Interaction Chromatography Research Articles

Metabolomics and serum pharmacochemistry revealed the preventive mechanism of Gushudan in kidney-yang-deficiency-syndrome rats.

Kidney-yang-deficiency-syndrome (KYDS) is a metabolic disease caused by neuroendocrine disorder. Gushudan (GSD) is a traditional Chinese medicine prescription with the effect of nourishing kidney and strengthening bones. In this study, the mechanism of preventive effect of GSD on KYDS was explored by integrating metabolomics and serum pharmacochemistry. Reversed-phase/hydrophilic interaction chromatography-ultra-high-performance liquid chromatography-Quadrupole-Orbitrap high-resolution mass spectrometry (RP/HILIC-UHPLC-Q-Orbitrap HRMS)-based serum metabolomics indicated metabolic disturbances of KYDS rats, and 50 potential biomarkers including l-threonine, succinic acid and phytosphingosine were obtained, which were mainly involved in alanine, aspartate and glutamate metabolism, citrate cycle (tricarboxylic acid cycle) and glycerophospholipid metabolism, among others. Serum pharmacochemistry identified 29 prototypical ingredients and 9 metabolites of GSD after administration, such as icaritin and xanthotoxol. The combination of 10 serum migration ingredients in GSD, including icaritin and osthole, with 7 important targets, including AKT serine/threonine kinase 1 (AKT1) and MAPK14, was found to be key for GSD to prevent KYDS in the network pharmacology study. This study provided a new idea for the research of pathogenesis of diseases and the pharmacodynamic mechanism of traditional Chinese medicine.

Two-dimensional tools for analyzing polymer microstructure; coupling non-aqueous ion-exchange chromatography to size-exclusion chromatography

Traditional liquid-chromatographic techniques, such as size-exclusion chromatography, (critical) interaction chromatography, and hydrodynamic chromatography, can all reveal certain aspects of polymers and the underlying distributions. The distribution of incorporated acid groups present in polyacrylates can be determined by non-aqueous ion-exchange chromatography, independent of other distributions present. The microstructural details on how this number of acid groups is incorporated in the polymer remains unknown. A low-molar mass polymer molecule with high acid content and a high-molar mass polymer with a low acid content may have the exact same number of acid groups. To take a next step towards understanding the polymer microstructure of water-borne resins, the distribution of incorporated acid groups over the molar-mass distribution has been investigated.For this purpose, an on-line coupling of non-aqueous ion-exchange chromatography (NAIEX) to size-exclusion chromatography (SEC) was established. Practical considerations regarding the system setup with respect to chromatography modes and column- and valve switching dimensionality are discussed. The orthogonality of NAIEX and SEC is demonstrated. Both liquid chromatography modes may be calibrated using polymer standards, yielding a calibrated separation plane. Cross-sectional data on either the molar mass distribution or the acid group distribution at a certain point of the separation plane is obtained. The value of the designed setup was demonstrated by the detailed characterization of the combined acid-group and molar-mass distribution in polymers with a low acid content, in the order of a few mass-%. Several stages of the emulsion polymerization process could be identified using the combined power of NAIEX and SEC.

In situphotoinitiated fabrication of phosphorylcholine-functionalized polyhedral oligomeric silsesquioxane hybrid monolithic column for mixed-mode capillary electrochromatography

A phosphorylcholine-functionalized POSS hybrid monolithic column was synthesizedviaUV curing. It exhibits hydrophilic interaction and weak cation exchange chromatography retention mechanism for the separation of typical polar and charged compounds.

Phase equilibria and conformational behavior of dendrimers in porous media: Towards chromatographic analysis of dendrimers

HypothesisThe intricate entropy-enthalpy interplay of dendrimers confined in pores affects their conformation and retention in the porous stationary phase. This work aims at providing important insights into its impacts on partitioning and chromatographic separation in both size-exclusion chromatography (SEC) and interaction chromatography (IC) regimes. SimulationsUsing Monte Carlo (MC) simulations, we investigated the bulk-pore phase equilibria and the conformational behavior of flexible dendrimers differing in generation, in spacer length and in fraction of modified terminal groups interacting differently with pore walls than the majority building units. FindingsWith increasing interaction strength, a distinct transition from a roughly spherical shape caused by simultaneous interactions with two walls to an ellipsoidal (or even disklike) conformation tenaciously adhering to only one wall was observed for moderately confined dendrimers. The strongly deformed dendrimers subjected to severe confinement gain high energy and the samples differing in the degree of modification become chromatographically discernable thanks to large energy differences. Consequently, our results suggest that the column fillings with fairly narrow pores which are ineffective in SEC, are highly efficient separation media for dendrimer studies by IC above the critical adsorption point (CAP). Overall, our simulations reveal useful information for advancing and optimizing experimental liquid chromatography studies of dendrimers.

A novel method of high-purity extracellular vesicle enrichment from microliter-scale human serum for proteomic analysis.

We have developed a rapid, low-cost, and simple separation strategy to separate extracellular vesicles (EVs) from a small amount of serum (i.e.,<100μL) with minimal contamination by serum proteins and lipoprotein particles to meet the high purity requirement for EV proteome analysis. EVs were separated by a novel polyester capillary channel polymer (PET C-CP) fiber phase/hydrophobic interaction chromatography (HIC) method which is rapid and can process small size samples. The collected EV fractions were subjected to a post-column cleanup protocol using a centrifugal filter to perform buffer exchange and eliminate potential coeluting non-EV proteins while minimizing EV sample loss. Downstream characterization demonstrated that our current strategy can separate EVs with the anticipated exosome-like particle size distribution and high yield (∼1×1011 EV particles per mL of serum) in approximately 15min. Proteome profiling of the EVs reveals that a group of genuine EV components were identified that have significantly less high-abundance blood proteins and lipoprotein particle contamination in comparison to traditional separation methods. The use of this methodology appears to address the major challenges facing EV separation for proteomics analysis. In addition, the EV post-column cleanup protocol proposed in the current work has the potential to be combined with other separation methods, such as ultracentrifugation (UC), to further purify the separated EV samples.

Amphipathic carbon quantum dots-functionalized silica stationary phase for reversed phase/hydrophilic interaction chromatography

Carbon quantum dots (CQDs) are considered as good chromatographic separation materials. However, due to the hydrophily of the synthesized CQDs, their applications in HPLC are limited to HILIC for separating strong polar compounds only. In this work, a novel amphipathic CQDs with both hydrophobicity and hydrophily is developed as mixed-mode stationary phase for RPLC/HILIC. To give CQDs certain hydrophobicity, 1,8-diaminooctane is chosen as one of the carbon sources for introducing alkyl chain into CQDs. The amphipathic CQDs modified silica (CQDs/SiO2) stationary phase has typical characteristic of RPLC/HILIC. Both hydrophobic and hydrophilic compounds including alkylbenzenes, polycyclic aromatic hydrocarbons, nucleosides and bases, amino acids, β-adrenoceptor blockers and agonists, sulfonamides, antibiotics and alkaloids obtain satisfactory separation on this CQDs/SiO2 column. 14 nucleosides and bases commonly existing in living organisms achieve good separation on this amphipathic CQDs/SiO2 column within 25 min and the resolutions reach 1.33–13.83 with an average column efficiency of 18,800. The retention mechanism of this novel CQDs/SiO2 column is investigated by linear solvation energy relationship model. It is found that hydrophobic interaction, π-π stacking, hydrogen-bonding and electrostatic interactions are main retention interactions under RPLC mode. This work provides a new approach for synthesis of amphipathic CQDs. Also, it indicates that amphipathic CQDs with versatile functional properties have great prospect in separation science.

Comprehensive Analysis of Polyethylene Graft Copolymers by Preparative Fractionation, Interaction Chromatography, and Thermal Analysis

The comprehensive analysis of complex copolymers is a challenge and requires the combination of results from different methods including size exclusion chromatography for molar mass and molecular s...

Fractionation of chain walking polyethylene and elucidation of branching, conformation and molar mass distributions

State-of-the-art liquid interaction chromatography (IC) allows separation of macromolecules according to chemical composition (CC), functionality or molecular topology in contrast to size-based approaches such as size exclusion chromatography (SEC), which has its limitations in distinguishing chemical structures. Yet, for macromolecules with multivariate molecular distributions, online identification and characterization of distinct components in bulk samples is a considerable challenge. A useful approach to narrow down the complexity of a given sample is preparative molar mass fractionation (pMMF) that provides fractions in mg or g scale, which can be subjected to further advanced analysis to elucidate the physical and structural properties of the sample. In this study, novel chain-walking polyethylenes (CWPE), which differ substantially in macromolecular architecture, are fractionated using pMMF. Using IC in temperature and solvent gradient modes (TGIC, SGIC), the pMMF fractions are separated according to branching topology. Quadruple-detector high temperature SEC is carried out to determine the physical properties and study the MM-dependent structural transformation of CWPE. A detailed branching analysis was carried out by means of nuclear magnetic resonance. Two-dimensional liquid chromatography (LC) hyphenating IC and SEC provides comprehensive bivariate correlations between MM and molecular topology.

Partitioning of polymers between bulk and porous media: Monte Carlo study of the effect of pore size distribution

In this paper we investigated the partitioning of polymer chains between bulk solvent and porous stationary phase under conditions appropriate for the chromatography under critical conditions (LCCC) close to the critical adsorption point (CAP). We addressed the concentration effect and the thermodynamic effect of pore-size dispersity (PSD) and their impacts on chromatography, i.e., the topics which surprisingly escaped from the interest of scientists in spite that the hydrodynamic effect of PSD has been a subject of numerous studies. The phase equilibria in narrow pores (as compared with the size of polymer coil) with attractive pores are complex and the enthalpy-to-entropy interplay is very intricate. The chains are attracted into pores and the partition coefficients are larger than 1, which corresponds to the interaction chromatography (IC), but they are strongly confined and deformed. The entropy plays important role and the elution volumes of chains differing in molar mass correspond to the size-exclusion chromatography (SEC). The study thus reveals a new chromatography regime which could be easily overlooked without the awareness of its existence. The unexpected findings are important not only for chromatography, but for understanding the phase equilibria of polymers in porous systems in general.

Glycan analysis of erythropoiesis-stimulating agents

Erythropoiesis stimulating agents (ESAs) are a group of therapeutic glycoproteins used to treat anaemia caused by chronic kidney disease or chemotherapy. A variety of ESA products are available in the European Union, including innovator, biosimilar and second-generation medicines. Glycosylation is a critical quality attribute of ESA products, as it has a crucial influence upon in vivo biological activity. In this study, a combination of chromatography and mass spectrometry analysis has been used to characterise and compare the glycosylation profiles of five ESA products; Eprex® (epoetin alfa), NeoRecormon® (epoetin beta), Binocrit® (epoetin alfa biosimilar), Silapo (epoetin alfa biosimilar) and Aranesp® (darbepoetin alfa). The methods utilised include mixed-mode anion-exchange/hydrophilic interaction chromatography (AEX/HILIC-MS) for N-glycan identification and quantitation, and HILIC-MS for O-glycan characterisation. The products exhibit notable differences in N- and O-glycosylation, including attributes such as sialic acid occupation, O-acetylation, N-acetyllactosamine extended antennae and sulphated/penta-sialylated N-glycans, which have the potential to cause divergence of therapeutic potencies. The study highlights the need for continued monitoring of ESA product glycosylation, ideally allied to pharmacological data, in order to ensure consistency and therapeutic equivalence between products and enhance our understanding of ESA structure-activity-relationships.

Critical parameters of liquid chromatography at critical conditions in context of poloxamers: Pore diameter, mobile phase composition, temperature and gradients

At the borderline between size exclusion chromatography (SEC) and interaction chromatography (IC) there is a special mobile phase composition and temperature at which polymer chains become “chromatographically invisible”. This point is termed as “chromatographic critical point” and chromatographic separations performed using these conditions are called “liquid chromatography at critical conditions” (LCCC). LCCC is a powerful technique in the analysis of functional polymers and block copolymers. At these so-called critical conditions molar mass discrimination of any specific homopolymer is suppressed rendering elution of whole range of molar mass at same elution volume. These conditions allow enhanced separation with regard to non-critical segment either in exclusion or interaction regime of the polymer chromatography. This article is intended to critically discuss different parameters that can be maneuvered to improve separation and in turn characterization of non-critical segment of block copolymers at LCCC. Different experimental parameters evaluated in this study include pore size of the column, mobile phase composition, temperature and gradients. These parameters can be adeptly adjusted to improve separation of non-critical segment while keeping the other segment close to critical conditions. Current study demonstrates that pore diameter and mobile phase are the only practical variable that can be used for improvement of characterization of non-critical block in the block copolymer while non-critical block is in exclusion regime. On the other hand, pore diameter of the column, temperature, solvent composition and gradients are important parameters that can be skillfully tuned for improvement of separation of non-critical block while non-critical block elutes in interaction regime. The above-mentioned variations are evaluated for di-block as well as tri-block copolymers of A-B-A and B-A-B type. Moreover, LCCC-IC is especially important for analysis of poloxamers.

1927-P: Metabolomics of Normal Weight vs. Overweight Diabetics

The majority of people living with type 2 diabetes mellitus (T2DM) are overweight or obese due to the additional burden of applying insulin to properly control blood sugar levels. However, some people, especially Asian populations, develop T2DM with a low BMI (normal weight). This discrepancy may be resulted from differences in body fat and muscle mass for a given BMI. In order to investigate the influences of body weight on the onset of T2DM, we collected plasma samples from 9 overweight and 17 normal-weight diabetic patients as a pilot study, then applied untargeted metabolomics profiling on these plasma samples. By combining Hydrophilic Interaction (HILIC) and Reverse-Phase Liquid Chromatography (RPLC)-Mass Spectrometry, we comprehensively detected and relatively quantified 5,469 metabolites. We then employed various pattern recognition algorithms (principal component analysis, hierarchical clustering, Welsh t-test, etc.), clearly separated overweight and normal-weight diabetic groups based on their plasma metabolite profiling. Furthermore, we statistically identified 52 significantly different metabolites between these two groups, mainly involved in lipid metabolism. These results not only demonstrate that untargeted metabolomics is able to serve as an efficient diagnostic tool to distinguish overweight and normal-weight diabetic patients, but also provide meaningful insights into molecular mechanisms about tight relationships between diabetes and obesity, further facilitate biomarker discovery for early diagnosis and differentiation of normal/overweight diabetes. Disclosure L. Faroqi: None.

Separation of polymers differing in their chain architecture by interaction chromatography: Phase equilibria and conformational behavior of polymers in strongly adsorbing porous media

Understanding the competition between adsorption on confining interfaces and conformational behavior of polymers in confined volumes is a necessary condition for correct interpretation of chromatographic curves. Accordingly, we investigated the conformational behavior of linear (L) and star (S) polymers and the impact of the chain architecture on phase equilibria in porous media using Monte Carlo (MC) simulations. We focused on interaction chromatography (IC) under conditions close to the critical adsorption point (CAP). We evaluated the concentration-dependent partition coefficients by MC and used them as an input for the solution of a time-discretized mass balance equation which predicts the shapes of the elution curves. The study elucidates the effects of chain architecture on the IC separation mechanism and shows that IC is a promising method for the separation of L and S polymers.

Pore size effect on the separation of polymers by interaction chromatography. A Monte Carlo study

When the polymers are studied by interaction chromatography (IC) in porous media, the IC separation mechanism competes with the size-exclusion chromatography (SEC) mechanism and under specific conditions close to the critical adsorption point (CAP), the elution times of monodisperse polymer samples nonmonotonically depend on pore sizes. We performed Monte Carlo (MC) simulations to elucidate this intriguing effect. By analyzing the behavior of self-avoiding and intersecting chains in two-dimensionally (2D)-confining square pores and in 1D-confining slits in good and Θ-solvents, we confirmed that the dimensionality of the confinement, more specifically, pore geometry, controls the chromatographic behavior. The nonmonotonic dependence of chromatographic characteristics on pore sizes occurs only in separations of self-avoiding chains on stationary phases composed of 2D-confining pores with strongly interacting walls. In agreement with experimental observations, the partition coefficient, K, increases with pore size, D, in narrow pores, peaks and then decreases in wider pores. The combination of thermodynamic and conformational analyses clearly showed that a complex interplay between enthalpy and entropy in 2D-confined media explains the nonmonotonic pore size dependence observed in the IC regime. The study specifies the region of conditions which endanger unambiguous interpretation of elution curves. Because the interplay of steric and adsorption effects takes place not only in chromatography, but also in other separation techniques (e.g., gel electrophoresis, nanofluidic techniques), the conclusions are generally relevant for all separations of large molecules in porous media.

Preparation of a monolithic column with a mixed‐mode stationary phase of reversed‐phase/hydrophilic interaction for capillary liquid chromatography

A monolithic capillary column with a mixed-mode stationary phase of reversed-phase/hydrophilic interaction chromatography was prepared for capillary liquid chromatography. The monolith was created by an in-situ copolymerization of a homemade monomer N,N-dimethyl-N-acryloxyundecyl-N-(3-sulfopropyl) ammonium betaine and a crosslinker pentaerythritol triacrylate in a binary porogen agent consisting of methanol and isopropanol. The functional monomer was designed to have a highly polar zwitterionic sulfobetaine terminal group and a hydrophobic long alkyl chain moiety. The composition of the polymerization solution was systematically optimized to permit the best column performance. The columns were evaluated by using acidic, basic, polar neutral analytes, as well as a set of alkylbenzenes and Triton X100. Very good separations were obtained on the column with the mixed-mode stationary phase. It was demonstrated that the mixed-mode stationary phase displayed typic dual retention mechanisms of reversed-phase/hydrophilic interaction liquid chromatography depending on the content of acetonitrile in the mobile phase. The method for column preparation is reproducible.

Review on the Application of Mixed-mode Chromatography for Separation of Structure Isoforms.

Proteins often generate structure isoforms naturally or artificially due to, for example, different glycosylation, disulfide scrambling, partial structure rearrangement, oligomer formation or chemical modification. The isoform formations are normally accompanied by alterations in charged state or hydrophobicity. Thus, isoforms can be fractionated by reverse-phase, hydrophobic interaction or ion exchange chromatography. We have applied mixed-mode chromatography for fractionation of isoforms for several model proteins and observed that cation exchange Capto MMC and anion exchange Capto adhere columns are effective in separating conformational isoforms and self-associated oligomers.

Two-Dimensional Liquid Chromatography Analysis of Polystyrene/Polybutadiene Block Copolymers.

A detailed characterization of a commercial polystyrene/polybutadiene block copolymer material (Styrolux) was carried out using two-dimensional liquid chromatography (2D-LC). The Styrolux is prepared by statistical linking reaction of two different polystyrene- block-polybutadienyl anion precursors with a multivalent linking agent. Therefore, it is a mixture of a number of branched block copolymers different in molecular weight, composition, and chain architecture. While individual LC analysis, including size exclusion chromatography, interaction chromatography, or liquid chromatography at critical condition, is not good enough to resolve all the polymer species, 2D-LC separations coupling two chromatography methods were able to resolve all polymer species present in the sample; at least 13 block copolymer species and a homopolystyrene blended. Four different 2D-LC analyses combining a different pair of two LC methods provide their characteristic separation results. The separation characteristics of the 2D-LC separations are compared to elucidate the elution characteristics of the block copolymer species.

Characterization and Morphological Distribution of Ethylene Content in Impact Propylene Copolymers

The effect of ethylene content, in a range between 8 and 11 mol%, and distribution on the solid‐state morphology of five impact polypropylene copolymers (IPC) have been studied via SEM, fractionation and microstructural characterization via gel permeation chromatography (GPC), high‐temperature thermal‐gradient interaction chromatography (HT‐TGIC), and differential scanning calorimetry (DSC). All copolymers display heterophasic morphology, with rubbery droplets randomly distributed in a predominant iPP matrix. While the mass fraction of amorphous ethylene‐propylene rubber molecules is very similar for all IPC studied, the size of the droplets increases from 0.6 to 1.1 μm with increasing ethylene content. This change in droplet size is correlated with changes in the composition of the crystalline ethylene‐propylene (CEP) component extracted via fractionation.

Living Anionic Polymerization – Part II: Further Expanding the Synthetic Versatility for Novel Polymer Architectures

Living Anionic Polymerization – Part II: Further Expanding the Synthetic Versatility for Novel Polymer Architectures

End‐Functionalized Chains via Anionic Polymerization: Can the Problems with Using Diphenylethylene Derivatives be Solved by using Bisphenol F?

AbstractThe controlled functionalization of polymers via anionic polymerization draws great attention not only because of the importance of introducing functionality into otherwise unfunctionalized polymers, but also because of the possibility to use the resulting macromonomers to make a variety of complex architectures. The versatile family of 1,1‐diphenylethylene (DPE) derivatives is widely used to produce many different functionalized (co)polymers. DPE can be added either as an end‐capping agent or be activated by butyllithium to initiate the polymerization. However, each approach faces potential problems in gaining precise control over the number of DPE moieties per chain. In this work, for the first time, the effectiveness of each approach is compared by the characterization of 1,1‐bis(4‐tert‐butyldimethylsiloxyphenyl)ethylene functionalized polystyrene, synthesized via both the procedures. A combination of NMR, size exclusion chromatography, matrix‐assisted laser desorption ionization‐time of flight (MALDI‐ToF) mass spectrometry, and interaction chromatography is used. To overcome the limitations of DPE derivatives, the use of a novel (protected) bisphenol F potassium initiator is proposed.