Preparation of a mixed-mode hydrophilic interaction/anion-exchange polymeric monolithic stationary phase for capillary liquid chromatography of polar analytes

A novel cationic hydrophilic interaction monolithic stationary phase based on the copolymerization of 2-(methacryloyloxy)ethyltrimethylammonium methyl sulfate (META) and pentaerythritol triacrylate (PETA) in a binary porogenic solvent consisting of cyclohexanol/ethylene glycol was designed for performing capillary liquid chromatography. While META functioned as both the ion-exchange sites and polar ligand provider, the PETA, a trivinyl monomer, was introduced as cross-linker. The monolithic stationary phases with different properties were easily prepared by adjusting the amount of META in the polymerization solution as well as the composition of the porogenic solvent. The hydrophilicity of the monolith increased with increasing content of META in the polymerization mixture. A typical hydrophilic interaction chromatography mechanism was observed when the content of acetonitrile in the mobile phase was higher than 20%. The poly(META-co-PETA) monolith showed very good selectivity for neutral, basic and acidic polar analytes. For polar-charged analytes, both hydrophilic interaction and electrostatic interaction contributed to their retention. Peak tailing of basic compounds was avoided and the efficient separation of benzoic acid derivatives was obtained.     

Novel highly hydrophilic methacrylate-based monolithic column with mixed-mode of hydrophilic and strong cation-exchange interactions for pressurized capillary electrochromatography

A novel highly hydrophilic polymethacrylate-based monolithic stationary phase based on the copolymerization of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and pentaerythritol triacrylate (PETA) was designed for pressurized capillary electrochromatography. A typical hydrophilic interaction chromatography mechanism could be observed when the content of acetonitrile (ACN) in the mobile phase exceeded 25%. Slight swelling or shrinking with mobile phases of different polarity was observed in permeability studies. Good retentions and efficient separations of polar analytes, such as neutral amides and phenols, were well achieved in hydrophilic interaction chromatography mode with only about 50% ACN content in the mobile phase. It was remarkably lower than the content of ACN (>90%) used on the hydrophilic polymethacrylate-based monoliths reported previously. Additionally, a mixed mode of hydrophilic interaction (HI) and strong cation-exchange (SCX) could be also obtained in the analysis of charged peptides, and high column efficiency up to 80,000 plates/m was achieved without peak tailing. The prepared hydrophilic stationary phase might provide a potential environmental friendly separation media for polar solutes as it consumes a low volume of organic solvents.     

Preparation of monolithic silica column with strong cation-exchange stationary phase for capillary electrochromatography

A monolithic silica based strong cation-exchange stationary phase was successfully prepared for capillary electrochromatography. The monolithic silica matrix from a sol-gel process was chemically modified by treatment with 3-mercaptopropyltrimethoxysilane followed by a chemical oxidation procedure to produce the desired function. The strong cation-exchange stationary phase was characterized by its substantial and stable electroosmotic flow (EOF), and it was observed that the EOF value of the prepared column remained almost unchanged at different buffer pH values and slowly decreased with increasing phosphate concentration in the mobile phase. The monolithic silica column with strong cation-exchange stationary phase has been successfully employed in the electrochromatographic separation of beta-blockers and alkaloids extracted from traditional Chinese medicines (TCMs). The column efficiencies for the tested beta-blockers varied from 210,000 to 340,000 plates/m. A peak compression effect was observed for atenolol with the mobile phase having a low phosphate concentration.     

A reversed phase/hydrophilic interaction/ion exchange mixed-mode stationary phase for liquid chromatography

A new stationary phase demonstrated effective separation towards polar analytes or their counterions within a single run.     

Monolithic column with double mixed-modes of hydrophilic interaction/cation-exchange and reverse-phase/cation-exchange stationary phase for pressurized capillary electrochromatography

A monolithic capillary column with double mixed-modes of hydrophilic interaction/cation-exchange and RP/cation-exchange stationary phase was prepared by in situ thermal polymerization and then hydrolyzed with hydrochloric acid. The polymerization solution containing glycidyl methacrylate (GMA), 3-sulfopropyl methacrylate potassium salt (SPMA), and ethylene dimethacrylate (EDMA) in a binary porogenic solvent consisting of dimethylformamide (DMF) and 1,4-butanediol was polymerized in a fused-silica capillary pretreated with 3-(trimetoxysilyl) propyl methacrylate. The epoxy groups on the surface were hydrolyzed to diol groups with hydrochloric acid to enhance the polarity of the stationary phase. By simply altering the ACN content in the mobile phase, two mixed-mode mechanisms could be achieved on the same monolithic column in different mobile phase condition. Hydrophilic interaction (or hydrophilic interaction/cation-exchange) was observed at high ACN content, as well as RP (or RP/cation-exchange) was observed at low ACN content. The monolithic column provided good selectivity and high efficiency for separation of neutral polar analytes and basic compounds. Phenols, anilines, alkaloids, nucleic acid bases, and narcotic pharmaceuticals have been successfully separated. Effects of salt concentration and ACN content on the separation have also been investigated. High column efficiencies of up to 352 000 plates/meter were obtained by the separation of narcotic pharmaceuticals.     

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.     

Capillary liquid chromatography using a hydrophilic/cation-exchange monolithic column with a dynamically modified cationic surfactant

A novel form of reversed-phase liquid chromatography (RPLC) by the dynamically modified hydrophilic interaction monolithic column has been described in this paper. A porous poly(SPMA-co-PETA) monolith with strong cation-exchange (SCX) was prepared and the resulting monolith showed a typical hydrophilic interaction chromatography (HILIC) mechanism at higher organic solvent content (ACN% > 50%). The good selectivity for neutral, basic and acidic polar analytes was observed in the HILIC mode. In order to increase the hydrophobic interaction, the monolith with SCX was dynamically modified with a long-chain quaternary ammonium salt, cetyltrimethylammonium bromide (CTAB), which was added to the mobile phase. CTAB ions were adsorbed onto the surface of the SCX monolithic material, and the resulting hydrophobic layer was used as the stationary phase. Using the dynamically modified SCX monolithic column, neutral, basic and acidic hydrophobic analytes were well separated with the RPLC mode.     

Physically adsorbed chiral stationary phase of avidin on monolithic silica column for capillary electrochromatography and capillary liquid chromatography

The physical adsorption method proposed previously has been successfully applied to a monolithic silica column. By virtue of the physical adsorption, a chiral stationary phase of avidin was prepared onto the silica monolith. The phase ratio of resulting stationary phase was evaluated with frontal analysis. The method proved to be comparable in phase ratio to the chemical bonding methods used in high-performance liquid chromatography (HPLC). Enantiomer separations were carried out in capillary electrochromatography (CEC) and capillary liquid chromatography (CLC) modes. Due to its larger phase ratio, the resulting column showed more powerful separation capability as compared to open-tubular CEC (OTCEC). Twelve chiral compounds were baseline-resolved. The resulting column showed high separation efficiency, with average theoretical plate numbers of 66 000/m for CLC and 122 000/m for CEC. Good reproducibility was observed, with RSD value less than 1.3% for retention time, retention factor and separation factor, and less than 6.6% for plate counts and resolution (n = 40). Fast separations were achieved with a short column. The test enantiomers were baseline-resolved within 4 min under CLC and CEC modes. In addition, field-enhanced sample injection (FESI) was coupled to CLC as well as CEC to improve the detection sensitivity.     

Preparation and evaluation of a neutral methacrylate-based monolithic column for hydrophilic interaction stationary phase by pressurized capillary electrochromatography

A polar and neutral polymethacrylate-based monolithic column was evaluated as a hydrophilic interaction capillary electrochromatography (HI-CEC) stationary phase with small polar–neutral or charged solutes. The polar sites on the surface of the monolithic solid phase responsible for hydrophilic interactions were provided from the hydroxy and ester groups on the surface of the monolithic stationary phase. These polar functionalities also attract ions from the mobile phase and impart the monolithic solid phase with a given zeta potential to generate electro-osmotic flow (EOF). The monolith was prepared by in situ copolymerization of a neutral monomer 2-hydroxyethyl methacrylate (HEMA) and a polar cross-linker with hydroxy group, pentaerythritol triacrylate (PETA), in the presence of a binary porogenic solvent consisting cyclohexanol and dodecanol. A typical HI-CEC mechanism was observed on the neutral polar stationary phase for both neutral and charged analytes. The composition of the polymerization mixture was systematically altered and optimized by altering the amount of HEMA in the polymerization solution as well as the composition of the porogenic solvent. The monoliths were tested in the pCEC mode. The resulting monoliths had different characteristics of hydrophilicity, column permeability, and efficiency. The effects of pH, salt concentration, and organic solvent content on the EOF velocity and the separation of nucleic acids and nucleosides on the optimized monolithic column were investigated. The optimized monolithic column resulted in good separation and with greater than 140,000 theoretical plates/m for pCEC.     

Imidazoline type stationary phase for hydrophilic interaction chromatography and reversed-phase liquid chromatography

An imidazoline was prepared by solvent-free microwave-assisted organic synthesis and immobilized on porous silica particles by polymerization. The resulting material was composed of both hydrophobic alkyl ester chains and hydrophilic imidazoline rings, which gave it both hydrophilic interaction and reversed-phase characteristics. The titration curve suggests that the new material has buffering capacity and acquires increasing positive charge over the pH range 9-4, and is "zwitterionic" in the upper part of this pH range. Through investigating the effect of column temperature, the water content, pH and ion strength of mobile phase on the retention time of polar compounds in highly organic eluents, it was found that the new material could be used as a hydrophilic interaction liquid chromatography (HILIC) stationary phase which involved a complex retention process consisting of partitioning, surface adsorption and electrostatic interactions. In addition, the retention behavior of aromatic compounds in different mobile phase conditions was also studied, which showed the new material mainly exhibited a partitioning mechanism in the reversed-phase liquid chromatography (RPLC) mode. The separation of six water-soluble vitamins and five aromatic compounds were achieved by using the new material in the HILIC and RPLC modes, respectively.     

Sulfoalkylbetaine‐based monolithic column with mixed‐mode of hydrophilic interaction and strong anion‐exchange stationary phase for capillary electrochromatography

A novel monolithic stationary phase with mixed mode of hydrophilic and strong anion exchange (SAX) interactions based on in situ copolymerization of pentaerythritol triacrylate (PETA), N,N‐dimethyl‐N‐methacryloxyethyl N‐(3‐sulfopropyl) ammonium betaine (DMMSA) and a selected quaternary amine acrylic monomer was designed as a multifunctional separation column for CEC. Although the zwitterionic functionalities of DMMSA and hydroxy groups of PETA on the surface of the monolithic stationary phase functioned as the hydrophilic interaction (HI) sites, the quaternary amine acrylic monomer was introduced to control the magnitude of the EOF and provide the SAX sites at the same time. Three different quaternary amine acrylic monomers were tested to achieve maximum EOF velocity and highest plate count. The fabrication of the zwitterionic monolith (designated as HI and SAX stationary phase) was carried out when [2‐(acryloyloxy)ethyl]trimethylammonium methylsulfate was used as the quaternary amine acrylic monomer. The separation mechanism of the monolithic column was discussed in detail. For charged analytes, a mixed mode of HI and SAX was observed by studying the influence of mobile phase pH and salt concentration on their retentions on the poly(PETA‐co‐DMMSA‐co‐[2‐(acryloyloxy)ethyl]trimethylammonium methylsulfate) monolithic column. The optimized monolith showed good separation performance for a range of polar analytes including nucleotides, nucleic acid bases and nucleosides, phenols, estrogens and small peptides. The column efficiencies greater than 192 000 theoretical plates/m for estriol and 135 000 theoretical plates/m for charged cytidine were obtained.     

Preparation of chitosan functionalized monolithic silica column for hydrophilic interaction liquid chromatography

A novel cationic hydrophilic interaction monolithic stationary phase based on the chemical modification of carboxymethyl chitosan (CMCH) to the monolithic silica skeleton using carbodiimide as an activation reagent was prepared for performing capillary liquid chromatography. The amino and hydroxy moieties of CMCH functioned as both the ion-exchange sites and polar providers. The performance of the column was studied by the separation of polar acidic compounds. The chitosan functionalized monolithic silica column showed good selectivity for nucleosides, nucleotides, aromatic acids and aliphatic acids. The mechanism for the separation of these compounds was also studied. The results showed that these compounds were separated primarily based on the hydrophilic interaction mechanism.     

Preparation of pH-responsive stationary phase for reversed-phase liquid chromatography and hydrophilic interaction chromatography

Novel pH-responsive polymer-grafted silica was successfully synthesized through the radical "grafting from" polymerization on azo initiator-immobilized silica. The immobilization of azo initiator onto the silica surface was achieved by the reaction of surface amino groups with 4,4'-azobis(4-cyanovaleric acid chloride). The polymer-grafted silica was prepared by stirring suspension of the azo initiator-immobilized silica in anhydrous dioxane containing acrylic acid (AAc) and butyl acrylate (BA). The resulting polymer-grafted silica was demonstrated to be pH responsive to hydrophobic/hydrophilic property by reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC). In RPLC mode, the retention of aromatic compounds decreased with the increase in the pH of mobile phase. However, the opposite result was obtained in HILIC mode; the retention of soybean isoflavones was stronger with the mobile phase at higher pH. Finally, the separations of sulfonamides and soybean isoflavones were carried out in RPLC mode and the separation of some nucleotides was achieved in HILIC mode.     

杂化硅胶整体材料研磨法制备混合型高效液相色谱固定相

以聚乙二醇（PEG）为致孔剂，四甲氧基硅烷（TMOS）和乙烯基三甲氧基硅烷（VTMS）为杂化硅胶前驱体，在乙酸催化作用下使硅烷发生水解，在尿素加热分解提供的碱性环境下水解的硅烷进一步缩聚得到杂化硅胶整体材料。将此整体材料用球磨机研磨，然后用三羟甲基氨基甲烷处理，并洗涤干燥得到粒径为3 μm左右的硅胶颗粒。探索了不同反应条件对硅胶颗粒的大小、比表面积和孔径、表面形貌和分散性的影响；当TMOS和VTMS体积比为3：1时可以得到孔径为7.5 nm和比表面积为245 m²/g的硅胶颗粒。通过对所制得的硅胶颗粒表面进行C18（十八烷基二甲基氯硅烷）键合修饰和巯基-烯点击反应，得到混合型高效液相色谱固定相。对此固定相的测试结果表明以上硅胶色谱填料的制备方法具有一定的实用性。     

Novel zwitterionic polyphosphorylcholine monolithic column for hydrophilic interaction chromatography

A novel porous zwitterionic monolith was prepared by thermal co-polymerisation of 2-methacryloyloxyethyl phosphorylcholine (MPC) and ethylene glycol dimethacrylate (EDMA) within 100 μm I.D. capillaries. Mercury intrusion porosimetry, scanning electron microscopy (SEM), micro-HPLC (μ-HPLC), elemental analysis and ζ-potential analysis were used to evaluate the monolithic structure. No evidence of swelling or shrinking of the monolith in different polarity solvents was observed. A typical hydrophilic liquid chromatography (HILIC) mechanism was observed at high organic solvent content (acetonitrile >60%). The phosphorylcholine (PC) functionality has both a positively charged quaternary ammonium and a negatively charged phosphate group. For charged analytes, a weak electrostatic interaction was also observed by studying the influence of mobile phase pH and salt concentration on their retentions on the poly(MPC-co-EDMA) monolithic column. The optimised poly(MPC-co-EDMA) monolith showed very good selectivities for a range of polar test analytes, especially small peptides. This might be ascribed to the good biocompatibility of PC functionality. At low organic solvent content, baseline separation was also observed for a test mixture of seven alkylphenones by a reversed-phase separation mechanism.     

Polymethacrylate monolithic and hybrid particle-monolithic columns for reversed-phase and hydrophilic interaction capillary liquid chromatography

We prepared hybrid particle-monolithic polymethacrylate columns for micro-HPLC by in situ polymerization in fused silica capillaries pre-packed with 3–5 μm C₁₈ and aminopropyl silica bonded particles, using polymerization mixtures based on laurylmethacrylate–ethylene dimethacrylate (co)polymers for the reversed-phase (RP) mode and [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl) zwitterionic (co)polymers for the hydrophilic interaction (HILIC) mode. The hybrid particle-monolithic columns showed reduced porosity and hold-up volumes, approximately 2–2.5 times lower in comparison to the pure monolithic columns prepared in the whole volume of empty capillaries. The elution volumes of sample compounds are also generally lower in comparison to packed or pure monolithic columns. The efficiency and permeability of the hybrid columns are intermediate in between the properties of the reference pure monolithic and particle-packed columns. The chemistries of the embedded solid particles and of the interparticle monolithic moiety in the hybrid capillary columns contribute to the retention to various degrees, affecting the selectivity of separation. Some hybrid columns provided improved separations of proteins in comparison to the reference particle-packed columns in the reversed-phase mode. Zwitterionic hybrid particle-monolithic columns show dual mode retention HILIC/RP behaviour depending on the composition of the mobile phase and allow separations of polar compounds such as phenolic acids in the HILIC mode at lower concentrations of acetonitrile and, often in shorter analysis time in comparison to particle-packed and full-volume monolithic columns.     

Covalently bonded polysaccharide-modified stationary phase for per aqueous liquid chromatography and hydrophilic interaction chromatography

The mixed sulfated/methacryloyl polysaccharide derivative was prepared and successfully immobilized onto the surface of porous silica particles by polymerization. Polysaccharide derivative was calculated as 10.33% in the stationary phase prepared. The new stationary phase (PMSP) showed both hydrophilic interaction (HILIC) and per aqueous liquid chromatography (PALC) characteristics. The effects of column temperature, the water content, pH and ion strength of mobile phase on the retention time of test compounds in highly aqueous eluents were investigated to evaluate the PALC features of PMSP. The column efficiency is about 31,000 plates/m for benzoic acid in water/ACN (97/3, v/v) mobile phase at a flow rate of 1.0 mL/min. Compared with C18 column, the PMSP had shorter retention time for weak polar and non-polar compounds, but also showed stronger retention for strong polar compounds. It indicated that PALC was a suitable mode of chromatography as replacement of HILIC and complementarity of reversed-phase liquid chromatography (RPLC).     

Silica based click amino stationary phase for ion chromatography and hydrophilic interaction liquid chromatography

A silica based amino stationary phase was prepared by immobilization of propargylamine on azide-silica via click chemistry. This readily prepared click amino stationary phase demonstrated good selectivity in separation of common inorganic anions under ion chromatography (IC) mode, and the triazole ring in combination with free amino group was observed to play a major role for separation of the anions examined. On the other hand, the stationary phase also showed good hydrophilic interaction liquid chromatography (HILIC) properties in the separation of polar compounds including nucleosides, organic acids and bases. The retention mechanism was found to match well the typical HILIC retention.