Chromatography Performance Research Articles

Discovery of broad-spectrum high-affinity peptide ligands of spike protein for the vaccine purification of SARS-CoV-2 and Omicron variants

To combat with emerging SARS-CoV-2 variants of concern (VOCs), we report the identification of a set of unique HWK-motif peptide ligands for the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein from a phage-displayed peptide library. These HWK-motif peptides exhibited nanomolar affinity for RBD. Among them, the peptide, HWKAVNWLKPWT (SP-HWK), had not only the highest affinities for RBD and trimer S protein, but also broad-spectrum affinities for RBDs from VOCs. Molecular dynamics simulations and competitive ELISA revealed a conserved pocket between the cryptic and the outer faces of RBD for SP-HWK binding, distinct from the human angiotensin-converting enzyme 2 receptor binding site. By coupling SP-HWK to agarose gel, the as-prepared affinity gel could efficiently capture RBD and trimer S from the ancestral strain and the Omicron variant, and the bound targets could be recovered by mild elution at pH 6.0. More importantly, the affinity gel presented excellent and stable chromatographic performance in the purification of inactivated SARS-CoV-2 and Omicron vaccines, affording high yields and purities, and strong HCP reduction. The results demonstrated the potential of SP-HWK as a broad-spectrum peptide ligand for developing a universal platform for the vaccine purification of SARS-CoV-2 and VOCs.

Cyano-modified molecular cage silica gel stationary phase: Multi-functional chromatographic performance by high-performance liquid chromatography

This study successfully prepared different loading levels of cyano-functionalized RCC3 molecular cage silica gel stationary phase (RCC3-CN@SiO2) through aldehyde-amine condensation reaction and subsequent modification strategies. Fourier transform infrared spectroscopy, thermogravimetric analysis, nitrogen adsorption-desorption, and scanning electron microscopy confirmed the successful synthesis of RCC3-CN@SiO2 chromatographic stationary phase. The research demonstrates that due to hydrophobic/hydrophilic interactions, π-π interactions, hydrogen bonding, and size-selective porous structure, the stationary phase effectively separates moderately polar and weakly polar compounds in reversed-phase liquid chromatography (RPLC) mode, exhibiting hydrophobic selectivity comparable to the commercial DaisoC18-RP columns. Additionally, the tertiary amine and cyanogen groups on the molecular cage surface enhance the interaction with polar compounds, successfully separating nucleosides, sulfonamides, amino acids, and sugars in hydrophilic interaction chromatography (HILIC) mode. Further applications in the separation analysis of acidic drugs, alkaline drugs, cinnamic acid natural products, and chiral compounds demonstrate the multifunctional chromatographic capabilities for diverse compound types. Compared to Unitary Diol commercial columns, the prepared stationary phase showed significant advantages in wide polarity range separation performance. Moreover, through nucleoside compound separation mode switching analysis, RCC3-CN@SiO2 stationary phase further validates its favorable performance in both RPLC and HILIC modes, demonstrating extensive potential applications in the field of analytical chemistry. Importantly, the stationary phase exhibits efficient separation of nucleoside compounds in pure water systems, aligning with the principles of green analysis.

Microchip gas chromatographic columns dedicated for space exploration: Stationary phase coating, setup optimization and evaluation of column performances

This work is the first part of a project aiming to specifically design gas chromatographic microcolumns for space exploration. In particular, this study explored the functionalization and characterization of silicon/glass microchip gas chromatographic columns designed with a serpentine-shaped channel having an internal square cross-section. This microcolumn can be connected using a robust fluidic manifold with removable capillary connections to a conventional gas chromatograph or implemented in a prototype instrument for space exploration. First, benchtop gas chromatographic system was optimized in terms of injector liner volume, internal diameter (I.D.) of the capillary connections and data frequency of detection to ensure an optimal evaluation of column efficiency. Junction capillaries of 100 μm I.D., a liner of 1.2 mm I.D. and a detection acquisition frequency of 100 Hz were found to be the optimal set-up and parameters. Moreover, the stationary phase coating velocity was slowed-down to increase column efficiency by 100 %. Then, the performances of a square cross-section capillary column were compared with those of a conventional circular cross-section column. The coating efficiencies were estimated at 55 % and 42 % for circular and square internal cross-section geometries respectively, demonstrating that the square section geometry did not affect significantly the column performances. Stationary phase films of different thicknesses, from 0.032 to 0.260 µm, were coated on different microchips of the same production batch to assess the influence of film thickness on the chromatographic performances. Microchips performance was evaluated through Golay's plots, and some of the microchips were also studied by optical microscopy. The efficiency and retention capacity of our microchip column is shown to be highly dependent on the film thickness (9,000 plates.m-1 with k = 0.12 for the thinnest film and up to almost 4,000 plates.m-1 with k = 0.93 for the thickest film). Finally, the coating process repeatability was validated by producing three identical microchips with RSD of 4.8 % for the average number of theoretical plates.

Bridged Ethylene Polyethylene Oxide Surfaces to Improve Packing Materials for Widepore Size Exclusion Chromatography.

Here, we describe the preparation of bridged ethylene polyethylene oxide (BE-PEO) surface-modified silica packing materials for size exclusion chromatography. BE-PEO surface-modified silica was hydrolyzed and subsequent 1H nuclear magnetic resonance analysis of hydrolysis products confirmed the successful formation of BE-PEO bonded surface. Silica particles exhibiting 3µm diameters and 1000 Å nominal pore diameters were selected as a base material for this work out of the critical need to improve analytical capabilities for the testing of cell and gene therapy drug products. Accelerated high pH aging study revealed significant enhancement in column stability. Multi-angle light scattering noise measurements showed inordinately lower baseline noise. Moreover, we evaluated the chromatographic performance of BE-PEO silica-packed columns through separations of a protein test mixture, DNA ladder, monoclonal antibody-based therapeutics, and adeno-associated viruses. BE-PEO silica columns demonstrated high resolution, high recovery separations that were confirmed to be reproducible and capable of extended column lifetimes and exhibited low ionic and hydrophobic secondary interactions. In summary, BE-PEO silica particles have yielded a new level of performance, improved base stability, and inherently lower baseline noise. These novel widepore particles will facilitate more sensitive size-based detection and characterization of large biologics in the form of advanced gene therapy products.

Chiral microporous organic network/covalent organic framework/silica composites: Powerful and versatile chromatographic separation materials

Nowadays, covalent organic framework (COF) has shown great potential in chromatographic separation, and enriching the design of novel COF based stationary phases and broadening their application prospects remains of great significance. Microporous organic network (MON) is a novel type of porous material with large specific surface area and excellent stability connected by covalent bonding, which is extremely similar to COF. Especially, nano-sized MON has the characteristics of easy binding with silica matrix and superior chromatographic separation performance. In this work, the combination of MON and COF as the surface modification materials of silica gel was first explored. The conjugated network structure of MON and COF can improve the permeability of silica stationary phase, and the presence of abundant functional groups in COF and MON can enhance the separation ability for a series of aromatic compounds. Moreover, hydrophilic L-cysteine containing thiol group was cleverly introduced into MON/COF@SiO2via thiol-ene/yne click reactions to balance the hydrophilicity and hydrophobicity of stationary phase. The L-cysteine modified chiral MON/COF@SiO2 (CMON/CCOF@SiO2) was used for effective separation of various chiral/hydrophilic/hydrophobic analytes, and the CMON/CCOF@SiO2 was observed to exhibit superior separation ability to the CCOF@SiO2. Multi-mode chromatographic separation mechanisms were explored, demonstrating that multiple interactions were involved in the separation process. Finally, the CMON/CCOF@SiO2 was applied to detect the environmental endocrine disruptors in the water sample and pyridoxine in the tablet sample, respectively, indicating a great potential for application in actual sample detection.

Per aqueous liquid chromatography of Radix hedysari polysaccharides and Au nanoparticles co-functionalized stationary phase and its application in the determination of iridoids and phenylethanols

BackgroundHydrophilic Interaction Liquid Chromatography (HILIC) is an outstanding strategy for the challenging analysis of hydrophilic and polar components. Nevertheless, analysis under HILIC mode typically consumes 70%–95 % acetonitrile with the disadvantage of high analytical costs, being environmentally unfriendly and causing biohazards, which is not in line with the concept of green chromatography. Research has shown that Per Aqueous Liquid Chromatography (PALC) simultaneously emphasizes efficient analytical performance for hydrophilic analytes and green analytical concepts. The development of new PALC stationary phases with superior performance is necessary. ResultsIn this paper, silanized silica was sequentially subjected to esterification reaction, polymerization reaction and covalent bonding through five steps to obtain SiO2-RHP-AuNPs material, which was prepared as a novel stationary phase for PALC. Comprehensive characterization of the materials by means of Fourier transform infrared spectroscopy, Transmission scanning electron microscope, Elemental analysis and Thermogravimetric analysis showed the successful bonding of the functionalized groups on the original silica. The polymeric stationary phase based on Radix hedysari polysaccharide and Au nanoparticles had higher density of hydroxyl and ester functionalized groups. The Au nanoparticles upgraded their mesoporous structure and thermal stability, providing exceptional chromatographic performance and selectivity for chromatographic analysis. The influence of mobile phase water content, salt concentration, pH and column temperature on the retention behavior was evaluated. The novel Column was found to exhibit a dual mechanism of hydrophobic interactions/ion exchange interactions in a mobile phase with high water content. Significance and noveltyThe separation efficiency and selectivity of SiO2-RHP-AuNPs columns for synthetic pigments and organic acids in PALC mode were superior to those of commercial HILIC and C18 columns. In addition, a method for the determination of seven active ingredients in Fructus Ligustri Lucidi by SiO2-RHP-AuNPs column in PALC mode was developed. The method had good stability, reproducibility and accuracy, which was capable of realizing the quality evaluation of Chinese Materia Medicas.

Optimized reversed phase liquid chromatography methodology for the determination of vonoprazan fumarate impurities: Towards Six Sigma quality standards and sustainability assessment

Vonoprazan fumarate (VPZ), a potent potassium-competitive acid blocker, holds great promise as a therapeutic option for addressing acid-related disorders. This study introduces a refined reversed phase liquid chromatography methodology tailored for the comprehensive analysis of eight related substances, including starting materials, byproducts, and degradants within VPZ. Our approach integrates response surface methodology and tolerance analysis to achieve six sigma quality standards in chromatographic performance. By embedding specifications into the optimization process, we ensure robustness during method development. Chromatographic separation was executed using an XSelect CSH Phenyl-Hexyl column under stepped gradient conditions, employing a mobile phase comprising 0.1 % trifluoroacetic acid aqueous solution and acetonitrile. The flow rate was maintained at 1.3 mL/min, with UV absorbance at 252 nm, and a column temperature set at 25 °C. To evaluate the stability indicating ability of the method, forced degradation studies were conducted. Importantly, identified degradants did not interfere with the accurate quantification of VPZ and its associated impurities. Validation of the method was achieved through accuracy profiles. A greenness assessment was conducted using National Environmental Methods Index (NEMI), carbon footprint analysis, Analytical Greenness Calculator (AGREE), and Complementary Green Analytical Procedure Index (Complex GAPI). Additionally, blueness and whiteness assessments were conducted using the Blue Applicability Grade Index (BAGI) and Red-Green-Blue 12 (RGB 12) algorithms, respectively. The proposed method exhibited a green profile in NEMI and Complex GAPI. The carbon footprint was calculated at 0.055 kg CO2 equivalent per sample. The AGREE score was 0.67, BAGI was 80.0, and the whiteness score from the RGB12 algorithm was 83.5.This methodological framework holds promise for utilization in process development and quality assurance of VPZ in bulk drug manufacturing, particularly in the absence of official monographs within recognized compendia.

Construction of amine-rich coating utilizing amine curing reaction of triglycidyl isocyanate with triethylenetetramine on the surface of poly(styrene-divinylbenzene) microspheres for preparation of anion exchange stationary phase.

Epoxy resins, as important thermosetting polymers, exhibit excellent adhesion to various substrates. In view of this, reticulate coating of triglycidyl isocyanate with triethylenetetramine was introduced onto the surface of poly(styrene-divinylbenzene) utilizing amine curing reaction to obtain poly(styrene-divinylbenzene)@triglycidyl isocyanate-triethylenetetramine composite microspheres. The amino groups and epoxy groups of triglycidyl isocyanate-triethylenetetramine endowed poly(styrene-divinylbenzene) with good reactivity, which could be quaternized under mild conditions to obtain an anion exchange chromatographic stationary phase. The quaternized poly(styrene-divinylbenzene)@triglycidyl isocyanate-triethylenetetramine was characterized by scanning electron microscope, Fourier-transform infrared spectroscopy, N2 adsorption-desorption experiment, etal. The chromatographic performance of the customized column was evaluated by separating seven conventional anions, organic weak acids, and carbohydrates. Poly(styrene-divinylbenzene)@triglycidyl isocyanate-triethylenetetramine possesses the uniform size of poly(styrene-divinylbenzene) microspheres and good reactivity of triglycidyl isocyanate-triethylenetetramine, which offers a flexible strategy for the preparation of anion exchange stationary phase. The column exhibits excellent chemical and mechanical stability and chromatographic performance. Finally, the column was successfully applied for the determination of nitrite in pickles.

Removal of signal peptide variants by cation exchange chromatography: A case study

Signal peptide (SP) is required for secretion of recombinant proteins and typically cleaved by signal peptidase at its C-region to generate the mature proteins. Miscleavage of the SP is reported occasionally, resulting in a truncated- or elongated-terminal sequence. In the present work, we demonstrated that cation exchange (CEX) chromatography is an effective means for removing SP variants with a case study. With the selected resin/conditions, the chromatographic performance is comparable between runs performed at the low end and high end of load density and elution range. The procedure described in this work can be used as a general approach for resin selection and optimization of chromatographic conditions to remove byproducts that bind more strongly than the product to the selected resin.

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.

Esterified styrene-maleic acid copolymer modified silica as mixed-mode polymer-brush stationary phases for chromatographic separation

Styrene-maleic acid (SMA) copolymer has received much attention for its excellent solubilization characteristics. In this work, SMA copolymer brush-based chromatographic stationary phases were exploited and developed for the first time. First, SMA copolymer brush was in situ grown on the surface of spherical silica via living/controlled reversible addition-fragmentation chain transfer (RAFT) polymerization method. Subsequently, as a proof-of-concept demonstration, the copolymer was esterified by diethylene glycol mono-2-ethylhexyl ether (DGME) and 2-(2-ethylhexyloxy) ethanol (EHOE), respectively. The obtained Sil-SMA-DGME and Sil-SMA-EHOE copolymer-brush chromatographic stationary phases were characterized by transmission electron microscopy, Fourier transform infrared spectrometer, X-ray photoelectron spectroscopy, and thermogravimetric analysis, respectively. The chromatographic retention mechanism indicated that both the two packed columns exhibited hydrophilic/reverse mixed-mode retention modes. The maximum column efficiency was up to 71,000 N/m. The chromatographic separation performance evaluation indicated that the novel kind of stationary phases had excellent separation capabilities for hydrophilic, hydrophobic compounds and phospholipid standards. In addition, by combination with mass spectrometry identification, the Sil-SMA-DGME column was further exploited for separation and identification of phospholipids in human lung cancer cells. Totally, 9 classes including 186 phospholipid species were successfully identified. The results demonstrated the promising application prospects of the novel kind of SMA copolymer-brush chromatographic stationary phases.

Versatile injector for inline renewable solid-phase extraction: Application to cyclodextrin-based bioaccessibility assessment in environmental solids

BackgroundSolid phase extraction (SPE) is a standard sample preparation technique in HPLC workflows. Inline cartridges are high-performance alternatives to manual or robotic systems but at long term, they suffer from irreversible sorption of matrix components and sorbent compaction. Bead injection (BI) is a niche fluidic technique that allows renewing a sorbent bed through the manipulation of its suspension. However, there is a need for a versatile and reliable tool in HPLC that can exchange the inline sorbent automatically, resorting to inexpensive and assorted bulk sorbents. ResultsWe present a new flow path for a liquid chromatographic injector to perform inline micro-solid phase extraction. The sample is processed at real time, trapping the analytes and discarding the matrix. Cleaning the matrix and injecting 10 μL of sample takes 70 s, comparable with the injection in commercial HPLC systems. If the aim is to preconcentrate the analytes, average enrichment factors of 250 have been obtained after processing sample volumes of 3200 μL in 16 min (interleavable with the chromatographic step), keeping the peak position and width independent of the injected volume (compared to large volume direct injection).The desired bed mass is automatically and pressure-driven manipulated in the valve, retained by an inline frit, and optionally, after the analysis, removed by forward flow.The chromatographic performance of the new design is compared to the standard 6-port, 2-position HPLC injector. As a case study, we have monitored the extraction kinetics of a cyclodextrin-based bioaccessibility extraction test of persistent organic contaminants in soil, by extracting several fractions in valve, process them with inline SPE with a balanced hydrophilic-hydrophobic reversed-phase sorbent, and inject the bioaccessible compounds into HPLC. Aiming at avoiding carryover, the sorbent bed (ca. 3 mg) is exchanged before every run. It should be noted that this contribution focuses on HPLC, but other non-separative techniques, such as Flow Injection Analysis, can equally benefit from this injection platform. SignificanceThis contribution reports the first use of inline BI-solid phase extraction in HPLC workflows, without heart-cut eluate injection, in which the sorbent can be exchanged automatically by forward flow. This performance is enabled by prototyping a valve that can autonomously swap sorbents in real-time for diverse samples, as a cartridge exchanger, but using cost-effective and environmentally friendlier bulk sorbents (bed masses from sub-mg to 5 mg) without requiring additional hardware.

Substantial benefits of an inert biphenyl column for the analysis of steroids and their phase II metabolites in biological samples.

Steroids can be used as biomarkers in clinical metabolomics and other fields related to human toxicology. This chemical group is known for its complexity, considering its number of isobaric compounds and the wide variety of phases I and II metabolic pathways that parent compounds can undergo. For a successful analysis of steroids in biological samples, liquid chromatography separation must be finely tuned. It is especially challenging for glucuronidated and sulfated steroids derivatives that bear polar heads and can be affected by non-specific adsorption. The benefits of a biphenyl stationary phase chemistry for the selectivity of the separation of steroids and their phase II metabolites and the extent to which nonspecific adsorption phenomena could degrade chromatographic performance were investigated. Replacing a conventional hardware by a passivated hardware allowed to considerably reduce peaks width and asymmetry of sulfated species. The addition of weak ion pairing agents in the mobile phase could also help to reduce non-specific adsorption but are detrimental to mass spectrometry detection. As confirmed by the successful detection of 52 steroids in plasma, the use of a biphenyl stationary phase complemented by a passivated column hardware is of great help for a successful biomedical analysis of steroids and their phase II metabolites.

Analytical and Functional Characterization of Plasmid DNA Topological Forms and Multimers.

Plasmid DNA (pDNA) is an essential tool in genetic engineering that has gained prevalence in cell and gene therapies. Plasmids exist as supercoiled (SC), open circular (OC), and linear forms. Plasmid multimerization can also occur during the manufacturing process. Even though the SC forms are thought to provide optimal knock-in (KI) efficiency, there is no strong consensus on the effect of the topological forms and multimers on the functional activity. In addition, the results obtained for conventional pDNAs (>5 kbp) do not necessarily translate to smaller pDNAs (∼3 kbp). In this study, a workflow was developed for the analytical and functional characterization of pDNA topological forms and multimers. An anion exchange chromatography (AEC) method was first developed to quantify the topological forms and multimers. Four AEC columns were initially compared, one of which was found to provide superior chromatographic performance. The effect of mobile phase pH, various salts, column temperature, and acetonitrile content on the separation performance was systematically studied. The method performance, including precision and accuracy, was evaluated. The final AEC method was compared to capillary gel electrophoresis (CGE) by analyzing several pDNA sequences and lots. A forced degradation study revealed unexpectedly high degradation of the SC forms. Finally, the KI efficiency was compared for the SC and OC forms, and the multimers.

Evaluation of analytical columns suitable for high-temperature liquid-chromatography-isotope-ratio-mass-spectrometry analysis of anabolic steroids

Isotope ratio mass spectrometry (IRMS) can be used to determine the carbon isotope ratio of anabolic steroids. For example, in sports doping and food safety control, it enables determining an endogenous or synthetic origin of anabolic steroids. Generally, the steroids of interest are purified by liquid chromatography (LC) and analysed by gas chromatography combustion IRMS. LC-IRMS is not used since only mobile phases without carbon atoms can be used. For analysing mid-to apolar compounds, heated water can be used as an eluent as it has a similar polarity to a weak polar organic solvent. The silica-based columns are not robust enough at elevated temperatures in aqueous conditions. However, modified silica particles, metal oxides coated with polymers, and porous graphitic carbon are promising column materials for high-temperature LC (HT-LC) applications. Here, the stability of the stationary phase is crucial, and their chromatographic performance needs to be evaluated under the conditions mentioned above for anabolic steroid separations. Six columns using temperatures up to 200 °C were assessed, and only two were found to be appropriate. The ZirChrom-PBD column can be used for HT-LC-IRMS research purposes but is not recommended for routine laboratory practice applications due to the substantial loss of retention and resolution over time at elevated temperatures. Sachtopore-RP columns are the only suitable option for routine HT-LC-IRMS applications, even though they suffer from peak broadening over time when operating at elevated temperatures

The role of the cation and anion in aqueous liquid chromatography with sodium dodecyl sulphate and imidazolium-based ionic liquids as mobile phase reagents

BackgroundIn reversed-phase liquid chromatography, solute retention is primarily influenced by interactions between a nonpolar stationary phase and a moderately polar hydro-organic mobile phase, based on the solute lipophilicity. However, challenges regarding retention and peak tailing can arise due to ionic interactions between positively charged analytes and free silanols present on silica-based stationary phases. To address these challenges, incorporating surfactants and ionic liquids (ILs) into the mobile phase offers an effective solution. These additives synergistically enhance chromatographic performance through electrostatic and lipophilic interactions, which enable fine-tuning of selectivity and improved separation efficiency. ResultsThis study explores the chromatographic behaviour of several basic compounds in aqueous mixtures containing the anionic surfactant sodium dodecyl sulphate (SDS), above its critical micellar concentration, combined with various 1-alkyl-3-methylimidazolium-based ionic liquids (ILs) featuring chloride, tetrafluoroborate, and hexafluorophosphate anions, all without the addition of organic solvents. Specifically, this research investigates the influence of different anion types within the ILs and considers the impact of the IL cations. Analysis of solute peak profiles reveals narrow and symmetrical peaks. By introducing tetrafluoroborate and hexafluorophosphate IL anions into a mobile phase that contains an anionic surfactant, the study sheds light on the interactions occurring within the chromatographic column. This enhanced understanding of the combined effects of surfactants and ILs contributes to refining chromatographic methodologies. SignificanceThis research highlights the importance of carefully selecting the appropriate IL when incorporating it into a micellar mobile phase alongside SDS. This combination results in practical retention times that surpass the performance achieved with either the surfactant or IL alone in the mobile phase. The study particularly emphasises the impact of the IL anion, especially in the absence of SDS and organic solvents. This unveils interactions that are otherwise obscured in micellar and hydro-organic media, providing new insights into chromatographic dynamics.

Hydrophilic interaction chromatographic evaluation of zwitterionic polymer grafted silica gel via multiple binding sites.

A novel zwitterionic polymer grafted silica stationary phase, Sil-PZIC, was prepared by bonding poly(ethylene maleic anhydride) molecules on the surface of silica via multiple binding sites, followed by ammonolysis of maleic anhydride through a nucleophilic substitution reaction with ethylenediamine. The stationary phase was characterized by solid-state 13C nuclear magnetic resonance, zeta potential, and elemental analysis and the results show the successful encapsulation of zwitterionic polymer on the surface of silica. The chromatographic performance of Sil-PZIC was investigated by using nucleosides and nucleic bases as test analytes The variation of retention and separation performance of these model compounds were investigated by varying the chromatographic conditions such as the components of mobile phase, salt concentration, and pH. The results show that the retention of the Sil-PZIC phase was dominated by a hydrophilic partitioning mechanism accompanied by secondary interactions such as electrostatic and hydrogen bonding. In addition, saccharides and Amadori compounds were also well separated on the Sil-PZIC, indicating that the Sil-PZIC column has potential application for separation of the polar compound.

Continuous stationary phase gradient preparation on planar chromatographic media using vapor phase deposition of silane

A new, versatile, and straightforward vapor phase deposition (VPD) approach was used to prepare continuous stationary phase gradients (cSPGs) on silica thin-layer chromatography (TLC) plates using phenyldimethylchlorosilane (PDCS) as a precursor. A mixture of paraffin oil and PDCS was placed at the bottom of an open-ended rectangular chamber, allowing the reactive silanes to evaporate and freely diffuse under a controlled atmosphere. As the volatile silane diffused across the length of the TLC plate, it reacted with the surface silanol groups thus functionalizing the surface in a gradient fashion. Characterization of the gradient TLC plates was done through UV visualization and diffuse reflectance spectroscopy (DRS). Visualizing the fluorescent gradient plates under UV radiation shows the clear presence of a gradient with the side closest to the vapor source undergoing the most modification. More quantitative characterization of the shape of the gradient was provided by DRS. The DRS showed that the degree of modification and shape of the gradient was dependent on the concentration of silane, VPD time, and relative humidity. To evaluate the chromatographic performance, a mixture of three aromatic compounds (acetaminophen (A), aspirin (As), and 3-hydroxy-2-naphthoic acid (3H)) was spotted on the high (GHP) and low phenyl (GLP) ends of the gradient TLC plates and the results compared to the separations carried out on unmodified and uniformly modified plates. The GHP TLC plates showed retention factors (Rf) of 0.060 ± 0.006, 0.391 ± 0.006, and 0.544 ± 0.006, whereas the unmodified plate displayed Rf values of 0.059 ± 0.006, 0.092 ± 0.003, and 0.037 ± 0.002 for the analytes A, As, and 3H, respectively. From the Rf values, it was observed that each modified plate exhibited different selectivity for the analytes. The GHP TLC plates exhibited better separation performance, and improved resolution compared to the GLP, unmodified, and uniformly modified plates. Overall, VPD is a new, cost-effective method for creating a gradient on the stationary phase which has the potential to advance chromatographic separation capabilities.

Zwitterionic sulfobetaine-based hypercrosslinked hydrophilic materials for bioanalysis

Analysis of bioactive polar molecules is crucial for different fields, including biology, clinic medicine, and separation science. However, the design of the separation materials remains a huge challenge due to their tedious preparation process and limited chromatographic performances of traditional hydrophilic materials. New-generation hydrophilic polymer materials prepared via a sole-monomer system exhibit excellent reproducibility and chromatographic efficiency; however, such polymers reported to date have a high swelling propensity and risk of low surface exposure of functional groups, which limit their application as separation materials. In this study, N,N-bis(methacryloxyethyl)-N-methyl-N-(3-sulfopropyl)ammonium betaines (BMMSB), a zwitterionic sulfobetaine, was used to rapidly fabricate the zwitterionic hypercrosslinked hydrophilic polymer materials via the sole-monomer system. The novel hypercrosslinked hydrophilic materials exhibited high porosity and permeability, superhydrophilicity, low protein adsorption, and excellent capillary column efficiency (up to 134,950 plates/m for thiourea). It was able to enrich 28 N-glycopeptides from hIgG tryptic digests with a higher efficiency than that of traditional poly(SPE-co-EDMA) monolith and commercial ZIC-HILIC materials. Additionally, the polyBMMSB hydrophilic materials demonstrated superior selectivity and column efficiency to the poly(SPE-co-EDMA) monolith for the separation of various small bioactive molecules, including nucleobases and nucleosides, phenol derivatives, benzoic acid derivatives, urea and allantoin. Especially, high performance bioanalysis of Alzheimer’s disease biomarkers plasma Aβ42 and Aβ40, or some steroid hormones in human plasma were also achieved by the polyBMMSB monolith combined with nano liquid chromatography-mass spectrometry. As such, these sulfobetaine-based zwitterionic polymers may serve as a prototypical next-generation zwitterionic hydrophilic materials for rapid and efficient bioanalysis.

Evaluation of RPLC stationary phases for tocopherol and tocotrienol positional isomer separation: Method development and profiling

Reversed-phase separation of tocopherols (Ts) and tocotrienols (Ts) using C18 stationary phases results in the coelution of β and γ positional isomers, leading to identification errors. This study investigates the potential of alternative stationary phase chemistries to effectively resolve tocochromanols, specifically focusing on the critical pair of β and γ positional isomers. Initial screening of seven different stationary phases (C18, C18-PFP, C30, PFP, 5PYE, πNAP, and RP-Amide) was conducted. Linear solvent strength (LSS) studies were performed to assess the impact of the organic modifier (methanol) and temperature on the chromatographic performance parameters. Five columns were found to be suitable for the tocochromanol separation and two different chromatographical conditions per column were proposed. Elution order of tocochromanols was unique for 5PYE, πNAP and C30 columns in comparison to RP-Amide and PFP. Method development for the quantitative analysis of four tocopherol and four tocotrienol homologues was performed. The optimised method employed the RP-Amide (150 × 4.6 mm, 2.6 μm dp) superficially porous particle column, mobile phase of methanol:water of 92:8, v/v, with a flow rate of 1.0 mL/min, column oven temperature of 40 °C and fluorescence detection (λex 295 nm, λem 330 nm). The analysis run time was 10.5 min with 13.6 MPa back pressure. The method was validated and the obtained LOQs were found to be 1.30-3.13 μg/mL. The method developed was successfully applied for the determination of tocochromanols in twenty samples with unique tocochromanol profiles. Principal component analysis illustrated three distinct groups based on the tocochromanol profile.