Mixed-mode Chromatographic Stationary Phase Research Articles

Green preparation of composite hydrogel coated hydrophobic long-chain carboxylic acid-bonded silica microspheres for mixed-mode chromatography

Combining composite hydrogel with silica gel can endow hydrogel with new application possibilities. In this work, polyvinyl pyrrolidone (PVP) and polyvinyl alcohol (PVA) were combined into a composite hydrogel, which was then wrapped on the surface of 1-undecenoic acid (UA) bonded silica microspheres. As a result, a novel PVP/PVA composite hydrogel-functionalized silica stationary phase (PVP/PVA@Sil-UA) was prepared by a green and facile method. Specifically, the thiol-ene click reaction was preferentially used to bond a hydrophobic long-chain carboxylic acid (UA) on the surface of thiolated silica gel in deep eutectic solvent, which was able to provide abundant hydrogen bonding sites for the further coating of PVP/PVA composite hydrogel. The hydrophobic long alkyl chain not only improved the separation selectivity, but also effectively alleviated the swelling of hydrogel. It was discovered that the prepared PVP/PVA@Sil-UA showed outstanding separation performance for a wide range of samples. The study of retention behavior proved that the PVP/PVA@Sil-UA was a mixed-mode chromatographic stationary phase, possessing hydrophilic and hydrophobic retention characteristics. In summary, an eco-friendly method was applied to realize the effective combination of composite hydrogel and SiO2 due to the fact that no harmful organic solvents were used in the preparation process, which provided a novel idea for the application of composite hydrogel in the field of chromatographic separation.

Performance evaluation of 2-undecylimidazole/propyl methacrylate bifunctional silica gel for mixed-mode reversed-phase/anion-exchange chromatography

In this work, 2-undecylimidazole (UI) and propyl methacrylate (PMA) dual-functionalized silica gel was prepared and applied as a mixed-mode chromatographic stationary phase. Successful preparation of this stationary phase (SiO2-UI-PMA) was proved through a range of characterization techniques. The chromatographic performance of the obtained SiO2-UI-PMA was investigated for the separation of environmental endocrine disruptors (phthalates, steroid hormones, bisphenols and alkylphenols), sulfonamides and food-borne stimulants in reversed-phase chromatography, as well as for the separation of inorganic anions in anion-exchange chromatography. The effect of column temperature, acetonitrile content, salt concentration and mobile phase pH on retention was studied to explore the separation mechanism, and the results indicated that this stationary phase provided multiple interactions with analytes. Compared with the commercial C18 column, the SiO2-UI-PMA demonstrated more preferable separation ability under the mobile phase conditions with a low content of acetonitrile, thereby acting as a good alternative candidate for the conventional reversed-phase stationary phase in chromatographic analysis. Furthermore, the SiO2-UI-PMA stationary phase exhibited high column efficiency (45 860 plates m−1 for progesterone) even though it was prepared from the non-serviceable silica based chromatographic packing materials.

Preparation and chromatographic evaluation of a mixed polymer brush-silica stationary phase with temperature-sensitive property.

In this study, a developed chromatographic stationary phase combines the high selectivity of mixed-mode retention with a temperature-responsive property to boost separation efficiency. Copolymer brushes were grafted onto silica gels through surface initiated-atom transfer radical polymerization by polymerizing two types of monomer, temperature-responsive vinylcaprolactam (VCl) and quinine (Qun) containing benzopyridine, a tertiary ammonium positive center, and hydroxyl groups. The obtained silica@poly(Qun-co-VCl) stationary phases were packed as a chromatographic column, and the retention behavior of hydrophobic polycyclic aromatics, highly polar nucleosides, charged organic acids and β-agonists was studied for this column under different separation modes. The ability to separate different types of analyte shows that the silica@poly(Qun-co-VCl) column provides multiple hydrophobic, hydrophilic and electrostatic interactions toward analytes, achieving the separation of various compounds in one column. In addition, temperature-dependent resolution of polycyclic aromatics, nucleosides, organic acids and β-agonists was investigated using modulation of the column temperature, and the column exhibited adjustable separation selectivity by simply changing the column temperature. These results demonstrate that the grafting of copolymer brushes on a silica surface, consisting of temperature-responsive poly-VCl and multifunctional groups of poly-Qun, is useful as a mixed-mode chromatographic stationary phase for thermally-modulated multiple interactions. Additionally, this column was also used for the quantitative detection of uridine and inosine from cordyceps.

Octadecylamine and serine-derived carbon dots-modified silica gel for reversed phase/hydrophilic interaction liquid chromatography

In order to meet the demand for the separation of complex samples, the development of mixed-mode chromatographic stationary phases has become one of the research priorities in chromatographic separation. In this work, carbon dots, Ser-OCDs, with amino, hydroxyl, carboxyl and alkyl chains were synthesized for the first time and modified on silica gel for RPLC/HILIC mixed-mode chromatography. The stationary phase, Sil-Ser-OCDs, enables the effective separation of hydrophobic and hydrophilic compounds such as alkylbenzenes, polycyclic aromatic hydrocarbons, nucleosides, bases and sulfonamides with better separation selectivity and higher column efficiency compared to classical C18 column. Polycyclic aromatic hydrocarbons, nucleosides, bases and sulfonamides can all be separated in less than 10 min, Moreover, the column efficiency (theoretical plates, N) of butylbenzene was even as high as 124,024 plates m−1. By investigating the effects of column temperature and concentration of buffer salt solution on retention and constructing the van't Hoff equation, hydrogen bonding, π-π conjugation, electrostatic repulsion, partitioning and hydrophobic interactions were found to be the main retention interactions. Sil-Ser-OCDs column was eluted for a long time at pH 3 and 8, and the relative retention remained basically unchanged, which proved that the stability of the column was excellent. This work broadens the variety of chromatographic stationary phase materials, at the same time, indicates that carbon dots have promising applications in mixed-mode chromatography.

Cooperative Retention of Nonsteroidal Anti-inflammatory Drugs on Polyamine-Based Mixed-Mode Chromatographic Stationary Phases

Cooperative Retention of Nonsteroidal Anti-inflammatory Drugs on Polyamine-Based Mixed-Mode Chromatographic Stationary Phases

High stability amino-derived reversed-phase/anion-exchange mixed-mode phase based on polysilsesquioxane microspheres for simultaneous separation of compound drugs

A series of amino-derived mixed-mode chromatographic stationary phases were synthesized based on porous mercaptopropyl-functionalized polysilsesquioxane mesoporous microspheres synthesized by a co-condensation of methyltrimethoxysilane (MTMS) and mercaptopropyltrimethoxysilane (MPTMS). Through controlling the ratio of MTMS and MPTMS, the modified stationary phases with different amino densities were prepared by a “thiol-ene” click chemistry reaction. The morphology, pore structure, and functional groups of the microspheres were characterized by scanning electron microscope (SEM), nitrogen adsorption-desorption test, Fourier transform infrared spectroscopy (FT-IR), elemental analysis, and zeta potential, respectively. The chromatographic behavior of the stationary phases was evaluated by using alkylbenzene homologs and inorganic anions as probes. The mixed-mode retention behavior and separation mechanisms for neutral, alkaline, and acidic drugs on the prepared column had been systematically studied by changing the value of pH, ionic, and solvent strength of the mobile phase. Compared with the silica-based amino-bonded column (S-NH2), the synthesized organosilica phase exhibited higher hydrothermal stability and longer service life under high alkaline conditions. The newly synthesized phase was successfully applied to the simultaneous separation of the multiple substances in compound drugs.

Particle packed mixed-mode chromatographic stationary phase for the separation of peptide in liquid chromatography.

A particle-based stationary phase has been prepared for the separation of five synthetic peptides and a mixture containing tryptic digest of cytochrome C in liquid chromatography. Particles originating from silica monolith were differentially sedimented to obtain 1-2 μm particles. A stationary phase was achieved by the coating of poly(styrene-methacrylic acid-N-phenylacrylamide) copolymer onto the particles via reversible addition-fragmentation chain transfer polymerization reaction. Stainless steel column (30cm long and 1mm internal diameter) was packed with stationary phase. Very high separation efficiency (ca. 351000 plates/m) was achieved for five commercial peptides with a percent relative standard deviation of less than 1%. Protocol for the synthesis and modification of silica monolith particles has been well optimized with a good reproducibility both in particle and pore size. The column resolved about 21 peptide components from a mixture containing tryptic digest of cytochrome C, under the elution conditions of acetonitrile/15mM ammonium format (65/35 v/v%) with a flow rate of 28 μL/min.

A new strategy for the preparation of core-shell MOF/Polymer composite material as the mixed-mode stationary phase for hydrophilic interaction/ reversed-phase chromatography

A facile method for efficient synthesis of core-shell composite material was proposed. In this method, the silica microspheres were co-modified with metal organic framework (MOF-235) and polyethylene glycol polymer (PEG) and used as mixed-mode stationary phase (MOF-235@PEG@silica) for high-performance liquid chromatography. Elemental analysis, scanning electron microscopy, X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy, and Brunauer-Emmett-Teller etc. methods were used to investigate the properties of the core-shell composite material. The MOF-235@PEG@silica stationary phase showed flexible selectivity for the separation of both hydrophilic and hydrophobic compounds especially for the separation of nine alkaloids, which showed superior hydrophilic separation performance than previous MOF-based composite stationary phases. Some factors including the pH of buffer salt, the ratio of organic phase and water phase in the mobile phase have been investigated, suggesting that the chromatographic retention mechanism of the column was a mixed mode of hydrophilic and reversed phase. The composite material also showed excellent chromatographic repeatability with the RSDs of the retention time found to be 0.2%–0.6% (n = 10) and the standard addition test in the actual sample proved that it can be used for practical sample analysis. In short, it provided a general way for preparing MOFs-based composites as mixed-mode chromatographic stationary phases, and changed the current status of MOF-based composite materials as single mode chromatographic stationary phases.

Preparation and chromatographic properties of mixed-mode chromatographic stationary phases modified by imidazole side-group functionalized poly(ionic liquids)

A novel mixed-mode chromatographic stationary phase was prepared via surface initiated atom transfer radical polymerization using an imidazole side-group functionalized ionic liquid monomer, 1-(4-vinylbenzyl)-3-cyanomethyl bromide. The structure of the stationary phase was characterized by infrared spectroscopy, elemental analysis, and thermogravimetric analysis. The stationary phase showed good separation ability. The retention mechanism of reversed-phase/ion exchange toward solutes on the stationary phase was verified by determining the effect of mobile phase pH on the retention of substances. Compared with octadecylsilyl-bonded silica stationary phase, it is confirmed that the poly(ionic liquid)-modified stationary phase provides π-π interaction toward the retention of the solutes. The results show that the functionalization of imidazole side groups is a feasible method for preparing new ionic liquid stationary phases.

A new strategy for the preparation of mixed-mode chromatographic stationary phases based on modified dialdehyde cellulose

A new strategy for the preparation of mixed-mode chromatographic stationary phases based on modified dialdehyde cellulose was proposed. Two novel mixed-mode chromatographic stationary phases, dicarboxyl cellulose-modified silica (DCC/SiO2) and (S)-α-phenylethylamine-bonded DCC/SiO2 ((S)-α-PEA/DCC/SiO2), were prepared by utilizing the easy functionalization characteristics of dialdehyde cellulose. The chromatographic evaluation showed that DCC/SiO2 column could be used in hydrophilic interaction liquid chromatography (HILIC) and ion exchange chromatography (IEC) modes, (S)-α-PEA/DCC/SiO2 column could be used in HILIC, IEC and chiral separation modes. The DCC/SiO2 column and (S)-α-PEA/DCC/SiO2 column exhibited excellent chromatographic performance by separating strongly polar compounds, phenylamines and chiral compounds in the above separation modes. The preparation method of modified dialdehyde cellulose-based mixed-mode chromatographic stationary phases was simple, and also provided a new idea for the development of the subsequent novel mixed-mode chromatographic stationary phases.

Preparation and evaluation of a molybdenum disulfide quantum dots embedded C18 mixed-mode chromatographic stationary phase.

Molybdenum disulfide quantum dots (MoS2 QDs) were chosen as a functional two-dimensional material to improve the separation performance of a traditional C18 column. In this work, MoS2 QDs were synthesized by the combination of sonication and solvothermal treatment of bulk MoS2. The prepared MoS2 QDs were characterized by transmission electron microscope (TEM), Zeta potential measurement, UV-visible absorption and fluorescence spectroscopy. Then, a novel MoS2 QDs embedded C18 (Sil-MoS2-C18) stationary phase was prepared for performing mixed-mode liquid chromatography. The results of elemental analysis (EA), thermogravimetric analysis (TGA), Fourier transform infrared spectrometry (FT-IR), X-ray photoelectron spectroscopy (XPS), scanning electron microscope (SEM) and Brunauer-Emmett-Teller (BET) measurements indicated the stationary phase was prepared successfully. Five types of compounds including alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs), nucleosides and nucleobases, anilines and flavonoids were utilized to evaluate reversed phase, weak cation exchange and hydrophilic interaction of the new column. To a certain extent, the column could achieve separation for different properties of samples on one column, with less organic solvent and shorter time than conventional alkyl and amino columns. Furthermore, the mechanism for separation was studied by investigating effects of mobile phase composition and pH on retentions. In summary, the Sil-MoS2-C18 stationary phase was deemed able to serve the performance of various types of phases, which revealed the prepared mixed-mode column could be potentially applied for the analysis of complex samples. Graphical abstract.

Controlled Fabrication of Silica@Covalent Triazine Polymer Core-Shell Spheres as a Reversed-Phase/Hydrophilic Interaction Mixed-Mode Chromatographic Stationary Phase.

The unique properties of covalent triazine-based organic framework/polymers, including large surface area, hydrophilic-lipophilic-balanced adsorption, and economical preparation, make it a promising candidate as a stationary phase for high-performance liquid chromatography. However, irregular shapes and wide size distributions of such particles hinder column packing, resulting in a low column efficiency or a high back pressure. Herein, we describe the fabrication of SiO2@ covalent triazine-based organic polymer (CTP) core-shell microspheres with a distinct sphere-coating-sphere appearance using aminosilica as the supporting substrate to grow the CTP shell. By adjusting the amount of reactants, the thickness of the CTP shell, which consists of triazine and 1,3,5-triphenylbenzene monomers, was easily controlled. The developed core-shell microspheres were characterized via scanning electron microscopy, energy dispersive X-ray spectroscopy, transmission electron microscopy, solid-state 13C nuclear magnetic resonance analysis, and N2 adsorption experiments. The synergism of the triazine and aromatic moieties on CTP provides the new stationary phase with multiple retention mechanisms, including hydrophobic, π-π, electron donor-acceptor, hydrogen-bonding interactions, and so forth. On the basis of these interactions, successful separation and higher shape selectivity were achieved among several analytes that vary in polarity under both reversed-phase and hydrophilic interaction liquid chromatography conditions. Therefore, SiO2@CTP microspheres combine the advantages of good column packing properties of the uniform monodisperse silica microspheres and the recognition performance of CTP, generating flexible selectivity and application prospect for both hydrophilic and hydrophobic analytes.

Epoxide-derived mixed-mode chromatographic stationary phases for separation of active substances in fixed-dose combination drugs.

A method for the preparation of novel mixed-mode reversed-phase/strong cation exchange stationary phase for the separation of fixed-dose combination drugs has been developed. An epoxysilane bonded silica prepared by vapor phase deposition was used as a starting material to produce diol, octadecyl, sulfonate, and mixed octadecyl/sulfonate groups bonded silica phases. The chemical structure and surface coverage of the functional groups on these synthesized phases were confirmed by fourier-transform infrared and solid-state 13 C NMR spectroscopy and elemental analysis. Alkylbenzene homologs, basic drugs, nucleobases and alkylaniline homologs were used as probes to demonstrate the reversed-phase, ion exchange, hydrophilic interaction and mixed-mode retention behaviors of these stationary phases. The octadecyl/sulfonate bonded silica exhibits pronounced mixed-mode retention behavior and superior retentivity and selectivity for alkylaniline homologs. The mixed-mode retention is affected by either ionic or solvent strength in the mobile phase, permiting optimization of a separation by fine tuning these parameters. The mixed-mode stationary phase was applied to separate two fixed-dose combination drugs: compound reserpine tablets and compound methoxyphenamine capsules. The results show that simultaneous separation of multiple substances in the compound dosage can be achieved on the mixed-mode phase, which makes multi-cycles of analysis for multiple components obsolete.

Recent advances in mixed-mode chromatographic stationary phases.

Mixed-mode phases have become very popular in the last decade, and the number of new mixed/multi-mode sorbents is growing fast. Unlike single-mode stationary phases, perfectly suited for the separation of the analytes possessing similar physicochemical properties, for instance reversed-phase chromatography for hydrophobic solutes, mixed-mode sorbents providing multimodal interactions can render better separation selectivity for complex mixtures of solutes differing significantly in their physicochemical characteristics. The most frequent modern mixed-mode stationary phases are di/tri-mode sorbents embracing the following interactions, hydrophobic, electrostatic (coulombic), and hydrophilic. According to their structures, it is possible to distinguish silica-based, polymer-based, hybrid, and monolithic mixed-mode stationary phases. Herewith, newly synthesized mixed-mode sorbents developed within the last two and half years are categorized, discussed, and summarized. The main attention is devoted to the description of the synthetic routes and characterization methods applied for the new stationary phases.

Preparation and evaluation of a reversed-phase/hydrophilic interaction/ion-exchange mixed-mode chromatographic stationary phase functionalized with dopamine-based dendrimers

In this work, a novel dendritic stationary phase was synthesized by the repeated grafting of 1,4-butanediol diglycidyl ether (BDDE) and dopamine (DA) on the surface of silica for performing mixed-mode high-performance liquid chromatography (MHPLC). Elemental analysis (EA), thermogravimetric analysis (TGA) and Fourier transform infrared spectrometry (FT-IR) showed the successful preparation of the dendritic stationary phase. The prepared stationary phase showed the retention mechanisms of reversed-phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC) and ion-exchange chromatography (IEC) under different mobile phase conditions. In detail, alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs) and hydrophobic positional isomers were separated successfully in the RPLC mode. The baseline separation of nucleobases, nucleosides and flavonoids was achieved under HILIC mode, respectively. Meanwhile, some acidic and basic analytes were used to evaluate the IEC mode. The effects of different chromatographic conditions, such as acetonitrile content, salt concentration and pH in the mobile phase, on the different chromatographic modes were also investigated. In addition, the application of the mixed-mode dendritic stationary phase was demonstrated by the analysis of traditional Chinese medicine (TCM), including Carthamus tinctorius L. and Abelmoschus manihot (Linn.) Medicus. Interestingly, the stationary phase also has the ability for the capture and separation of boric acids. These meaningful applications confirmed that the mixed-mode dendritic stationary phase can be potentially applied in the analysis of complex samples.

Synthesis and Characterisation of a New Hydrophilic Interaction/Reversed Phase Mixed-Mode Chromatographic Stationary Phase

A novel multifunctional stationary phase based the amide-silica was synthesised starting from L-isoleucine and 2-aminoacetanilide. Hereby, compounds 1, 2, 3 and 4 were synthesised for the first time and 1, 2 and 4 were identified. The stationary phase was synthesised by a reliable and repeatable method and characterized by elemental analysis, solid state 13C NMR, scanning electron microscope (SEM), and Brunauer, Emmett and Teller (BET). This stationary phase possess four amide groups as polar cites, phenyl ring as aromatic non-polar cite and isopropyl as an aliphatic non-polar cite. Therefore, it can act as both hydrophilic interaction (HILIC) and reversed phase (RPLC) chromatographic stationary phase. The stationary phase has also chirality and can be used in enantioseparation of racemic compounds in normal phase chromatography (NPLC).

Novel reversed-phase/weak-cation-exchange mixed-mode chromatographic packing materials applied in mass-spectrometry-based proteomics

High efficiency separation plays a key role in deep-coverage proteomics studies. In this study, we investigate a mixed-mode chromatographic packing material and use “thiol-ene” click chemistry to prepare a novel reversed-phase/weak-cation-exchange mixed-mode chromatographic packing material. First, we used a mixture of chemical compounds with low molecular weights to characterize the packing material and the results showed that it has both reversed-phase and weak-cation-exchange chromatographic retention mechanisms. Next, we used the reversed-phase/weak-cationic-exchange mixed-mode chromatographic stationary phase in the separation of a mixture of six standard peptides with similar hydrophobicities but with different theoretical isoelectric points. The experimental results showed that the packing materials have a mixed-mode retention performance. We also found that the hydrophilic properties of the embedded functional groups play an important role in decreasing the hydrophobicity of the packing material and therefore reduces the loss of some of the peptides due to the strong adsorption of very high hydrophobic peptides. Lastly, we selected an off-line RP/WCX column with a pH gradient elution as the first dimension for pre-separation and employed reversed-phase liquid chromatography (RPLC)-mass spectrometry analysis as the second dimension for the analysis of the protein mixture of 100-µg HepG2 cells. In total, we identified 5924 proteins. These results are similar to those obtained by the RPLC-mass spectrometry analysis commonly used in proteome research, which suggests that this mixed-mode chromatographic packing material can be used in proteomics research.

Controllable preparation of a reverse-phase/hydrophilic interaction mixed-mode chromatographic stationary phase with adjustable selectivity

A mixed-mode stationary phase prepared by grafting block copolymers via ATRP could adjust separation selectivity by simply tuning the ratios of copolymer chains.

Preparation and evaluation of diblock copolymer-grafted silica by sequential surface initiated-atom transfer radical polymerization for reverse-phase/ion-exchange mixed-mode chromatography.

A novel approach that involved the grafting of diblock copolymer with two types of monomer onto substrate by sequential surface initiated-atom transfer radical polymerization was proposed to prepare a mixed-mode chromatographic stationary phase. The distinguishing feature of this method is that it can be applied in the preparation of various mixed-mode stationary phases. In this study, a new reverse-phase/ion-exchange stationary phase was prepared by grafting hydrophobic styrene and cationic sodium 4-styrenesulfonate by the proposed approach onto silica surface. The chromatographic properties of the prepared stationary phase were evaluated by the separation of benzene derivatives, anilines, and β-agonists, and by the effect of pH values and acetonitrile content on the retention. Compared with typical RP columns, the prepared stationary phase achieved the better resolution and higher selectivity at a shorter separation time and lower organic content. Moreover, the application of the prepared column was proved by separating widely distributed polar and charged compounds simultaneously.

Controllable preparation of a hydrophilic/ion-exchange mixed-mode stationary phase by surface-initiated atom transfer radical polymerization using a mixture of two functional monomers.

Mixed-mode chromatographic stationary phases require functionalization with at least two functional groups to yield multiple interactions with analytes. Departing from reported methods, a mixture of two different monomers, glycidyl methacrylate and 2-dimethylaminoethylmethacrylate, was grafted onto the surface of silica by a one-step surface-initiated atom transfer radical polymerization to prepare a novel hydrophilic interaction/anion-exchange mixed-mode chromatographic stationary phase. The grafted amounts of functional groups were controlled via varying the ratio of monomers in the polymerization system. The influences of water content, salt concentration and pH in the mobile phase were investigated to illustrate the mixed interaction between the stationary phase and analytes. The retention of various solutes on three columns, especially acidic and basic solutes, showed an obvious dependence on the ratio of the two monomers in the polymerization system. The results indicated that the strategy proposed in this work was beneficial to develop various types of mixed-mode chromatographic stationary phases with adjustable selectivity to meet the needs of complex samples. Finally, the column was successfully employed in the isolation of melamine in liquid milk.