Separation Of Complex Mixtures Research Articles

Separation of triacylglycerol (TAG) isomers by cyclic ion mobility mass spectrometry

Triacylglycerols (TAGs), a major lipid class in foods and the human body, consist of three fatty acids esterified to a glycerol backbone. They can occur in various isomeric forms, including sn-positional, cis/trans configurational, acyl chain length, double bond positional, and mixed type isomers. Separating isomeric mixtures is of great interest as different isomers can have distinct influence on mechanisms, such as digestibility, oxidative stability, or lipid metabolism. However, TAG isomer separation remains challenging with established analytical methodologies such as liquid-chromatography coupled to mass spectrometry (LC-MS). In this study, we developed a method with cyclic ion mobility mass spectrometry (cIMS-MS) for the separation and identification of all types of TAG isomers. First, the influence of different adducts (Li+, NH4+, Na+, and K+) on the separation was studied. Overall, it was concluded that the sodium adduct is the best choice to efficiently separate all types of TAG isomers. In addition, trends were found in the influence of specific structural features on the drift time order. An order of relative influence (from high to low) was established; (1) degree of unsaturation of the fatty acid(s) on an exterior position (if the total degree of unsaturation(s) is equal in both TAGs), (2) acyl chain length on the exterior positions, (3) cis/trans configuration, and (4) double bond (DB)-position. Finally, various cIMS-MS strategies were developed for the separation of mixtures containing four, five, and six isomers. To conclude, the developed methods can be used for separation of complex mixtures of TAG isomers and have great potential to be expanded to isomers of similar types of lipids such as di- and monoacylglycerols. This study also shows the potential of cIMS-MS to be used for the application on real TAG samples.

Nanofractionation Analytics for Comparing MALDI-MS and ESI-MS Data of Viperidae Snake Venom Toxins.

Worldwide, it is estimated that there are 1.8 to 2.7 million cases of envenoming caused by snakebites. Snake venom is a complex mixture of protein toxins, lipids, small molecules, and salts, with the proteins typically responsible for causing pathology in snakebite victims. For their chemical characterization and identification, analytical methods are required. Reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (RP-LC-ESI-MS) is a widely used technique due to its ease of use, sensitivity, and ability to be directly coupled after LC separation. This method allows for the efficient separation of complex mixtures and sensitive detection of analytes. On the other hand, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is also sometimes used, and though it typically requires additional sample preparation steps, it offers desirable suitability for the analysis of larger biomolecules. In this study, seven medically important viperid snake venoms were separated into their respective venom toxins and measured by ESI-MS. In parallel, using nanofractionation analytics, post-column high-resolution fractionation was used to collect the eluting toxins for further processing for MALDI-MS analysis. Our comparative results showed that the deconvoluted snake venom toxin masses were observed with good sensitivity from both ESI-MS and MALDI-MS approaches and presented overlap in the toxin masses recovered (between 25% and 57%, depending on the venom analyzed). The mass range of the toxins detected in high abundance was between 4 and 28 kDa. In total, 39 masses were found in both the ESI-MS and/or MALDI-MS analyses, with most being between 5 and 9 kDa (46%), 13 and 15 kDa (38%), and 24 and 28 kDa (13%) in size. Next to the post-column MS analyses, additional coagulation bioassaying was performed to demonstrate the parallel post-column assessment of venom activity in the workflow. Most nanofractionated venoms exhibited anticoagulant activity, with three venoms additionally exhibiting toxins with clear procoagulant activity (Bothrops asper, Crotalus atrox, and Daboia russelii) observed post-column. The results of this study highlight the complementarity of ESI-MS and MALDI-MS approaches for characterizing snake venom toxins and provide a complementary overview of defined toxin masses found in a diversity of viper snake venoms.

2D-HPLC-MS Technology Combined with Molecular Network for the Identification of Components in Tibetan medicine Aconitum pendulum.

In this study, a comprehensive approach was employed, utilizing 2D-HPLC-MS technology in conjunction with the molecular network to unravel the intricate chemical composition of the Tibetan medicinal plant APB. Through the implementation of 2D-HPLC, enhanced separation of complex mixtures was achieved, enabling the isolation of individual compounds for subsequent analysis. The molecular network approach further aided in elucidating structural relationships among these compounds, contributing to the determination of potential bioactive molecules. This integrated strategy efficiently identified a wide array of chemical components present within the plant. The findings revealed a diverse spectrum of chemical constituents within APB, including alkaloids, among others. This research not only advances understanding of the phytochemical profile of this traditional Tibetan medicine but also provides valuable insights into its potential therapeutic properties. The integration of 2D-HPLC-MS and molecular network proves to be a powerful tool for systematically exploring and identifying complex chemical compositions in herbal medicines, paving the way for further research and development in the field of natural product discovery.

Comparison of System Constants of Thermostable Polysiloxane Stationary Liquid Phases of Different Origins

The expansion of possible areas of use of modern gas chromatography is largely determined by the thermal stability of stationary liquid phases (SLP), which allows the separation of complex mixtures of high-boiling compounds. A way to increase the thermal stability of nonpolar and weakly polar polysiloxane SLPs is to introduce rigid fragments into the polysiloxane chain. Currently, there is an extensive set of columns with thermostable SLPs, which, in some cases, have the index “ms”. However, in most cases, the nature of these phases and the methods for preparing the columns are not disclosed. In this work, using the linear free energy relationship (LFER) model, we compared the selectivity of a number of proprietary columns with in-house columns in which the siloxane–silarylene phase and the column preparation methods themselves are known. Various types of intermolecular interactions are considered within the framework of the LFER model.

Data-driven predictions of complex organic mixture permeation in polymer membranes

Membrane-based organic solvent separations are rapidly emerging as a promising class of technologies for enhancing the energy efficiency of existing separation and purification systems. Polymeric membranes have shown promise in the fractionation or splitting of complex mixtures of organic molecules such as crude oil. Determining the separation performance of a polymer membrane when challenged with a complex mixture has thus far occurred in an ad hoc manner, and methods to predict the performance based on mixture composition and polymer chemistry are unavailable. Here, we combine physics-informed machine learning algorithms (ML) and mass transport simulations to create an integrated predictive model for the separation of complex mixtures containing up to 400 components via any arbitrary linear polymer membrane. We experimentally demonstrate the effectiveness of the model by predicting the separation of two crude oils within 6-7% of the measurements. Integration of ML predictors of diffusion and sorption properties of molecules with transport simulators enables for the rapid screening of polymer membranes prior to physical experimentation for the separation of complex liquid mixtures.

Dialkyl Carbonates as Green Solvents for Polyvinylidene Difluoride Membrane Preparation

Membrane processes are employed in a wide variety of industrial applications such as separation of complex mixtures, hydrogen isolation, CO2 removal, wastewater treatment, etc. Their use allows energy savings on the production cost compared to other traditional separation technologies. Nevertheless, the preparation of membranes not always fulfills sustainability obligations, especially when considering the commonly employed solvents, i.e., N-methyl-2-pyrrolidone and N,N-dimethylformamide, to mention just a few. Dialkyl carbonates (DACs) are well-known green solvents and reagents that have been extensively investigated as safe alternatives to chlorine-based compounds and media such as alkyl halides, phosgene, and chlorinated solvents. Following our recent study on a scale-up procedure to non-commercially available or expensive DACs, herein we report for the first time the application of organic carbonates as green media for membrane preparation. Theoretical thermodynamic studies were first carried out to predict the solubilities in DACs of different polymers commonly employed for membranes preparation. As a result, the use of selected organic carbonates as media for polyvinylidene difluoride membrane preparation was investigated by nonsolvent-induced phase separation (NIPS) and a combination of vapor-induced phase separation (VIPS)-NIPS techniques. Membranes obtained with custom-made DACs displayed greater structural resistances and smaller pore sizes compared to the ones achieved using commercially available cyclic organic carbonates. Data collected showed that it was possible to achieve a wide variety of dense and porous membranes by using a single family of compounds, highlighting once again the great versatility of DACs as green solvents.

High-Performance Chromatographic Separation of Cerebrosides.

High-performance thin layer chromatography (HPTLC) is a very robust, fast, and inexpensive technique that enables separation of complex mixtures. Here, we describe the analytical separation of glucosylceramide and galactosylceramide by HPTLC. This technique can be used for quantitation purposes but also with small modification for subsequent mass spectrum analysis for structural determination.

Flash Chromatography and Semi-Preparative HPLC: Review on the Applications and Recent Advancements over the Last Decade

Flash chromatography and Semi-Preparative High Performance Liquid Chromatography have showed themselves as promising separation tools. These techniques assist in the process of isolation, fractionation and purification of chemical, biological and pharmaceutical substances. The present article describes the recent advancements made in flash chromatography and semi-preparative HPLC techniques in the last decade with specific focus on natural products. This article highlights the basics, instrumentation, current advancements made to facilitate the separation, advantages and applications of these two techniques. Flash chromatography is a versatile tool for the rapid but efficient separation and purification of analytes in relatively pure form. Nowadays, there are even functionalized silica pre-packed cartridges with different silica weights and particle sizes that are available to use. Flash separation using dual columns can be used to enhance separation of complex mixtures and can be applied to numerous classes of compounds. The semipreparative techniques, despite their high cost, can be helpful in high purity separation and production of reference standards for the pharmaceutical industry. This comprehensive review presents a brief analysis of all the recent research employing these two techniques for varied applications. This review will help chromatography specialists to make the decision of applying these two techniques in order to accelerate their research and development journey.

High-Resolution Liquid Chromatography-Mass Spectrometry for Lipidomics.

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is a powerful tool for identification and classification of lipids. Ultra-high performance liquid chromatography (UHPLC) allows for robust separations of complex mixtures, while high-resolution mass spectrometry (HRMS) identifies compounds with efficiency and accuracy (Zullig T and Kofeler HC, Mass Spectrom Rev 40:162-176, 2021). The high specificity and sensitivity of mass spectrometry makes it the method of choice when analyzing lipids (Kofeler HC, J Lipid Res 62:100138, 2021). Untargeted mass spectrometry identifies all lipids within a sample and is useful for identification and further discovery. This chapter describes the use of a Q Exactive mass spectrometer to perform an untargeted LC-MS/MS lipidomics analysis.

Liquid Membranes in Catalysis

: The supported ionic liquid (SIL) membranes have demonstrated huge potential for numerous applications in current separation science and catalysis. Membrane technology allows for the separation of complex mixtures of gases, vapors, liquids and /or solids below trivial conditions. Simultaneous chemical transformations can also be achieved in membranes by using catalytically active materials comprising the membrane or embedded catalysts in the custom-built membrane reactors. In the present editorial, the remarkable contribution of liquid membranes in catalysis is highlighted. Some recent applications are presented and compared with conventional methods. In addition, SILs and their applications in catalysis, catalytic membranes, and recent advances in membrane separation processes are briefly described.

Continuous chromatography applied to the purification of sugars: a proven separation technology with multiple industrial applications

Low-pressure chromatography is a widely implemented purification technique in the sugar industry. It allows for the separation of complex mixtures with different affinities based on a stationary phase, composed of ion exchange resins, and a mobile phase, which is generally water. Industrially, it is implemented in columns with volumes that can reach several hundred cubic meters. The real-life performance for the purification of 2G sugars and organic acids and the advantage of coupling chromatography and nanofiltration for the production of high purity fructose will be explained further in this article.

Trapping-Enrichment Multi-dimensional Liquid Chromatography with On-Line Deuterated Solvent Exchange for Streamlined Structure Elucidation at the Microgram Scale.

At the forefront of chemistry and biology research, development timelines are fast-paced and large quantities of pure targets are rarely available. Herein, we introduce a new framework, which is built upon an automated, online trapping-enrichment multi-dimensional liquid chromatography platform (TE-Dt-mDLC) that enables: 1) highly efficient separation of complex mixtures in a first dimension (1 D-UV); 2) automated peak trapping-enrichment and buffer removal achieved through a sequence of H2 O and D2 O washes using an independent pump setup; and 3) a second dimension separation (2 D-UV-MS) with fully deuterated mobile phases and fraction collection to minimize protic residues for immediate NMR analysis while bypassing tedious drying processes and minimizing analyte degradation. Diverse examples of target isolation and characterization from organic synthesis and natural product chemistry laboratories are illustrated, demonstrating recoveries above 90 % using as little as a few micrograms of material.

Trapping‐Enrichment Multi‐dimensional Liquid Chromatography with On‐Line Deuterated Solvent Exchange for Streamlined Structure Elucidation at the Microgram Scale

AbstractAt the forefront of chemistry and biology research, development timelines are fast‐paced and large quantities of pure targets are rarely available. Herein, we introduce a new framework, which is built upon an automated, online trapping‐enrichment multi‐dimensional liquid chromatography platform (TE‐Dt‐mDLC) that enables: 1) highly efficient separation of complex mixtures in a first dimension (1D‐UV); 2) automated peak trapping‐enrichment and buffer removal achieved through a sequence of H2O and D2O washes using an independent pump setup; and 3) a second dimension separation (2D‐UV‐MS) with fully deuterated mobile phases and fraction collection to minimize protic residues for immediate NMR analysis while bypassing tedious drying processes and minimizing analyte degradation. Diverse examples of target isolation and characterization from organic synthesis and natural product chemistry laboratories are illustrated, demonstrating recoveries above 90 % using as little as a few micrograms of material.

Microchip Free-Flow Electrophoresis for Bioanalysis, Sensing, and Purification.

The separation of complex mixtures is ubiquitous throughout molecular biology, and techniques such as gel-based electrophoresis are common laboratory practice. Such methods are not without their drawbacks, however, which include non-specific interactions between analyte and the separation matrix, poor yields in purification and non-continuous analyte throughput. Microfluidic techniques, which exploit physical phenomena unique to the microscale, promise to improve many aspects of traditional laboratory procedures. These methods offer a quantitative, solution-based alternative to traditional gel electrophoresis, with rapid measurement times enabling the analysis of transient or weak biomolecular interactions that would be challenging to observe with traditional methods. Here, we present a protocol for the lithographic fabrication and operation of microfluidic chips capable of free-flow electrophoretic (FFE) fractionation and analysis of biological analytes. We demonstrate the efficacy of our approach through a protein-sensing methodology based on FFE fractionation of DNA-protein mixtures. In addition, the FFE technique described here can be readily adapted to suit a variety of preparative and analytical applications, providing information on the charge, zeta-potential, and interactions of analytes.

A versatile method to separate complex lipid mixtures using 1-butanol as eluent in a reverse-phase UHPLC-ESI-MS system

Simple, robust and versatile LC–MS based methods add to the rapid assessment of the lipidome of biological cells. Here we present a versatile RP-UHPLC-MS method using 1-butanol as the eluent, specifically designed to separate different highly hydrophobic lipids. This method is capable of separating different lipid classes of glycerophospholipid standards, in addition to phospholipids of the same class with a different acyl chain composition. The versatility of this method was demonstrated through analysis of lipid extracts of the bacterium Escherichia coli and the archaeon Sulfolobus acidocaldarius. In contrast to 2-propanol-based methods, the 1-butanol-based mobile phase is capable of eluting highly hydrophobic analytes such as cardiolipins, tetraether lipids and mycolic acids during the gradient instead of the isocratic purge phase, resulting in an enhanced separation of cardiolipins and extending the analytical range for RPLC.

Multiple Heart-Cutting Two-Dimensional Liquid Chromatography: Recent Developments and Applications

Background: Over the last few decades, there has been a growing interest in Two- Dimensional Liquid Chromatography (2D-LC). This advanced technology has become a relevant solution for solving all the analytical challenges encountered in the pharmaceutical industry. The primary purpose of this review is to give a brief account of the developing techniques notable in 2D-LC. Methods: Review of recent literature on advanced separation techniques for complex mixtures revealed that 2D-LC is the most widely used technique in the pharmaceutical laboratory for the separation of complex samples. Among the various techniques used in 2D-LC, comprehensive 2D-LC and heart-cutting 2D-LC are highly used techniques. To enhance the resolution, further development has been made to heart-cutting 2D-LC called multiple heart-cutting 2D-LC, which has recently become a very popular technique in the pharmaceutical industry. Therefore research articles mentioning the use of multiple heart-cutting 2D-LC for separation of complex mixtures were reviewed. Results: The most crucial benefit of multiple heart-cutting 2D-LC is its reduced effort in method development and also the storage of multiple sample sections for further analysis. The multiple heartcutting 2D-LC is suited, preferably for method development and impurity analysis of pharmaceutical substances, fine chemicals and offers better peak separation, which is otherwise not possible by comprehensive 2D-LC. Conclusion: The advantage of two-dimensional liquid chromatography over one-dimensional liquid chromatography is its high resolving power. Further improvement in resolution was achieved by using the advanced multiple heart-cutting 2D-LC which appears to have an imperative role in pharmaceutical and food analysis.

Development of comprehensive two-dimensional low-flow liquid-chromatography setup coupled to high-resolution mass spectrometry for shotgun proteomics

Bottom-up proteomics provides often small amounts of highly complex samples that cannot be analysed by direct mass spectrometry (MS). To gain a better insight in the sample composition, liquid chromatography (LC) and (comprehensive) two-dimensional liquid chromatography (2D-LC or LC × LC) can be coupled to the MS. Low-flow separations are attractive for HRMS analysis, but they tend to be lengthy. In this work, a low-flow, online, actively modulated LC × LC system, based on hydrophilic-interaction liquid chromatography (HILIC) in the first dimension and reversed-phase liquid chromatography (RPLC) in the second dimension, was developed to separate complex mixtures of peptides. Miniaturization permitted the analysis of small sample amounts (1–5 μg) and direct coupling with micro-ESI MS (1 μL min−1). All components were focused and automatically transferred from HILIC to RPLC using stationary-phase-assisted active modulation (C18 traps) to deal with solvent-incompatibility or dilution issues. Optimization of the setup was performed for the HILIC columns and the RPLC columns to provide a more efficient separation and higher identification rates than obtained using one-dimensional (1D) LC. A 60% increase in peak capacity was obtained with the 2D setup compared to a 1D-RPLC separation and a 17–34% increase in the number of proteins identified was achieved for the samples analysed (2D-yeast-8280 peptides and 2D-kidney tissue-8843 peptides), without increasing the analysis time (2 h).

THE USE OF PFEIFFER CHROMATOGRAPHY AS A TOOL FOR EVALUATING AGROECOLOGICAL MANAGEMENT IN AN AGRICULTURAL PROPERTY IN THE MUNICIPALLY OF BOQUEIRÃO – PB

Pfeiffer Circular Chromatography is seen as a tool used for physical separation of complex mixtures with the potential to holistically portray the biological attributes of the soil, providing the monitoring of agroecological managements. The objective here was to evaluate the soil quality of a family-based agroecosystem in the municipality of Boqueirão - PB, in order to guide decision-making with regard to the managements used. The design of the research is in accordance with (Pfeiffer, 1980), and has been adapted to the conduction optics for the peasantry. Soil samples were collected at a depth of 0-20cm, with Dutch auger dried in the shade and screened. Two solutions were used in different stages of conduction: dispersant (NaOH) and photoreactive (AgNO3). JPLAB 15cm filter papers were used, which were prepared for the production of chromatograms. The samples went through a stirring process interspersed with a rest period: 15min, 1h and 6h. The results showed good revelations of the chroma, which allowed us to infer the main indicators of each area. From the interpretations that chromatography allows, it was found that chroma C presents the best results in terms of soil health, since it expressed high enzymatic activity. On the other hand, chroma A and B did not express good conditions, evidencing the need for changes in the management condition.

Modern Trends in Mixed-Mode Liquid Chromatography (LC) Columns

Commercialization of columns that provide multiple modes of chromatographic separations have recently been on the rise. For example, combinations of retention modes, such as ion-exchange and reversed-phase, often enable the separation of complex mixtures of analytes not possible using single-mode columns. In this work, recent trends in what is often referred to as “mixed-mode” phase are investigated. Particular attention is paid to recent fundamental research, stationary phase development and design, and areas of application.

Mapping the Separation Landscape in Two-Dimensional Liquid Chromatography: Blueprints for Efficient Analysis and Purification of Pharmaceuticals Enabled by Computer-Assisted Modeling.

Recent developments in two-dimensional liquid chromatography (2D-LC) now make separation and analysis of very complex mixtures achievable. Despite being such a powerful chromatographic tool, current 2D-LC technology requires a series of arduous method development activities poorly suited for a fast-paced industrial environment. Recent introductions of new technologies including active solvent modulation and a support for multicolumn 2D-LC are helping to overcome this stigma. However, many chromatography practitioners believe that the lack of a systematic way to effectively optimize 2D-LC separations is a missing link in securing the viability of 2D-LC as a mainstay for industrial applications. In this work, a computer-assisted modeling approach that dramatically simplifies both offline and online 2D-LC method developments is introduced. Our methodology is based on mapping the separation landscape of pharmaceutically relevant mixtures across both first (1D) and second (2D) dimensions using LC Simulator (ACD/Labs) software. Retention models for 1D and 2D conditions were built using a minimal number of multifactorial modeling experiments (2 × 2 or 3 × 3 parameters: gradient slope, column temperature, and different column and mobile phase combinations). The approach was first applied to online 2D-LC analysis involving achiral and chiral separations of complex mixtures of enantiomeric species. In these experiments, the retention models proved to be quite accurate for both the 1D and 2D separations, with retention time differences between experiments and simulations of less than 3.5%. This software-based concept was also demonstrated for offline 2D-LC purification of drug substances.