Elution Chromatography Research Articles

A New Megastigmane Nor-sesquiterpene Isolated from Pourouma Guianensis by High-Performance Countercurrent Chromatography

Introduction: Pourouma guianensis (Urticaceae) is widespread throughout Brazil and is commonly known as “mapati”. Terpenes are common in Urticaceae species. However, few studies have reported the isolation and characterisation of these compounds in Pourouma genus. Method: The aim of this study was to separate substances from stem barks of P. guianensis using a high-performance countercurrent chromatography gradient elution method. The dichloromethane extract of the P. guianensis stem bark was submitted to silica gel column chromatography, yielding three fractions. Fraction 3 was submitted to a gradient elution system in high-performance countercurrent chromatography using two solvent systems, hexane/ethyl acetate/methanol/water (1:2:x:1, x=1.0 and 1.5). Six compounds were isolated. Results: A new megastigmane nor-sesquiterpene named 2-acetyl-4,7-epoxy-3-oxy-5,5,9- trimethylhexahydrocoumaran (1) was obtained together with five known terpenes: vomifoliol (2), 2α,3β,19α,23-tetrahydroxyurs-12-en-28-oic acid (3), 3-β-D-arabinopyranoside-19-hydroxyurs-12-en- 28-oic acid (4), arjunolic acid (5), and tormentic acid (6). Fractions 1 and 2 were purified by binary gradient elution from hexane to ethyl acetate. Conclusion: This process isolated three known compounds: friedelin (7) and 3β-friedelanol (8) from fraction 1, and β-sitosterol (9) from fraction 2. Gradient elution in countercurrent chromatography was a suitable alternative, exploiting variations in the partition coefficient between substances with small structural differences.

A method for suppressing pH excursion during elution in ion-exchange chromatography

A simple method for reducing pH excursion during salt-induced elution in cation exchange chromatography is presented in this paper. Connecting a preequilibrated anion exchange cartridge in series in front of the cation exchange device just prior to the elution step significantly reduced the transient decrease in pH that is typically observed during elution. The working principle of the proposed method is hypothesized. Bind and elute chromatography experiments showed that the suppression of pH excursion during elution of a monoclonal antibody from a cation exchange chromatography device resulted in significantly sharper and narrower eluted protein peaks. Improvement in pH control during elution would not only result in better reproducibility and efficiency in separation but would also ensure greater stability of pH sensitive proteins during their purification using ion exchange chromatography.

Integration of deep neutral network modeling and LC-MS-based pseudo-targeted metabolomics to discriminate easily confused ginseng species

Metabolomics covers a wide range of applications in life sciences, biomedicine, and phytology. Data acquisition (to achieve high coverage and efficiency) and analysis (to pursue good classification) are two key segments involved in metabolomics workflows. Various chemometric approaches utilizing either pattern recognition or machine learning have been employed to separate different groups. However, insufficient feature extraction, inappropriate feature selection, overfitting, or underfitting lead to an insufficient capacity to discriminate plants that are often easily confused. Using two ginseng varieties, namely Panax japonicus and P. japonicus var. major, containing the similar ginsenosides, we integrated pseudo-targeted metabolomics and deep neural network (DNN) modeling to achieve accurate species differentiation. A pseudo-targeted metabolomics approach was optimized through data acquisition mode, ion pairs generation, comparison between multiple reaction monitoring (MRM) and scheduled MRM, and chromatographic elution gradient. In total, 1980 ion pairs were monitored within 23 min, allowing for the most comprehensive ginseng metabolome analysis. The established DNN model demonstrated excellent classification performance (in terms of accuracy, precision, recall, F1 score, area under the curve, and receiver operating characteristic) using the entire metabolome data and feature-selection dataset, exhibiting superior advantages over random forest (RF), support vector machine (SVM), extreme gradient boosting (XGBoost), and multilayer perceptron (MLP). Moreover, DNNs were advantageous for automated feature learning, nonlinear modeling, adaptability, and generalization. This study confirmed practicality of the established strategy for efficient metabolomics data analysis and reliable classification performance even when using small-volume samples. This established approach holds promise for plant metabolomics and is not limited to ginseng.

Hybrid organic monolithic column containing MIL-68(Al) for the separation of small molecules by capillary HPLC

A hybrid organic monolithic column made of poly(lauryl methacrylate-co-1,6-hexanediol dimethacrylate) and the metal-organic framework MIL-68(Al) was prepared for the first time. The column was used in capillary liquid chromatography, both in isocratic and gradient elution modes. Separation performance towards small molecules of different chemical nature (polycyclic aromatic hydrocarbons, alkylbenzenes, phenols, etc.) was studied. Monte Carlo simulations were made to both select the proper precursors to obtain empty metal-organic framework micropores in the monolithic polymer and also, to analyze the potential free access of the studied analytes into the micropores (necessary to improve mass transfer and column efficiency). The hereby synthesized metal-organic framework microcrystals allowed obtaining homogeneous hybrid monolithic columns. Adding of MIL-68(Al) (1030 m2 g-1 BET specific surface area) increased the surface area from 3.9 m2 g-1 for the parent monolith to 18.2 m2 g-1 for the hybrid column containing 8 mg mL-1 of the microcrystals. Chromatographic performance of this new column was evaluated by studying retention factors, resolution, and plate counts at room temperature. Different compounds, not completely resolved in the parent monolith, were partially or completely separated after metal-organic framework addition. Using the monolithic column with only 2 mg mL-1 of MIL-68(Al), five alkylbenzenes were completely separated with very symmetrical peak shapes, resolution factors up to 3.60 and plate counts of 4300 plates m-1 for n-hexylbenzene. This value is higher than those obtained by other authors who used organic monolithic columns with embedded metal-organic frameworks to perform separations at room temperature. Additionally, nine polycyclic aromatic hydrocarbons were partially or completely resolved in gradient elution mode. The hybrid monolithic columns exhibited very good intra-day (%RSD=1.9), inter-day (%RSD=2.6), and column-to-column (%RSD=4.3) reproducibility values. Easy and fast column preparation, and versatility to efficiently separate several compounds of different chemical nature in isocratic and gradient mode, makes this new hybrid column a very good option for the analysis of small molecules in capillary (or nano) HPLC.

Enantiomeric Separation of Triacylglycerols Consisting of Three Different Fatty Acyls and Their Chiral Chromatographic Elution Behavior.

Chromatographic separation of triacylglycerol (TG) enantiomers is a highly challenging task of analytical chemistry because of the similar physicochemical properties. The analysis of chiral TGs is crucial for improving the knowledge of lipid biochemistry and for understanding the nutritional properties of fats and oils. Thus, this study aimed to systematically investigate the chiral resolution of TGs consisting of three different fatty acyls (FAs). Thirty-three asymmetric TG enantiopairs, including 49 synthesized enantiopure TGs and racemic TGs, were analyzed with a recycling chiral HPLC system. Twenty-six enantiopairs were successfully separated. Overall, having both unsaturated and saturated FAs in the outer positions or a difference in carbon chain length between two saturated FAs at the outer positions favored the separation of enantiomers. The retention time at separation correlated negatively with the sn-3 carbon number of the early eluting enantiomer and positively with the carbon number difference between sn-1 and sn-3. When the samples were studied in separate groups based on unsaturation and regioisomers, both the acyl carbon number and the degree of unsaturation of FAs in all three positions influenced the separation and elution behavior of chiral TGs, indicating an active role of both intermolecular interactions and steric hindrances. This is the first systematic study of the chiral separation of TGs consisting of three different FAs using a large number of enantiopairs. The novel findings on the behavior of TG enantiomers in a chiral environment provide important guidance and reference for a stereospecific study of the chemistry and biochemistry of natural lipids.

Nanoplastic Contamination in Freshwater Biofilms Using Gel Permeation Chromatography and Plasmonic Nanogold Sensor Approaches.

The worldwide contamination of aquatic ecosystems by plastics is raising concern, including their potential impacts on the base of the food chain, which has been poorly documented. This study sought to examine, for the first time, the presence of nanoplastics (NPs) in biofilms from freshwater streams/rivers. They were collected at selected polluted sites, such as the industrial sector for plastic recycling and production, miscellaneous industries, agriculture, municipal wastewaters/effluents and road runoffs. In parallel, the functional properties of sampled biofilms were determined by proteins, lipids, esterase (lipase), viscosity and oxidative stress. The results revealed that biofilms collected at the plastic industries and road runoffs contained the highest NP levels based on size exclusion chromatography, fluorescence detection and a new nanogold sensor visualization method. Examination of the chromatographic elution profiles showed increased abundance and size of NPs in the 10-150 nm size range at the polluted sites. Biofilms from the plastic industry site had elevated levels of aldehydes (oxidative stress) and lipids compared to the other sites. Biofilms collected at the municipal sites had elevated levels of proteins and esterases/lipases, with a decrease in total lipids. Biofilms collected at agriculture sites had the lowest levels of NPs in this campaign, but more samples would be needed to confirm these trends. In conclusion, biofilms represent an important sink for plastics in freshwater environments and display signs of distress upon oxidative stress.

To Study Force Degradation Of Ozenoxacin Using RP-HPLC

In this study a simple, robust and accurate RP-HPLC was developed and validated for the force degradation study of ozenoxacin as per ICH guidelines. Chromatographic separation and elution of peak was achieved by isocratic elution on agilant – 1260 infinite II HPLC system. An analysis C18X150X4.6mm 5mm, eclipse XDB column was used at flow rate 1ml/min in the mobile phase ratio methanol: 10 mM (adjusted pH -3 in the ratio of 42:52 %v/v at wavelength 250 nm and the temperature 25ºc. The force degradation study was performed on ozenoxacin under acidic, basic, oxidation, thermal, photodegradation,. The result obtained from the experiments was proved that the developed method suitable for routine analysis.

Quantitative and equipment-free paper-based agglutination assay of bacterial cells.

Background: point-of-care (POC) tests are useful for bedside/home applications, emergencies, frequent follow-ups, and resource-limited areas. Limited quantitative and equipment-free POC assays have been reported. This study aims to develop, validate, and apply a simple, quantitative, paper-based POC assay. Methods: wax-channeled paper treated with specific anti-Brucella and anti-Salmonella antibodies was used for distance-based chromatographic elution of stained bacterial cell agglutinations. Results: a qualitative paper-based agglutination POC test was developed using color intensity, tail appearance, and "+/-" signs that clearly distinguish the positive and negative results. The optimization of the test for paper type, microfluidic channel design, antibody and bacterial cell concentrations, and elution methods was carried out. Quantitative assay transformation was successfully developed using the color intensity of the original reaction zone, intensity of elution tail, and distance-based migration that correspond to bacterial agglutination size. The migration distance of eluted bacterial agglutination bands corresponds to the target concentration with good linearity and minimal variability. Reporting of colored band migration with numbers using microfluidic patterns was used to enhance non-technical end-user applications. A distance-based POC assay prototype was then successfully used for the accurate detection of known and unknown samples in comparison with standard assays. Conclusions: the migration distance of an eluted stained bacterial agglutination correlated with anti-bacterial antibody concentrations. A simple, cheap, quantitative, and equipment-free paper-based POC assay of bacterial cell agglutination was developed. This test can be used for simple "+/-" results, thermometer-like quantification, or text reporting with numbers corresponding to target concentrations. The assay has extended applications to different human disease biomarkers.

Theoretical Investigation of Dual-Mode Gradient Elution Chromatography Considering Simultaneous Variations in the Column Temperature and Solvent Composition

Theoretical Investigation of Dual-Mode Gradient Elution Chromatography Considering Simultaneous Variations in the Column Temperature and Solvent Composition

Lentiviral vector determinants of anion-exchange chromatography elution heterogeneity.

The demand for Lentiviral Vector (LV) drug substance is increasing. However, primary capture using convective anion-exchange chromatographyremains a significant manufacturing challenge. This stems from a poor understanding of the complex adsorption behaviors linked to LVs intricate and variable structure, such as high binding heterogeneity which is typically characterized by a gradient elution profile consisting of two peaks. Understanding which LV structural components drive these phenomena is therefore crucial for rational process design. This work identifies the key LV envelope components responsible for binding to quaternary-amine membrane adsorbents. Eliminating the pseudotype protein (Vesicular Stomatitis Virus G glycoprotein [VSV-G])did not impact the heterogenous two-peak elution profile, suggesting it is not a major binding species. Digestion of envelope glycosaminoglycans (GAGs), present on proteoglycans, leads to a dramatic reduction in the proportion of vector eluted in peak 2, decreasing from 50% to 3.1%, and a threefold increase in peak 1 maximum. Data from reinjection experiments point towards interparticle envelope heterogeneity from discrete LV populations, where the two-peak profile emerges from a subpopulation of LVs interacting via highly charged GAGs (peak 2) along with a weaker binding population likely interacting through the phospholipid membrane and envelope protein (peak 1).

Real-time monitoring of the column chromatography process of Ginkgo biloba using near-infrared and Raman spectroscopy combined with spectral fusion strategy

In the column chromatography elution process of traditional Chinese medicine (TCM), the real-time monitoring of active components plays an important role in product quality control. In this study, the column chromatography elution process in preparing of Ginkgo biloba leaf extract was performed in bench scale, where near-infrared (NIR) spectroscopy and Raman spectroscopy were collected to determine the concentration changes of several critical quality attributes. Based on single spectral data and fused spectral data, partial least squares and support vector machine models were established, respectively. The results indicated that for most analytes, models based on Raman spectra were superior to NIR spectral models, and fusion models established by the mid-level data fusion approach were superior to the single spectral models. The coefficients of determination (Rt2) of the optimal Raman and mid-level data fusion models were all higher than 0.96. The concentration profiles predicted by the mid-level data fusion quantitative models were in better agreement with the real trends defined by measured values. This is the first-ever study reporting the real-time monitoring of TCM column chromatography process by Raman spectroscopy combined with NIR spectroscopy, having vital reference values for process monitoring and understanding of the column chromatography for other TCM.

Research progress on the detection of water-soluble vitamins in food using liquid chromatography

Currently, liquid chromatography is widely used for the analysis and determination of water-soluble vitamins (WSVs) in functional foods. However, due to the diversity of sample characteristics and the requirement for purification accuracy, the chromatographic conditions and specific methods used vary. This paper reviews the chromatographic conditions of liquid chromatography techniques for detecting WSVs in food, as well as the selection of liquid chromatography methods in different application fields. The chromatographic conditions include chromatographic columns, column temperature, mobile phase, flow rate, and elution gradient, and the application scope of different chromatographic conditions is compared. Liquid chromatography methods include high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry, and discussions are made on the applications and prospects of these two methods under different chromatographic conditions. Therefore, this review provides research insights into different chromatographic conditions and methods for food quality inspection and biochemical analysis research.

Discontinuous Galerkin finite element method for solving non-linear model of gradient elution chromatography

Discontinuous Galerkin finite element method for solving non-linear model of gradient elution chromatography

Abstract 114: Inhibition Of Lecithin Cholesterol Acyl Transferase By A Novel Anti-apolipoprotein A1 Antibody Fragment

Apolipoprotein A1 (APOA1), the most abundant protein in high-density lipoproteins (HDL), is the primary activator of lecithin cholesterol acyl transferase (LCAT). LCAT’s cholesteryl esterification activity is key for maturing nascent HDL discs into the spherical forms commonly found in plasma. However, the molecular mechanism for APOA1 activation of LCAT is poorly understood. Using phage display selection against nanodiscs (lipid complexes stabilized by an APOA1 variant commonly used in transmembrane protein structural biology), we have identified, isolated, and expressed a novel Fab fragment (Fab16). Size exclusion chromatography elution shift experiments showed that Fab16 readily bound reconstituted HDL (rHDL) particles containing human purified plasma APOA1 with nearly quantitative binding at a 1:1 molar ratio of Fab16 to APOA1. Negative stain electron microscopy (NSEM), confirmed that 1-2 Fab16 bound to each rHDL particle in a specific pattern around the disc periphery. Additionally, Fab16 effectively bound both large and small spherical HDL particles isolated from human plasma. Chemical cross-linking / mass spectrometry experiments indicated that Fab16 binds an APOA1 epitope between residues 94 and 118 (i.e., within amphipathic helix 4). As our previous work has shown that APOA1 helix 4 is important for LCAT activation, we hypothesized that Fab16 would inhibit LCAT activity. Using a novel gas chromatography cholesterol esterification assay, we found that Fab16 applied at a 1.5:1 molar ratio to APOA1 inhibited LCAT cholesterol esterification activity by 56±7%. The symmetrical nature of both discoidal and spherical HDL particles has hampered the application of cryogenic electron microscopy (cryo EM) for understanding HDL structure. Fab16 may provide a valuable ‘landmark’ to assist in deriving a high-resolution structure of the complex. Work to optimize conditions for collecting high quality cryo EM datasets of discoidal and spherical HDL in the presence and absence of LCAT is ongoing. Deriving a high-resolution understanding of HDL structure and its interaction with remodeling factors such LCAT has been a long-standing problem but is crucial for deriving new strategies to manipulate HDL function for therapeutic benefit.

Numerical Approximation of a Nonequilibrium Model of Gradient Elution Chromatography Considering Different Functional Relationships between Model Parameters and Solvent Composition.

In this paper, a rigorous theoretical study is conducted to analyze the influence of varying solvent compositions on the retention characteristics of elution profiles within a fixed-bed liquid chromatographic column. In gradient chromatography, the propagation speed of elution profiles is manipulated through a progressive variation in the mobile-phase composition. Consequently, enhanced separation of the mixture components can be achieved together with a reduction in the requisite recycling times for subsequent injections. In other words, both the efficiency and the selectivity of the column can be enhanced. The lumped kinetic model coupled with the convection-diffusion equation for the volume fraction of the solvent is applied to simulate the process. The resulting nonlinear model equations are numerically solved by applying a semidiscrete second-order finite-volume method. The numerical solutions are utilized to quantify the effects of gradient starting and ending times, solvent composition, solvent strength parameters, and gradient slope on the concentration profiles. Additionally, temporal numerical moments are plotted versus the starting and ending times of the gradient, and standard performance criteria are presented for evaluating the process performance. The outcomes of this investigation will contribute to further enhancements in gradient elution chromatography.

Model-based design of gradient elution in liquid-liquid chromatography: Application to the separation of cannabinoids

Liquid-liquid chromatography (LLC) is a separation technique that utilizes a biphasic solvent system as the mobile and stationary phases. The components are separated solely due to their different distributions between the two liquid phases. Gradient change in the mobile phase composition during the chromatographic process is a powerful method for improving the resolution of separation or shortening the process time. Gradient elution readily applies to LLC with biphasic solvent systems in which the stationary phase composition remains nearly constant when the mobile phase composition changes. This work proposes a model-based approach to optimize gradients in LLC and circumvent tedious trial-and-error experiments. The solutes’ distribution constant depends on the mobile phase composition. Thus, the distribution constants were described as a function of the content of one of the solvents (= modifier) in the mobile phase. The dispersive and mass-transfer effects in the tubing and the column are modeled with a stage model. Only a few experiments are required to determine the model parameters. After the validation of the model and its parameters, the model can be used for LLC gradient optimization. The proposed approach was demonstrated for a gradient LLC separation of a mixture of four cannabinoids. Two different gradient shapes, one-step and linear gradient, were considered. For a pre-selected minimal purity requirement, the gradient was optimized for maximum process efficiency, defined as the product of productivity and yield. An experiment conducted with the optimized gradient conditions was in good agreement with the simulation, showing the potential of the proposed method.

Exclusion liquid chromatography of carboxymethylchitin and carboxymethylchitosan from crustacean <i>Artemia Parthenogenetica</i> of the Aral Sea

The article deals with the synthesis of water-soluble derivatives of chitin and chitosan from cysts of crustaceans of the genus Artemia Parthenogenetica of the Aral Sea by carboxymethylation of OH and NH2 groups of their chains. The elution, viscosity and polyelectrolyte properties in size exclusion liquid chromatography of carboxymethylchitin and carboxymethylchitosan were studied. The suppression of electrostatic effects and the implementation of the exclusion mechanism for the separation of these polysaccharides were achieved by using an aqueous solution of sodium nitrate with a concentration of 0.1 mol/l as an eluent, which was recommended for determining molecular weight characteristics.

Land-use classification of Malaysian soils by ultra-high performance liquid chromatography (UHPLC)-based untargeted data combined with chemometrics for forensic provenance

Soil has become the most precious trace evidence attributed to its transference and persistence, but little is known about the capability of non-volatile organic fractions of soil in predicting land-use class. Hence, this study devoted to discriminating soils into four land-use classes based on chromatographic data and predictive modelling. Nine sites representing four different land-use classes were selected for collecting 73 soil samples via a grid method. Then, all the soil samples were dried, homogenized, sieved and eventually extracted using acetonitrile. The extracts were further analyzed via an isocratic elution in an ultra-high performance liquid chromatography (UHPLC) system. The pixel-level chromatograms were carefully assessed via diverse data preprocessing (DP) methods including baseline correction, normalization and derivative algorithms. Furthermore, the retention time (RT) window was also segmented into several sub-windows and thoroughly evaluated. Prediction accuracy of classification and regression tree (CART) modelling as estimated using 100 subsets of training samples on the corresponding testing and blind samples were ranked using TOPSIS method for identifying the most desired sub-window and DP strategy. The sub-RT window covering 0–5 min preprocessed via modified polynomial fitting algorithm followed by vector normalization emerged to be the most desired sub-dataset. The best sub-dataset modelled using CART and partial least squares-discriminant analysis (PLS2-DA) algorithms achieved 79.5 % and 97.4 % accuracy in predicting the land-use classes of 13 mixed samples. In conclusion, UHPLC-based fingerprint technique coupled with predictive modelling shows great potential in inferring land-use class of trace amount of soil.

Development of an On-Column Trace Enrichment Method for the Determination of Sub-μg/L Bisphenol A in Bottled Water by RP-HPLC with Fluorescent Detection.

A simple extraction-free, on-column trace enrichment liquid chromatographic method for the determination of trace levels of bisphenol A (BPA) in bottled water samples has been developed. It was found possible to determine ng/L (ppt) levels of BPA by the direct introduction of 6 mL of sample water to the HPLC column utilising fluorescence detection (Exλ = 274 nm, Emλ = 314 nm). Following the loading of the sample and the chromatographic focusing of the BPA on the analytical column, a simple switch from the aqueous sample to the isocratic chromatographic elution step of 50% acetonitrile/deionised water was undertaken. Using a BPA concentration of 0.596 μg/L the effect of sample volume was investigated over the range 1.0 to 12 mL. A linear relationship with the sample volume introduced to the HPLC column and the resulting peak height for BPA was found over the entire range investigated (R2 = 0.999). Using a sample volume of 6.0 mL, a well-defined chromatographic peak was recorded for BPA over the concentration range of 0.1 μg/L to 6.25 μg/L (R2 = 0.9998). A limit of detection of 0.058 μg/L for BPA was calculated based on 3 δ. A mean recovery of 100% with an associated %CV of 7.6% (n = 5) was obtained for a bottled spring water sample fortified with 1.25 μg/L BPA. Samples can be processed in under 12 minutes, much faster than that commonly reported for conventional offline extraction and chromatographic-based methods. The results show that the optimised method holds promise for the determination of BPA in such samples.

Enhanced efficiency of MS/MS all-ion fragmentation for non-targeted analysis of trace contaminants in surface water using multivariate curve resolution and data fusion

Data-independent acquisition–all-ion fragmentation (DIA-AIF) mode of mass spectrometry can facilitate wide-scope non-target analysis of contaminants in surface water due to comprehensive spectral identification. However, because of the complexity of the resulting MS2 AIF spectra, identifying unknown pollutants remains a significant challenge, with a significant bottleneck in translating non-targeted chemical signatures into environmental impacts. The present study proposes to process fused MS1 and MS2 data sets obtained from LC-HRMS/MS measurements in non-targeted AIF workflows on surface water samples using multivariate curve resolution-alternating least squares (MCR-ALS). This enables straightforward assignment between precursor ions obtained from resolved MS1 spectra and their corresponding MS2 spectra. The method was evaluated for two sets of tap water and surface water contaminated with 14 target chemicals as a proof of concept. The data set of surface water samples consisting of 3506 MS1 and 2170 MS2 AIF mass spectral features was reduced to 81 components via a fused MS1-MS2 MCR model that describes at least 98.8% of the data. Each component summarizes the distinct chromatographic elution of components together with their corresponding MS1 and MS2 spectra. MS2 spectral similarity of more than 82% was obtained for most target chemicals. This highlights the potential of this method for unraveling the composition of MS/MS complex data in a water environment. Ultimately, the developed approach was applied to the retrospective non-target analysis of an independent set of surface water samples.Graphical abstract