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• Investigated the adsorption and retention mechanisms of a cyano-based sorbent. • Saturation adsorption amount is sensitive to the molecular structure of adsorbates. • Heterogeneity of adsorption is a key concept for understanding adsorption mechanisms. • Adsorption enthalpy shows a saturation curve relationship with mobile phase concentration. • A model was proposed to describe the van't Hoff enthalpic curves. This study investigated the adsorption mechanisms in a normal-phase system using a cyano-based stationary phase as the sorbent. The minor disturbance method was used to measure the adsorption isotherms of acetone and alcohols with various structures. Excluding data in pure n-hexane revealed that the adsorption behaviors on cyano sites were well described by the Langmuir model. The adsorption equilibrium constants, ranging from 8.86 to 11.15 at 25 °C, showed no significant differences across alcohol structures and decreased with increasing temperature. The saturation adsorption concentration decreased with increasing alcohol molecule size, with branched-chain alcohols showing a lower saturation adsorption amount compared to straight-chain alcohols. The standard state adsorption enthalpies and entropies calculated from the equilibrium constants for various alcohols ranged from −29 to −22 kJ/mol and −78 to −55 J/K·mol, respectively, showing enthalpy-entropy compensation. A discrepancy was observed between these adsorption enthalpies and those obtained from the retention factors of alcohols using pure n-hexane as the mobile phase. This discrepancy may result from the affinity energy distribution of the adsorbent. In pure n-hexane, the adsorption behaviors of adsorbates were considerably affected by high-affinity sites. Moreover, acetone and these alcohol molecules were used as solvent modifiers to investigate the relationship between the retention factor, modifier concentration, and temperature for various solutes with distinct functional groups. The retention curves were converted to enthalpic curves using the van't Hoff equation. A theoretical model was proposed to describe the relationship between the van't Hoff enthalpy change and mobile phase composition. The proposed model effectively described the enthalpic curves, indicating that the enthalpy change follows a saturation curve with increasing modifier concentration. This trend is primarily due to competitive adsorption and complexation behaviors between the solute and modifier molecules. [ABSTRACT FROM AUTHOR]

• High molecule hydrophobicity causes chromatography-induced aggregation of aBsAb. • Arg-HCl retards aBsAb elution from Protein A column rather than facilitating. • High molecular flexibility leads to different hydrodynamic sizes of aBsAb molecule. • Flexibility of the aBsAb molecule is pH dependent. • aBsAb purity was improved from 29 % to 93 % by one-step Protein A with HMW<5 %. Appended bispecific antibody (aBsAb) with two single chain variable fragments (scFv) linked at the c-terminus of its heavy chains is one of the promising formats in bispecific therapeutics. The presence of hydrophobic and flexible scFv fragments render aBsAb molecules higher molecule hydrophobicity and structural flexibility compared to monoclonal antibody (mAb), thus making its purification more challenging. We set out to investigate how the unique molecular properties of aBsAb affect its performance on Protein A chromatography. We showed that aBsAb has a high propensity for chromatography-induced aggregation due to its high molecule hydrophobicity, and this couldn't be improved by the addition of common chaotropic salts. Moreover, the presence of chaotropic salts, such as arginine hydrochloride (Arg-HCl), retarded aBsAb elution during Protein A chromatography rather than facilitating which was widely observed in mAb Protein A elution. Nevertheless, we were able to overcome the aggregation issue by optimizing elution condition and improved aBsAb purity from 29 % to 93 % in Protein A eluate with a high molecular weight (HMW) species of less than 5 %. We also showed that the high molecular flexibility of aBsAb leads to different hydrodynamic sizes of the aBsAb molecule post Protein A elution, neutralization, and re-acidification, which are pH dependent. This is different from mAbs where their sizes do not change post neutralization even with re-exposure to acid. The above unique observations of aBsAb in Protein A chromatography were clearly explained from the perspectives of its high molecular hydrophobicity and structural flexibility. [ABSTRACT FROM AUTHOR]

• Sequential diethylaminoethyl dextran-grafting and diethylaminoethyl modification. • The strategy breaks through the limitation of ionic capacity of DEAE dextran-media. • The strategy significantly improves the adsorption performance. Ion exchangers with high adsorption capacity, fast mass transfer, and high salt-tolerance synchronously are highly desired for high-performance protein purification. Here, we propose a sequential diethylaminoethyl dextran-grafting and diethylaminoethyl chloride modification strategy to achieve high-performance anion exchangers. The advantages of the double-modification strategy lie in: (1) the introduction of diethylaminoethyl in the second modification has no diffusion limitation due to the small molecular size, thus a high ionic capacity; (2) the grafting ligands not only provide three-dimensional adsorption space for high adsorption capacitybut alsofacilitate surface diffusion of protein by chain delivery. The maximum adsorption capacity of the obtained anion exchangers for bovine serum albumin reaches 333 mg/mL, the ratio of effective pore diffusivity (D e) to free solution diffusivity (D 0) reaches 0.69, and the adsorption amount reaches 97 mg/mL even in 100 mmol/L NaCl concentration,. All these results demonstrate the proposed sequential modification strategy are promising for the preparation of high-performance ion exchangers. [ABSTRACT FROM AUTHOR]

• Phosphorothioate oligonucleotide therapeutics exist as mixture of diastereomers. • Distinct bioactivities of diastereomers demand batch-to-batch reproducibility testing. • Ion-pair RPLC with hydrophilic alkylamines shows good diastereoselectivity. • Polysaccharide chiral column with RPLC conditions provides complementary selectivity. • Sequential selective comprehensive chiral × ion-pair RPLC 2D-LC doubles separated diastereomers. In recent years, many nucleic acid-based pharmaceuticals have been approved and entered the market, and even a larger number are in late stage clinical trials. Conventional oligonucleotides are facing issues in vivo like fast renal clearance and nuclease degradation. Therefore, to increase their stability, phosphorothioation is a frequent modification of therapeutic oligonucleotides (ONs) which also leads to improved binding affinity facilitating cell internalization and intracellular distribution. At the same time, by replacing a phosphodiester linkage with a phosphorothioate group, a phosphorous stereogenic center is generated which causes the formation of R p - and S p -diastereomers. It increases the structural diversity. For example, with 15 of those phosphorothioate (PS) linkages, 32,768 different diastereomers are expected. Since the phosphorothioate is introduced non-stereoselectively, the molecular complexity of the resultant phosphorothioate ON products is tremendously increased impeding the chromatographic separation in the course of quality control. Since distinct phosphorothioate diastereomers have different bioactivities and pharmacological properties, there is increasing interest in implications of stereoisomerism of phosphorothiate oligonucleotides. From a quality and regulatory viewpoint, batch-to-batch reproducibility of the diastereomer profile may be of significant concern. In order to address this issue, this study investigates the stereoselectivity of LC methods for two phosphorothioate oligonucleotide (PSO) compounds differing in their molecular size and numbers of PS linkages. Diastereoselectivity of ion-pairing reversed-phase liquid chromatography (IP-RPLC), RPLC without ion-pairing agents and LC with chiral polysaccharide-based column were evaluated for model PSOs and an active pharmaceutical ingredient (API) of PSO with trivalent N -acetylgalactosamine (GalNAc) conjugate. Due to the structural complexity of PSOs, the separation power for the diastereomer mixture was increased by using sequential selective comprehensive two-dimensional chromatography with an amylose tris(α-methylbenzylcarbamate)-immobilized chiral stationary phase (CSP) in the first dimension and ion-pair RPLC with ethylammonium acetate in the second dimension. Improved diastereomer selectivity was obtained and a larger number of peaks could be separated. [ABSTRACT FROM AUTHOR]

• DOSY can provide the size of individual molecules from the hydrodynamic radius. • Online HPLC-NMR can separate mixtures and deliver structures of the analytes. • The new coupling of HPLC and DOSY provides also the sizes of molecules in addition. • Diffusion coefficients can also be used for the molar mass determination. The aim of this study was to introduce a powerful coupling of Liquid Adsorption Chromatography (LAC) and Diffusion-Ordered Spectroscopy (DOSY) for comprehensive structure analysis. This new hyphenation approach facilitated the simultaneous separation of a polymer mixture and the determination of molar masses within a single 3D experiment. The online coupling of High-Performance Liquid Chromatography (HPLC) and two-dimensional DOSY-NMR will be called 3D-LAC-NMR-DOSY experiment. Our methodology enabled the chromatographic separation of analytes based on their chemical heterogeneity, and provided accurate molar masses of the analytes through 2D-DOSY. This new method was demonstrated on a polystyrene oligomer mixture. In this case, the oligostyrenes could be separated with LAC according to their tacticity and chain length in protonated acetonitrile as eluent and DOSY measurements provided the molar masses of each oligomer. In order to show the power of the 3D-LAC-NMR-DOSY method, the comparison to 2D-DOSY, 3D-DOSY and LAC-NMR was separately evaluated. Furthermore, the recently published solvent-independent molar mass calibration of diffusion coefficients was also successfully applied in our LAC-DOSY studies for molar mass predictions of the oligomers in acetonitrile. The predicted molar masses were in good agreement with the LAC-DOSY measurements and were verified by calibrations of diffusion coefficients and mass spectrometry. Finally, this pioneering 3D technique offers a powerful new tool for advancing structure analysis and enhancing our understanding of complex systems such as oligostyrenes. [ABSTRACT FROM AUTHOR]

• Relations between gradient retention time and block copolymer structure are derived. • Block copolymers elute close to stronger adsorbing homopolymer. • Results were verified by experiment. • Elution behavior of block copolymer is similar to appropriate homopolymer. The retention of binary diblock copolymers in solvent gradient interaction chromatography (SGIC) was investigated theoretically and compared with experimental results. The isocratic and gradient elution behavior of binary block copolymers can be described by the equations of an equivalent homopolymer having a suitable critical eluent composition and size to pore size ratio. For diblock copolymers the adsorption-desorption transition occurs at a limiting interaction energy, which is characterized by a limiting interaction parameter c A,lim , like the critical interaction parameter of homopolymers. Both, the limiting interaction parameter and the critical interaction parameter are independent of pore size. However, while for homopolymers the critical energy is also independent of molecular size, the limiting interaction parameter of block copolymers depends on the size and composition of the macromolecule. Therefore, even for block copolymers of a fixed composition a molar mass independent elution cannot be realized at isocratic conditions. Since binary block copolymers in solvent gradients elute very close to the stronger adsorbing homopolymer, separation of the block copolymer from the stronger adsorbing homopolymer becomes difficult. A slightly better separation is expected to occur in larger pores. The close elution of block copolymers and homopolymers in gradient elution was confirmed for poly(methy methacrylate)-block-poly(styrene)s in chloroform-THF gradients on a bare silica column. [ABSTRACT FROM AUTHOR]

We report novel capillary gel electrophoresis (CGE) with poly(ethylene glycol) (PEG)-based hydrogels for the effective separations of biomolecules containing sugars and DNAs based on a molecular size effect. The gel capillaries were prepared in a fused silica capillary modified with 3-(trimethoxysilyl)propylmethacrylate using a variety of the PEG-based hydrogels. After the fundamental evaluations in CGE regarding the separation based on the molecular size effect depending on the crosslinking density, the optimized capillary provided the efficient separation of glucose ladder (G1 to G20). In addition, another capillary showed the successful separation of DNA ladder in the range of 10–1100 base pair, which is superior to an authentic acrylamide-based gel capillary. For both glucose and DNA ladders, the separation ranges against the molecular size were simply controllable by alteration of the concentration and/or units of ethylene oxide in the PEG-based crosslinker. Finally, we demonstrated the separations of real samples, which included sugars carved out from monoclonal antibodies, mAbs, and then the efficient separations based on the molecular size effect were achieved. [ABSTRACT FROM AUTHOR]

• The ingress and egress kinetics of biomolecules studied in sub-3 μ m particles • A rigorous analytical solution is derived for a rectangular concentration change. • The kinetics are reported for mAb, dsDNA, and virus-like particles in fast UHPLC. • BEH Particles are out of equilibrium with mobile phase for dsDNA and viruses. • Egress kinetics is very slow suggesting using non-porous separation materials. The increasing demand for the characterization of large biomolecules such as monoclonal antibodies, double-stranded deoxyribonucleic acid (dsDNA), and virus-like particles (VLPs) is raising fundamental questions pertaining to their absorption (ingress) and escape (egress) kinetics from fully porous particles. The exact expression of their concentration profiles is derived as a function of time and radial position across a single sub-3 μ m Bridge-Ethylene-Hybrid (BEH TM) Particle present in size exclusion chromatography (SEC) columns. The boundary condition at the external surface area of the particle is a rectangular concentration profile mimicking the passage of the chromatographic zone. Four different BEH Particles were considered in the calculations depending on the molecular size of the analyte: 2.0 μ m 100 Å BEH Particles for small molecules, 2.0 μ m 200 Å BEH Particles for monoclonal antibodies, 2.0 μ m 300 Å BEH Particles for dsDNA (100 base pairs), and 2.5 μ m 900 Å BEH Particles for virus-like particles (VLPs). The calculated concentration profiles of small molecules and monoclonal antibodies confirm that all BEH Particles present in the column reach quasi-instantaneously thermodynamic equilibrium with the bulk mobile phase during the passage of the chromatographic band. This is no longer the case for larger biomolecules such as dsDNA or VLPs, especially when the SEC particle is located near the column inlet and for high velocities. The kinetics of biomolecule egress is slower than its kinetics of ingress leading to pronounced peak tailing. The mean concentration of the largest biomolecules in the SEC particles remains always smaller than the maximum bulk concentration. This persistent and transient intra-particle diffusion regime has direct implications on the theoretical expressions of the observed retention factors and plate heights. Classical theories of chromatography assume uniform spatial distribution of the analyte in the particle volume: this hypothesis is not verified for the largest biomolecules. These results imply that non-porous particles or monolithic structures are the most promising stationary phases for the separation and purification of the largest biomolecules in life science. [ABSTRACT FROM AUTHOR]

• AF4 facilitated purification of long ssRNA and dsRNA molecules in native conditions. • AF4 preserved native RNA conformation and enabled detection of RNA multimers. • New information on solution properties of RNA was obtained. • Use of semipreparative AF4 channel allowed milligram RNA loads. • AF4 purified dsRNA molecules triggered RNAi-mediated gene silencing in aphids. Robust RNA purification and analysis methods are required to support the development of RNA vaccines and therapeutics as well as RNA interference -based crop protection solutions. Asymmetrical flow field-flow fractionation (AF4) is a gentle native purification method that applies liquid flows to separate sample components based on their hydrodynamic sizes. We recently showed that AF4 can be utilized to separate RNA molecules that are shorter than 110 nucleotides (nt), but the performance of AF4 in the analysis and purification of longer RNA molecules has not been previously evaluated. Here, we studied the performance of AF4 in separation of single-stranded (ss) and double-stranded (ds) RNA molecules in the size range of 75–6400 nt. In addition, we evaluated the power of AF4 coupling to different detectors, allowing separation to be combined with data collection on yield as well as molecular weight (MW) and size distribution. We show that AF4 method is applicable in RNA purification, quality control, and analytics, and results in good recoveries of ssRNA and dsRNA molecules. In addition, our results demonstrate the utility of AF4 multidetection platforms to study biophysical properties of long RNA molecules. [ABSTRACT FROM AUTHOR]

We investigate the impact of polydispersity of the sample molecules on the separation process and on the efficiency of size-exclusion chromatography. Polydispersity was integrated into the molecular (stochastic) model of chromatography; the characteristic function, the band profile and the most important moments of the elution profiles were calculated for several kind of pore structures. We investigated the parameters affected by polydispersity on the separation for a number of pore shapes. Our results demonstrate that even a small distribution in the molecular size (i.e. polydispersity) can contribute substantially to the total width of the chromatographic peak. The pure effect of polydispersity can only be investigated via mathematical modeling, because its contribution to an experimental chromatogram cannot be separated from other band-broadening effects. [ABSTRACT FROM AUTHOR]

• A MIP column was prepared by packing polymyxin E-imprinted microspheres. • MIP column showed excellent stability, efficiency and selectivity to polypeptide antibiotics. • In-line MISPE allowed the screening of polypeptide antibiotics in animal tissue by LC-UV. • A LC-MS/MS method based on in-line MISPE for the analysis of polypeptide antibiotics was developed. • Obtained recoveries ranged from 66.7 to 94.5% and limits of detection were 3.0 ∼ 8.0 μg/kg. Effective purification and enrichment of polypeptide antibiotics in animal tissues is always a challenge, due to the co-extraction of other endogenous peptides which usually interfere their final determination. In this study, a molecularly imprinted column was prepared by packing polymyxin E-imprinted particles into a 100 mm × 4.6 mm i.d. HPLC column. The as-prepared imprinted columns were able to tolerate 100% aqueous phase and exhibited good stability and high column efficiency. Polypeptides antibiotics with similar molecular size or spatial structure to polymyxin E were well retained by the imprinted column, suggesting class selectivity. After optimization of mobile phase conditions of imprinted column, polypeptide antibiotics in animal tissue extracts were enriched and cleaned up by in-line molecularly imprinted solid-phase extraction, allowing the screening of target analytes in complex samples at low concentration levels by UV detection. Eluate fraction from the imprinted column was collected, and further dried and re-dissolved with methanol-0.5% formic acid aqueous solution (80:20, v/v) for final LC-MS/MS analysis. Analysis was accomplished using multiple reaction monitoring (MRM) in positive electrospray ionization mode and analytes quantified using the matrix-matched external calibration curves. The results showed high correlation coefficients for target analytes in the linear range of 2 ∼ 200 μg kg−1. At four different concentration levels (limit of quantification, 50, 100 and 200 μg kg−1), recoveries of four polypeptide antibiotics in swine, cattle and chicken muscles ranged from 66.7 to 94.5% with relative standard deviations lower than 16.0%. The limits of detection (LOD) were 2.0 ∼ 4.0 μg/kg, depending upon the analyte and sample. Compared with a conventional pretreatment method, the imprinted column was able to remove more impurities and to significantly reduce matrix effects, allowing the accurate analysis of polypeptide antibiotics. [ABSTRACT FROM AUTHOR]

Highlights: [•] A new model has been proposed to evaluate the effective pore diffusion. [•] This model takes into account that particle tortuosity depends on the molecule size. [•] Better agreement between model and data obtained with the peak parking method. [ABSTRACT FROM AUTHOR]

Highlights: [•] Phenolic antioxidants were separated at 60–100°C on a polydentate C8 column. [•] The temperature effects on retention are well described by the linear van’t Hoff model. [•] A simple retention model describes combined effects of temperature and mobile phase. [•] Molecular size and solute hydrogen-bond acceptor capacity control the retention. [•] High-temperature solvent gradients are superior to temperature programming. [ABSTRACT FROM AUTHOR]

Highlights: [•] We examine the LC separation of RNAs on macro-porous polystyrene-divinylbenzene resins. [•] The method separates RNAs of 20–8000 nucleotides with high sensitivity. [•] The separation occurs essentially according to the molecular size of RNA. [•] The method allows for the separation of various RNAs from biological sources. [•] The isolated RNAs can be characterized by nanoflow LC–tandem mass spectrometry. [ABSTRACT FROM AUTHOR]

Highlights: [•] We studied the effect of pressure on retention for different reversed-phase columns. [•] Solute molecular size has the greatest influence on retention increases produced. [•] C18 column nature (monomeric or polymeric) has relatively little influence. [•] Solute polarity and planarity influence pressure effect on retention. [•] Increased pressure increases shape selectivity of polymeric columns. [ABSTRACT FROM AUTHOR]

Abstract: In protein chromatography, the size of the protein determines which fraction of pores it can access within a resin and at which rate of diffusion. Moreover, in the presence of grafted polymers like in advanced materials, adsorbed proteins and electrolytes complicate the interaction pore-protein. In this study, we evaluated in a comparative way the behavior of Fractogel® EMD SO3 (M) and (S), “tentacle”-type, strong cation exchangers, as well as a reference material without tentacles, all of which are commonly used for protein purification. ISEC experiments were carried out with a set of Dextran tracers of largely different molecular size covering the typical range of protein sizes. Experimental values of porosity (internal and external to the particles) as well as of pore diffusion coefficients have been measured at different NaCl concentrations and under protein loading. These results provide useful insights into the complex interplay among mentioned factors: first, the presence of tentacles induces size exclusion selectivity in the materials; second, the salt induces conformational changes of the tentacles, leading to porosities larger than expected in tentacle materials; third, protein adsorption mainly leads to a reduction of porosity due to pore space occupied by the protein and to a decrease of pore diffusion coefficient. [Copyright &y& Elsevier]

• A generic stability indicating IP-RPLC method for gRNA analysis was developed. • Wide pore size column is required for the 100-mer gRNA separation. • Charge interaction is the key separation mechanism of IP-RPLC separation of gRNA. • Reduction in alkali metal ions leads to good LC-MS characterization of gRNA. Guide ribonucleic acid (gRNA) is a critical reagent in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. The single stranded guide RNA (sgRNA) is the most commonly used gRNA in application. Evaluation of the impurity profile of synthetic sgRNA is important for any CRISPR genome editing experiments. However, the large molecular size, complex impurity profile and unique secondary structure pose many challenges in the analysis of sgRNA by ion pairing reversed-phase liquid chromatography (IP-RPLC), the commonly used method. In this work, we developed a generic IP-RPLC method for guide RNA analysis. We found that large pore size of stationary phase was the most critical column parameter to achieve high resolution separation of sgRNA while particle structure, particle size and surface chemistry had less impact. Our results indicated that charge interaction was the most critical mechanism for retention and mass transfer had less impact on the performance of separation. An IP-RPLC/mass spectrometry (MS) method was also developed with a specific practice to reduce adducts and enable intact MS analysis of sgRNAs. The generic IP-RPLC method demonstrates its feasibility to serve as a release, stability, characterization and in-process control testing method for synthetic sgRNA products. [ABSTRACT FROM AUTHOR]

• Developed NPLC × UHPLC-ELSD methodology for the characterization of propoxylates. • Analysis of mutually dependent functionality-type and molecular-weight distributions. • Fast screening method (<30 min) for unknown samples was developed. Understanding the relation between chemical characteristics and properties of synthetic polymers is one of the challenges faced by analytical chemists in industry. This is a complex task, as polymers are not synthesized as single molecule, but are populations of chemically similar compounds with distributions over several properties. The latter include, for example, molecular weight, nature of end-groups (functionality), and chemical composition. In this paper, comprehensive two-dimensional liquid chromatography was used to determine the combined functionality-type and molecular-weight distributions of hydroxy‑functionalized propoxylates. Propoxylates derived from different initiators (one up to eight terminal hydroxyl groups) were separated in the first dimension using a gradient normal-phase LC separation (NPLC). In the second dimension ultra-high pressure size-exclusion chromatography separation (UHPSEC), further speciating distributions based on molecular size. The developed NPLC × SEC method with evaporative light-scattering detection can be used for the fast screening (< 30 min) of mutually dependent functionality-type and molecular-weight distributions of unknown propoxylates. [ABSTRACT FROM AUTHOR]

• The separation efficiency of HIC was assessed in isocratic and gradient mode. • The optimal flow rate yielding maximum peak capacity decreases with gradient time. • Effects of particle diameter and column coupling on resolving power is demonstrated. • A peak capacity of 240 was achieved using a 10 cm column packed with 2.3 µm particles. This study reports on the assessment of the separation performance of hydrophobic interaction chromatography for intact protein analysis using non-porous butyl polymethacrylate phases. The maximum peak capacity in inverse gradient mode was reached at a volumetric flow rate which was significantly (10–20 times) higher than the flow rate yielding the minimum plate height in isocratic mode, as the gradient volume dominates the peak-capacity generation. The flow rate yielding the maximum peak capacity increased with decreasing gradient volume, i.e. , steeper gradients, and also depends on the magnitude of the mass-transfer contribution to peak dispersion (affected by particle size and molecular diffusion coefficient of proteins) at these high flow rates. The maximum peak capacity using a 100 mm long column packed with 4 µm particles for steep 7.5 min gradients was determined to be 60. Increasing the column length by coupling columns leads to better gradient performance than increasing the gradient duration for gradients of 60 min and longer. Using a coupled column system (2 × 100 mm long columns packed with 4 µm particles), the maximum peak capacity was determined to be 105, which was 33% higher compared to that of a single column while applying a similar gradient volume. Decreasing the particle size to 2.3 µm leads to higher peak capacities even though the column was operated at lower volumetric flow rate. The maximum peak capacity obtained with the 2.3 µm column was 128% higher than was obtained with the coupled column. Even at suboptimal conditions, the 2.3 µm column yields a higher peak capacity (14%) than when using two coupled columns packed with 4 µm at optimal conditions (gradient time of 120 min and a flow rate of 0.5 mL/min). [ABSTRACT FROM AUTHOR]

• ANNs including either β-CD (mM) or β-CD A (μM) showed remarkable performances • ANN's performances were the most influenced by CATS2D_08_PL and RDF075m • RDF075m showed the most pronounced effect on retention in β-CD modified RP-HPLC • Retention is governed by steric factors, molecular size and shape and lipophilicity • The saturation of stationary phase with β-CD leads to reduction in retention times The quantitative structure-retention relationship (QSRR) models are not only employed in retention behaviour prediction, but also in an in-depth understanding of complex chromatographic systems. The goal of the present research is to enable the comprehensive understanding of retention underlying the separation in β-cyclodextrin (CD) modified reversed-phase high performance liquid chromatography (RP-HPLC) systems, through the development of mixed QSRR models. Moreover, the amount of β-CD adsorbed on the stationary phase surface (β-CD A) is added as the model's input in order to evaluate its contribution to both model performances and retention. Nuclear magnetic resonance (NMR) experiments were conducted to confirm the predicted inclusion complex structures and support the application of in silico tools. The most significant descriptors revealed that retention is governed by the steric factors 7.5 Å distant from the geometrical centre of a molecule, 3D arrangement of atoms determining the molecular size and shape, lipophilicity indicated by topological distances, as well as the unbound system's energy, related to the inclusion complex formation. In addition, a notable effect of the pH of the aqueous phase on the retention of ionizable analytes was shown. In the case of pH of the aqueous phase and β-CD A the change in retention behaviour of the studied analytes was observed only at the highest β-CD A value (5.17 μM/m2), but it was not related to the ionization state of analytes. When the analytes did not change the ionization form across the investigated studied pH range, and the acetonitrile content in the mobile phase was 25% (v/v), the retention factor had low values regardless of the β-CD A ; under these circumstances the retention is probably acetonitrile driven. [ABSTRACT FROM AUTHOR]

• new application of nitrogen-doped carbon dots (NCDs) as buffer additives for electrokinetic-based separations • differential interaction of NCDs with ssDNA based on molecule size: longer ssDNA interacts more weakly; shorter ssDNA interacts more strongly with these carbon nanomaterials • NCD-modified capillary transient isotachophoresis (ctITP) method allows for the resolution of ssDNA molecules ranging in size from 32- to 100-bases under free-solution separation conditions • NCDs are easily synthesized via a hydrothermal method; they are stable and water soluble; and when employed as separation additives at low concentration, they permit the quantitative determination of FAM-labeled ssDNA samples by laser-induced fluorescence detection. This study demonstrates a novel application of nitrogen-doped carbon dots (NCDs) to enable the separation of different lengths of single-stranded DNA (ssDNA) by eletrokinetic means. Carbon dots have recently found widespread application in the fields of sensing, diagnostics, and healthcare due to their biocompatibility and low toxicity. In light of growing interest in the use of ssDNA aptamers over antibodies in the fields of biosensor development and drug delivery, it is important to establish a simple and effective method for aptamer separation. In this study, we employed NCDs as buffer additives in a capillary electrophoresis (CE)-based method, giving rise to the separation of FAM-labeled ssDNA samples ranging from 32 to 100 bases in length, with resolutions ranging from 1.30 – 1.77. In particular, we adopted a capillary transient isotachophoresis (ctITP) system with laser-induced fluorescence (LIF) detection, with both the separation and sample buffers modified by the addition of 30 μg/mL NCDs. These nanomaterials were prepared by a simple hydrothermal method from a mixture of citric acid and ethylenediamine. The NCDs themselves are highly fluorescent and photostable. As components in the background electrolyte, they did not interfere with the fluorescence emission of the FAM-labeled DNA samples. Under the conditions employed, no separation could be achieved in the absence of the NCDs nor with undoped CDs. The results show that NCDs function as buffer additives capable of enhancing electrokinetic-based separations of ssDNA, and hence, provide a new application for these carbon nanomaterials. [ABSTRACT FROM AUTHOR]

• The applications of AF4 for the analysis of polysaccharide from different sources were reviewed. • The separation of polysaccharide can be completed by AF4. • The effect of operating conditions of AF4 on analysis of polysaccharides was discussed. • More information on polysaccharide can be obtained by AF4 coupled with multidetector. • AF4 will be a state-of-the-art technology for studying polysaccharide. Polysaccharides are the most abundant natural biopolymers on the earth and are widely used in food, medicine, materials, cosmetics, and other fields. The physicochemical properties of polysaccharides such as particle size and molecular weight often affect their practical applications. In recent years, asymmetrical flow field-flow fractionation (AF4) has been widely used in the separation and characterization of polysaccharides because it has no stationary phases or packing materials, which reduces the risk of shear degradation of polysaccharides. In this review, the principle of AF4 was introduced briefly. The operation conditions of AF4 for the analysis of polysaccharides were discussed. The applications of AF4 for the separation and characterization of polysaccharides from different sources (plants, animals, and microorganisms) over the last decade were critically reviewed. [ABSTRACT FROM AUTHOR]

• Structural and adsorptive properties of multimodal anion exchangers are characterized • Adsorption rates are controlled by pore diffusion • Mass transfer of large proteins is highly restricted in agarose-based resins • Polymer-based resins provide higher dynamic binding capacity at short residence times The effect of bead and ligand structure on protein adsorption was investigated for multimodal anion exchangers combining a quaternary ammonium ion group with hydrophobic moieties: Nuvia aPrime 1 and aPrime 2, based on a 54 μm diameter polymeric bead, and Capto Adhere ImpRes and Capto Adhere, based on agarose beads 51 and 78 μm diameter, respectively. Bovine serum albumin (BSA) monomer, BSA dimer, and thyroglobulin (Tg) were used as model proteins. Based on TEM imaging and iSEC, the Nuvia resins have a microgranular structure and large pores (110 nm radius), while the Capto resins have a fibrous structure and smaller pores (32-36 nm radius). Comparable binding capacities (80-110 mg/mL), decreasing as salt is added, are observed for all three proteins on the Nuvia resins. Higher capacities (110-130 mg/mL), also decreasing as salt is added, are observed for BSA monomer and dimer on the Capto resins. However, the Tg binding capacity is very low in this case and increases as salt is added. Confocal laser scanning microscopy show that the kinetics are controlled by pore diffusion for all four resins, but with diffusivities that decrease as the protein size increases especially for the Capto resins. For Tg at low salt, binding is restricted to a thin shell close to the bead surface for both Capto resins. The ratio of effective and free diffusivity is about 0.30, 0.18, and 0.08 for BSA monomer, BSA dimer, and Tg, respectively, on the Nuvia resin. These values decrease to about 0.11, 0.04, and 0.01, respectively, for the Capto resins as a result of diffusional hindrance. Dynamic binding capacities are consistent with the equilibrium and rate behaviors. [ABSTRACT FROM AUTHOR]