leading to complex liquid mixtures made of hundreds of oxygenated analytes. A large part of the released compounds belong to the carbohydrates family. To overcome the complexity of such samples, a comprehensive on-line two-dimensional reversed-phase liquid chromatography hyphenated to high-resolution mass spectrometry (RPLC × RPLC-HRMS) was dedicated to the separation of carbohydrates and more specifically oligomers coming from pretreated lignocellulosic biomass. The first part of this study consisted in the optimization of such hyphenation (i.e. selection of stationary phases, mobile phases, sampling time, etc.). Then, the analytical method was applied to an industrial aqueous biomass product coming from the sulfuric acid-based pretreatment of a wheat straw. Around 70 well-resolved chromatographic peaks corresponding to oligomers were obtained. Occupation of the separation space between each chromatographic dimension was estimated to 75%. In the last part of this study, the interest of ion mobility-mass spectrometry in addition to RPLC × RPLC was discussed. Some examples highlighted the additional separation that can bring ion mobility to RPLC × RPLC-IMS-HRMS method. Using this four-dimensional hyphenation method, each analyte was described by two retention times, the collisional cross section and the molecular formula allowing to reach a level of detail never seen for biomass sample compositions.