Incorporation of ionic liquid into porous polymer monoliths to enhance the separation of small molecules in reversed‐phase high‐performance liquid chromatography

An ionic liquid was incorporated into the porous polymer monoliths to afford stationary phases with enhanced chromatographic performance for small molecules in reversed‐phase high‐performance liquid chromatography. The effect of the ionic liquid in the polymerization mixture on the performance of the monoliths was studied in detail. While monoliths without ionic liquid exhibited poor resolution and low efficiency, the addition of ionic liquid to the polymerization mixture provides highly increased resolution and high efficiency. The chromatographic performances of the monoliths were demonstrated by the separations of various small molecules including aromatic hydrocarbons, isomers, and homologues using a binary polar mobile phase. The present column efficiency reached 27 000 plates/m, which showed that the ionic liquid monoliths are alternative stationary phases in the separation of small molecules by high‐performance liquid chromatography     

Towards porous polymer monoliths for the efficient, retention-independent performance in the isocratic separation of small molecules by means of nano-liquid chromatography

We have investigated the free-radical copolymerization dynamics of styrene and divinylbenzene in the presence of micro- and macro-porogenic diluents in 100 μm I.D. sized molds under conditions of slow thermal initiation leading to (macro)porous poly(styrene-co-divinylbenzene) monolithic scaffolds. These specifically designed experiments allowed the quantitative determination of monomer specific conversion against polymerization time to derive the porous polymer scaffold composition at each desirable copolymerization stage after phase separation. This was carried out over a time scale of 3h up to 48 h polymerization time, enabling the efficient and repeatable termination of the polymerization reactions. In parallel, the porous and hydrodynamic properties of the derived monolithic columns were thoroughly studied in isocratic nano-LC mode for the reversed-phase separation of a homologous series of small retained molecules. At the optimized initiator concentration, polymerization temperature and time, the macroporous poly(styrene-co-divinylbenzene) monoliths show a permanent mesoporous pore space, which was readily observable by electron microscopy and indicated by nitrogen adsorption experiments. Under these conditions, we consistently find a polymer scaffold composition which suggests a high degree of cross-linking and thus minimum amount of gel porosity. These columns reveal a retention-insensitive plate height in the separation of small retained molecules which only slightly decreases at increased linear mobile phase velocity. As the polymerization progresses, a build-up of less-densely cross-linked material occurs, which is directly reflected in the observed consistent increase in retention and plate heights. This leads to a significant deterioration in overall isocratic separation performance. The decrease in performance is ascribed in particular to the increased mass transfer resistance governing the monoliths' performance over the whole linear chromatographic flow velocity range at polymerization times significantly higher than that of phase separation. The performance of the optimized monoliths only becomes limited by fluid dispersion due to the poorly structured macroporous pore space.     

Porous polymer monoliths for small molecule separations: advancements and limitations

Porous polymer monoliths are considered to be one of the major breakthroughs in separation science. These materials are well known to be best suited for the separation of large molecules, specifically proteins, an observation most often explained by convective mass transfer and the absence of small pores in the polymer scaffold. However, this conception is not sufficient to explain the performance of small molecules. This review focuses in particular on the preparation of (macro)porous polymer monoliths by simple free-radical processes and the key events in their formation. There is special focus on the fluid transport properties in the heterogeneous macropore space (flow dispersion) and on the transport of small molecules in the swollen, and sometimes permanently porous, globule-scale polymer matrix. For small molecule applications in liquid chromatography, it is consistently found in the literature that the major limit for the application of macroporous polymer monoliths lies not in the optimization of surface area and/or modification of the material and microscopic morphological properties only, but in the improvement of mass transfer properties. In this review we discuss the effect of resistance to mass transfer arising from the nanoscale gel porosity. Gel porosity induces stagnant mass transfer zones in chromatographic processes, which hamper mass transfer efficiency and have a detrimental effect on macroscopic chromatographic dispersion under equilibrium (isocratic) elution conditions. The inherent inhomogeneity of polymer networks derived from free-radical cross-linking polymerization, and hence the absence of a rigid (meso)porous pore space, represents a major challenge for the preparation of efficient polymeric materials for the separation of small molecules.     

Recent advances in the design of organic polymer monoliths for reversed-phase and hydrophilic interaction chromatography separations of small molecules

Owing to their favorable porous structure with pore size distribution shifted towards large flow-through pores, organic polymer monoliths have been mainly employed for the separation of macromolecules in gradient elution liquid chromatography. The absence of significant amounts of small pores with a stagnant mobile phase and the resulting low surface area were considered as the main reason for their poor behavior in the isocratic separation of small molecules. Several recent efforts have improved the separation power of organic polymer monoliths for small molecules offering column efficiency up to tens of thousands of plates per meter. These attempts include optimization of the composition of polymerization mixture, including the variation of functional monomer, the cross-linking monomer, and the porogen solvents mixture, adjustment of polymerization temperature, and time. Additionally, post-polymerization modifications including hypercross-linking and the use of carbon nanostructures showed significant improvement in the column properties. This review describes recent developments in the preparation of organic polymer monoliths suitable for the separation of small molecules in the isocratic mode as well as the main factors affecting the column efficiency.     

有机聚合物整体柱的制备与应用的研究进展

整体柱具有通透性能良好和传质速度快等特点,可实现快速、高效、高通量的分离,近年来已引起人们的热切关注。聚合物整体柱是其中应用最为广泛的一种,它是由单体、交联剂、致孔剂和引发剂等通过原位聚合得到的连续均一的棒状聚合物,具有取材广泛,使用pH范围比较宽,生物兼容性好等特点,通过化学修饰,可以用作多种色谱模式的固定相。该文主要综述了2003年至2006年期间有关聚合物整体柱制备和应用的研究进展。     

Micro-bore titanium housed polymer monoliths for reversed-phase liquid chromatography of small molecules

A new method for the fixation of polymethacrylate monoliths within titanium tubing of up to 0.8 mm I.D. for use as a chromatographic column under elevated temperatures and pressures is described. The preparation of butyl methacrylate–ethylene dimethacrylate-based monolithic stationary phases with desired porous structures was achieved within titanium tubing with pre-oxidised internal walls. The oxidised titanium surface was subsequently silanised with 3-trimethoxysilylpropyl methacrylate resulting in tight bonding of butyl methacrylate porous monolith to the internal walls, providing stationary phase stability at column temperatures up to 110 °C and at operating column pressure drops of >28 MPa. The titanium housed monoliths exhibited a uniform and dense porous structure, which provided peak efficiencies of up to 59,000 theoretical plates per meter when evaluated for the separation of small molecules in reversed-phase mode, under optimal conditions (achieved at 15 μL/min and temperature of 110 °C for naphthalene with a retention factor, k = 0.58). The developed column was applied to the reversed-phase isocratic separation of a text mixture of pesticides.     

Chromatographic evaluation of reversed-phase/anion-exchange/cation-exchange trimodal stationary phases prepared by electrostatically driven self-assembly process

This work describes chromatographic properties of reversed-phase/cation-exchange/anion-exchange trimodal stationary phases. These stationary phases were based on high-purity porous spherical silica particles coated with nano-polymer beads using an electrostatically driven self-assembly process. The inner-pore area of the material was modified covalently with an organic layer that provided both reversed-phase and anion-exchange properties while the outer surface was coated with nano-sized polymer beads with strong cation-exchange characteristics. This design ensured spatial separation of the anion-exchange and the cation-exchange regions, and allowed reversed-phase, anion-exchange and cation-exchange retention mechanisms to function simultaneously. Chromatographic evaluation of ions and small molecules suggested that retention of ionic analytes was influenced by the ionic strength, pH, and mobile phase organic solvent content, and governed by both ion-exchange and hydrophobic interactions. Meanwhile, neutral analytes were retained by hydrophobic interaction and was mainly affected by mobile phase organic solvent content. Depending on the specific application, selectivity could be optimized by adjusting the anion-exchange/cation-exchange capacity ratio (selectivity), which was achieved experimentally by using porous silica particles with different surface areas.     

Are we approaching a post‐monolithic era?

Thirty years after their introduction, monolithic stationary phases are an important member of chromatographic phases. When compared to conventional particulate materials, the continuous internal structure of both inorganic silica and organic polymer monoliths allows some hydrodynamic and analytical possibilities that are not provided by conventional particulate stationary phases. Polymer‐based monolithic stationary phases offer simple preparation and straightforward surface modification, which makes them very versatile materials that are applicable, for example, as chromatographic stationary phases, sample enrichment units, enzymatic reactors, and external trigger‐responding materials. On the other hand, current polymer monoliths cannot compete with efficiency provided by superficially porous and sub 2 µm particles. In this highlight article, I take advantage of the 30th anniversary of their introduction to discuss several concerns related to polymer‐based monolithic stationary phases. Particularly, I focus on preparation repeatability, porous properties, swelling of the polymers in organic solvents, column efficiency for small molecules, and heterogeneity of dominant flow‐through pores. In the end, I offer three possible approaches on how to overcome drawbacks related to stationary phases heterogeneity to further increase the applicability of polymer‐based monolithic stationary phases.     

Linear Solvation Energy Relationships in the Determination of Specificity and Selectivity of Stationary Phases

The retention of fifty structurally different compounds has been studied using linear solvation energy relationships. Investigations were performed with the use of six various stationary phases with two mobile phases (50/50?% v/v methanol/water and 50/50?% v/v acetonitrile/water). Packing materials were home-made and functionalized with octadecyl, alkylamide, cholesterol, alkyl-phosphate and phenyl molecules. This is the first attempt to compare all of these stationary phases synthesized on the same silica gel batch. Therefore, all of them may be compared in more complex and believable way, than it was performed earlier in former investigations. The phase properties (based on Abraham model) were used to the classification of stationary phases according to their interaction properties. The hydrophilic system properties s, a, b indicate stronger interactions between solute and mobile phase for most of the columns. Both e and v cause greater retention as a consequence of preferable interactions with stationary phase by electron pairs and cavity formation as well as hydrophobic bonds. However, alkyl-phosphate phase has different retention properties, as it was expressed by positive sign of s coefficient. It may be concluded that most important parameters influencing the retention of compounds are volume and hydrogen bond acceptor basicity. The LSER coefficients showed also the dependency on the type of organic modifier used as a mobile phase component.     

Preparation of Cyclodextrin Type Stationary Phase Based on Graphene Oxide and Its Application in Enantioseparation and Hydrophilic Interaction Chromatography

Graphene oxide (GO) was covalently coupled to the surface of amino silica gel by amide bond. β-cyclodextrin (β-CD) was further chemically bonded with GO to prepare a novel chiral stationary phase. The resulting material was characterized by Fourier transform-infrared (FT-IR) spectra, scanning electron microscopy (SEM), transmission electron microscopy (TEM), elemental analysis and thermogravimetric analysis (TGA). The separation of seven enantiomers was improved in varying degrees. Meanwhile, the stationary phase showed typical characteristics of hydrophilic interaction chromatography (HILIC), and four small nucleoside molecules were separated with the mobile phase of methanol-acetonitrile-water (45:45:10, V/V) in the HILIC mode. In addition, the separation mechanism of the stationary phase was further explored by studying the effects of mobile phase composition, temperature and pH value on the analyte retention. The low temperature was conducive to the separation of analytes at 20–60 °C. The addition of protonated solvent methanol significantly decreased the retention time of the four analytes. The change of pH affected the degree of protonation of the analyte, the interaction between analytes and the stationary phase, and retention time of analytes. The results showed that GO and β-CD played a synergistic effect in the chiral resolution of the chromatographic stationary phase. The retention of analytes in HILIC was attributed to their mixed-mode retention mechanisms including hydrophilic interaction, electrostatic interaction, hydrogen bonding, π-π stacking and so on.     

Preparation of monodispersed vinylpyridine–divinylbenzene porous copolymer resins and their application to high-performance liquid chromatographic separation of aromatic amines

For the separation of aromatic amines, two types of monodispersed porous polymer resins were prepared by the copolymerization of 2-vinylpyridine and 4-vinylpyridine with divinylbenzene in the presence of template silica gel particles (particle size 5 μm), followed by dissolution of the template silica gel in an alkaline solution. The transmission electron micrographs and the scanning electron micrograph revealed that these templated polymer resins have a spherical morphology with a good monodispersity and porous structure. Using these monodispersed polymer resins, the high-performance liquid chromatographic separation of aromatic amines in the mobile phases of pHs 2.0, 2.9, 4.1, 7.2 and 11.7 were carried out. The 2-vinylpyridine–divinylbenzene copolymer resins showed slightly stronger retentions for aromatic amines than the 4-vinylpyridine–divinylbenzene copolymer resins. Under acidic conditions (around pH 2.0), aniline and the toluidines showed no retention on these copolymer resins due to the repulsion between the cationic forms of these amines and pyridinium cations in the stationary phase, whereas less basic aromatic amines or non-basic acetanilide showed slight retentions. Above pH 4.1, the separation of aromatic amines with these polymer resins showed a typical reversed-phase mode separation. Therefore, the separation patterns of aromatic amines are effectively tunable by changing the pH value of the mobile phases. A good separation of eight aromatic amines was achieved at pH 2.9 using the 2-vinylpyridine–divinylbenzene copolymer resins.     

Recent strategies to enhance the performance of polymer monoliths for analytical separations

This review summarizes recent developments made in the incorporation of functional materials into organic polymer monoliths, together with new monolithic forms and formats, which enhance their application as supports and stationary phase materials for sample preparation and chromatographic separations. While polymer monoliths are well‐known supports for the separation of large molecules, recent developments have been made to improve their features for the separation of small molecules. The selectivity and performance of organic polymer monoliths has been improved by the incorporation of different materials, such as metal‐organic frameworks, covalent organic frameworks, or other types of nanostructured materials (carbon nanohorns, nanodiamonds, polyoxometalates, layered double hydroxides, or attapulgite). The surface area of polymer monoliths has been significantly increased by polymer hypercrosslinking, resulting in increased efficiency when applied to the separation of small molecules. In addition, recent exploration of less conventional supports for casting polymer monoliths, including photonic fibres and 3D printed materials, has opened new avenues for the applications of polymer monoliths in the field of separation science. Recent developments made in these topics are covered, focusing on the strategies followed by the authors to prepare the polymer monoliths and the effect of these modifications on the developed analytical applications.     

Optimization of the porous structure and polarity of polymethacrylate-based monolithic capillary columns for the LC-MS separation of enzymatic digests

The porous structure as well as the polarity of methacrylate ester-based monolithic stationary phases has been optimized to achieve the separation of various peptides originating from enzymatic digestion. The porous structure, determined by the size of both pores and microglobules, was varied through changes in the composition of porogenic solvents in the polymerization mixture, while the polarity was controlled through the incorporation of butyl, lauryl, or octadecyl methacrylate in the polymer backbone. Both the morphology and the chemistry of the monoliths had a significant effect on the retention and efficiency of the capillary columns. The best resolution of peptidic fragments obtained by digestion of Cytochrome c with trypsin in solution was obtained in a gradient LC-MS mode using a monolithic capillary column of poly(lauryl methacrylate-co-ethylene dimethacrylate) featuring small pores and small microglobules. Raising the temperature from 25 to 60 degrees C enabled separations to be carried out at 40% higher flow rates. Separations carried out at 60 degrees C with a steeper gradient proceeded without loss of performance in half the time required for a comparable separation at room temperature. Our preparation technique affords monolithic columns with excellent column-to-column and run-to-run repeatability of retention times and pressure drops.     

离子色谱整体固定相的最新进展

整体固定相是近年来新兴的一种多孔性固定相介质,它在离子态及极性化合物的分离中得到了越来越广泛的重视。本文就离子色谱领域整体固定相的发展以及最新的研究动向进行了综述,讨论了离子色谱整体固定相的优点、分类以及在分离分析离子态物质方面的应用等。     

System Maps for Retention of Small Neutral Compounds on a Superficially Porous Ethyl-Bridged,Octadecylsiloxane-Bonded Silica Stationary Phase in Reversed-Phase Liquid Chromatography

The system constants of the solvation parameter model are used to prepare system maps for the retention of small neutral compounds on an ethyl-bridged, ocatadecylsiloxane-bonded superficially porous silica stationary phase (Kinetex EVO C18) for aqueous mobile phases containing 10–70% (v/v) methanol or acetonitrile. Electrostatic interactions (cation-exchange) are important for the retention of weak bases with acetonitrile–water but not methanol–water mobile phase compositions. Compared with a superficially porous octadecylsiloxane-bonded silica stationary phase (Kinetex C18) with a similar morphology but different topology statistically significant differences in selectivity at the 95% confidence level are observed for neutral compounds that vary by size and hydrogen-bond basicity with other intermolecular interactions roughly similar. These selectivity differences are dampened with acetonitrile–water mobile phases, but are significant for methanol–water mobile phase compositions containing <30% (v/v) methanol. A comparison of a totally porous ethyl-bridged, octadecylsiloxane-bonded silica stationary phase (XBridge C18) with Kinetex EVO C18 indicated that they are effectively selectivity equivalent.     

Separation of drugs by high-performance liquid chromatography with porous polymer resins.

The separation of cold drugs and neuroleptics by high-performance liquid chromatogarphy with the porous polymer resin DVB-MCL-O (or 11-30-0), which is a styrene-divinylbenzene-methyl methacrylate copolymer substituted with hydroxymethyl groups, was studied. This copolymer was compared with the commercial porous polymers Hitachi gel 3011, 3011-0 and 3030. A very small theoretical plate height was obtained by using DVB-MCL-O and methanol-ammonia solution (99:1) as the stationary and mobile phases, respectively. This combination was found to be the most suitable for the rapid separation of condensed aromatic ring compounds.     

Applications of monolithic columns in gas chromatography and supercritical fluid chromatography

Porous monoliths are well‐known stationary phases in high‐performance liquid chromatography and capillary electrochromatography. Contrastingly, their use in other types of separation methods such as gas or supercritical fluid chromatography is limited and scarce. In particular, very few studies address the use of monolithic columns in supercritical fluid chromatography. These are limited to silica‐based monoliths and will be covered in this review together with an underlying reason for this trend. The application of monoliths in gas chromatography has received much more attention and is well documented in two reviews by Svec and Kurganov published in 2008 and 2013, respectively. The most recent studies, covered in this review, build on the previous findings and on further understanding of the influence of preparation conditions on porous properties and chromatographic performance of poly(styrene‐co‐divinylbenzene), polymethacrylate, and silica‐based monolithic columns while expanding to polymer‐based monoliths with incorporated metal organic frameworks and to vinylized hybrid silica monoliths. In addition, the potential application of porous layer open tubular monolithic columns in low‐pressure gas chromatography will be addressed.     

Influence of the polymerisation time on the porous and chromatographic properties of monolithic poly(1,2-bis(p-vinylphenyl))ethane capillary columns

In order to elucidate the effect of the polymerisation time on the morphology of styrene based monolithic support materials, continuous poly(1,2-bis(p-vinylphenyl))ethane (BVPE) rods were synthesised in 1.0 ml glass vials by thermally initiated free radical polymerisations of BVPE in the presence of porogens (toluene, decanol) and a,a′-azoisobutyronitrile (AIBN) as initiator at 65 °C for different polymerisation times (60, 90, 150, 300 and 600 min). Porosity parameters like pore-size-distribution and total porosity were investigated by mercury intrusion porosimetry, while the specific surface area of the BVPE monolithic supports was determined by N₂-adsorption (BET) measurements. An untypical bimodal pore-size-distribution comprising a high fraction of both mesopores (2–50 nm) and macropores (mainly flow-channels in the micrometer range) was observed as a result of the stepwise decrease of the polymerisation time. In consequence of the significant changes of the pore-size-profile, shortening the polymerisation time also resulted in enhanced total porosity due to enlarged flow-channel diameters and increased surface area according to the presence of a considerable amount of mesopores. Results upon the porosity profile of the support are further confirmed by SEM images of monoliths polymerised for different time periods. Since mesoporosity and high surface area of the chromatographic support material play key roles in the interaction and thus retention of low-molecular-weight compounds, polymerisation time should also affect the chromatographic properties and applicability of these polymers. To study the influence of the polymerisation time towards the separation efficiency of small molecules on BVPE capillary columns (200 μm I.D., 8 cm), a mixture of homologous alkylbenzenes was chosen for column evaluation. In accordance with the observations of the porous properties of BVPE stationary phases, the rapid and high resolution separation of a range of low-molecular-weight compounds on monolithic BVPE supports were successfully realised. The methodical reduction of the polymerisation time has been demonstrated to be a simple and effective tool to tailor the porous properties of organic monoliths to provide novel polymer-based stationary phases with porous properties adequate for the rapid and high resolution chromatography of small organic molecules.     

Temperature effects on retention and efficiency of butyl and lauryl acrylate porous polymer monoliths in capillary electrochromatography

The development of organic porous polymer monoliths represents an alternative approach to stationary phase design. The use of these materials has helped to rekindle interest in capillary electrochromatography. Although a large number of investigations have explored different monolith recipes, polymerization conditions, and application challenges, few investigations have addressed the fundamentals of this separation mode with this type of material. This study addresses the thermodynamics of the reversed phase retention mechanism on 100% butyl acrylate and 1:3 butyl:lauryl acrylate (volume/volume ratio) porous polymer monoliths used in a capillary electrochromatography mode. Linear van't Hoff plots yield enthalpies of retention of ?3.9 to ?14.3 kJ/mol on two different, but related columns for five selected hydrophobic analytes across a thirty degree temperature range. Minimum plate heights were only moderately impacted over this temperature range.     

Insights into the Retention Mechanism of Small Neutral Compounds on Octylsiloxane-Bonded and Diisobutyloctadecylsiloxane-Bonded Silica Stationary Phases in Reversed-Phase Liquid Chromatography

The system constants of the solvation parameter model are used to prepare system maps for the retention of small neutral compounds on an octylsiloxane-bonded (Kinetex C8) and diisobutyloctadecylsiloxane-bonded (Kinetex XB-C18) superficially porous silica stationary phases for aqueous mobile phases containing 10–70% (v/v) methanol or acetonitrile. Electrostatic interactions (cation-exchange) are important for the retention of weak bases with acetonitrile–water but not for methanol–water mobile phases. Compared with an octadecylsiloxane-bonded silica stationary phase (Kinetex C18) retention is reduced due to a less favorable phase ratio for both the octylsiloxane-bonded and diisobutyloctadecylsiloxane-bonded silica stationary phases while selectivity differences are small and solvent dependent. Selectivity differences for neutral compounds are larger for methanol–water but significantly suppressed for acetonitrile–water mobile phases. The selectivity differences arise from small changes in all system constants with solute size and hydrogen-bond basicity being the most important due to their dominant contribution to the retention mechanism. Exchanging the octadecylsiloxane-bonded silica column for either the octylsiloxane-bonded or diisobutyloctadecylsiloxane-bonded silica column affords little scope for extending the selectivity space and is restricted to fine tuning of separations, and in some cases, to obtain faster separations due to a more favorable phase ratio. For weak bases larger differences in relative retention are expected with acetonitrile–water mobile phases on account of the additional cation exchange interactions possible that are absent for the octadecylsiloxane-bonded silica stationary phase.