Synthesis and evaluation of a maltose‐bonded silica gel stationary phase for hydrophilic interaction chromatography and its application in Ginkgo Biloba extract separation in two‐dimensional systems

Maltose covalently bonded to silica was prepared by using carbonyl diimidazole as a cross‐linker and employed as a stationary phase for hydrophilic interaction liquid chromatography. The column efficiency and the effect of water content, buffer concentration, and pH value influenced on retention were investigated. The separation or enrichment selectivity was also studied with nucleosides, saccharides, amino acids, peptides, and glycopeptides. The results indicated that the stationary phase processed good separation efficiency and separation selectivity in hydrophilic interaction liquid chromatography mode. Moreover, a two‐dimensional hydrophilic interaction liquid chromatography× reversed‐phase liquid chromatography method with high orthogonality was developed to analyze the Ginkgo Biloba extract fractions. The development of this two‐dimensional chromatographic system would be an effective tool for the separation of complex samples of different polarities and contents.     

Separation behavior of basic compounds on unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases with reversed‐phase eluents

Unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases have been evaluated and compared for the separation of basic compounds of differing molecular weight, pK_a, and log D using aqueous/organic mobile phases. The influences of percentage of organic modifier, buffer pH, and concentration in the mobile phase on base retention were investigated on unbonded silicon oxynitride and silica phases. The results confirmed that unbonded silicon oxynitride and silica phases demonstrated excellent separation performance for model basic compounds and both the unbonded phases examined possessed a hydrophobic/adsorption and ion‐exchange character. The silicon oxynitride stationary phase exhibited high hydrophilicity compared with silica with a reversed‐phase mobile phase. An ion‐exclusion‐type mechanism becomes predominant for the separation of three aimed bases on the silicon oxynitride column at pH 2.8. Different from silicon oxynitride stationary phase, no obvious change for the retention time of three model bases on silica stationary phase at pH 2.8 can be observed.     

Tetra‐proline‐modified calix[4]arene‐bonded silica stationary phase for simultaneous reversed‐phase/hydrophilic interaction mixed‐mode chromatography

A new water‐soluble tetra‐proline‐modified calix[4]arene‐bonded silica stationary phase was prepared straightforwardly by an indirect method and characterized by elemental analysis, energy dispersive Spectrometry, solid‐state ¹³C NMR spectroscopy, Fourier‐transform infrared spectroscopy, and thermogravimetric analysis. Due to the simultaneous introduction of polar tetra‐proline and nonpolar calix[4]arene, the developed column possessing a double retention mode of reverse‐phase liquid chromatography and hydrophilic interaction liquid chromatography. A series of hydrophobic and hydrophilic test samples, including nucleosides and nucleotides, amines, monosubstituted benzenes, chiral compounds, and phenols, were used to evaluate the developed stationary phase. A rapid separation capability, high separation efficiency, and selectivity were achieved based on the multiple interactions between solutes and tetra‐proline‐modified calix[4]arene‐bonded silica stationary phase. Moreover, the developed stationary phase was further used to detect and separate hexamethylenetetramine in rice flour. All the results indicated the potential merits of the developed stationary phase for simultaneous separation of complex hydrophobic and hydrophilic samples with high selectivity.     

三元醇丙烯酰胺键合色谱固定相的制备及评价

利用巯基与乙烯基的"点击化学"反应合成了一种新型含多羟基的硅烷偶联剂,再将其与硅胶反应制得含多羟基的亲水固定相。经过元素分析表征证明多羟基官能团已成功键合到硅胶表面。采用一系列不同性质的标准物质考察了亲水色谱模式下固定相的溶质保留机理。由于固定相结构中既具有极性多羟基官能团,也有短的疏水碳链,因此固定相兼具疏水性与亲水性。将此固定相成功应用于亲水与反相色谱两种模式,并对比了两种模式下流速对柱效的影响。最后将固定相应用于烷基苯、水溶性维生素以及核苷的分离中,取得了较好的分离效果,证明了固定相良好的应用前景。     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.     

Effect of water on the retention on diol and amide columns in hydrophilic interaction liquid chromatography

The amount of water adsorbed on polar columns plays important role in hydrophilic interaction liquid chromatography. It may strongly differ for the individual types of polar columns used in this separation mode. We measured adsorption isotherms of water on an amide and three diol‐bonded stationary phases that differ in the chemistry of the bonded ligands and properties of the silica gel support. We studied the effects of the adsorbed water on the retention of aromatic carboxylic acids, flavonoids, benzoic acid derivatives, nucleic bases, and nucleosides in aqueous‐acetonitrile mobile phases over the full composition range. The graphs of the retention factors versus the volume fraction of water in mobile phase show “U‐profile” characteristic of a dual hydrophilic interaction–reversed phase retention mechanism. The minimum on the graph that marks the changing retention mechanism depends on the amount of adsorbed water. The linear solvation energy relationship model suggests that the retention in the hydrophilic interaction liquid chromatography mode is controlled mainly by proton–donor interactions in the stationary phase, depending on the column type. Finally, the accuracy of hydrophilic interaction liquid chromatography gradient prediction improves for columns that show a high water adsorption.     

一种聚合型弱阳离子交换/亲水相互作用色谱固定相的制备及色谱性能

通过亲核取代反应将聚乙烯马来酸酐键合到氨基硅胶表面,然后将残余的马来酸酐水解,制备了一种弱阳离子交换/亲水相互作用高效液相色谱固定相(Sil-PolyCOOH),通过固体核磁、ζ-电势及元素分析对固定相进行了表征。选取核苷和核酸碱为模型化合物,通过考察流动相组成,离子强度和pH等因素对溶质保留的影响,探讨了固定相的分离性能和保留机理,结果表明,该固定相的保留机理同时涉及亲水分配相互作用和多重主客体作用力。该固定相还对糖类、敌草快与百草枯等化合物具有良好的分离性能。上述研究结果表明该固定相在极性化合物的分离上具有良好的应用前景。     

Poly(l‐lactic acid)‐modified silica stationary phase for reversed‐phase and hydrophilic interaction liquid chromatography

Poly(l ‐lactic acid) is a linear aliphatic thermoplastic polyester that can be produced from renewable resources. A poly(l ‐lactic acid)‐modified silica stationary phase was newly prepared by amide bond reaction between amino groups on aminopropyl silica and carboxylic acid groups at the end of the poly(l ‐lactic acid) chain. The poly(l ‐lactic acid)‐silica column was characterized in reversed‐phase liquid chromatography and hydrophilic interaction liquid chromatography with the use of different mobile phase compositions. The poly(l ‐lactic acid)‐silica column was found to work in both modes, and the retention of test compounds depending on acetonitrile content exhibited “U‐shaped” curves, which was an indicator of reversed‐phase liquid chromatography/hydrophilic interaction liquid chromatography mixed‐mode retention behavior. In addition, carbonyl groups included into the poly(l ‐lactic acid) backbone work as an electron‐accepting group toward a polycyclic aromatic hydrocarbon and provide π–π interactions.     

Preparation of a novel carboxyl stationary phase by “thiol‐ene” click chemistry for hydrophilic interaction chromatography

A novel carboxyl‐bonded silica stationary phase was prepared by “thiol‐ene” click chemistry. The resultant Thiol‐Click‐COOH phase was evaluated under hydrophilic interaction liquid chromatography (HILIC) mobile phase conditions. A comparison of the chromatographic performance of Thiol‐Click‐COOH and pure silica columns was performed according to the retention behaviors of analytes and the charged state of the stationary phases. The results indicated that the newly developed Thiol‐Click‐COOH column has a higher surface charge and stronger hydrophilicity than the pure silica column. Furthermore, the chromatographic behaviors of five nucleosides on the Thiol‐Click‐COOH phase were investigated in detail. Finally, a good separation of 13 nucleosides and bases, and four water‐soluble vitamins was achieved.     

A Sulfonic‐Azobenzene‐Grafted Silica Amphiphilic Material: A Versatile Stationary Phase for Mixed‐Mode Chromatography

A novel sulfonic‐azobenzene‐functionalized amphiphilic silica material was synthesized through the preparation of a new sulfonic azobenzene monomer and its grafting on mercaptopropyl‐modified silica by a surface‐initiated radical chain‐transfer reaction. The synthesis was confirmed by infrared spectra, elemental analysis, and thermogravimetric analysis. This new material was successfully applied as a new kind of mixed‐mode stationary phase in liquid chromatography. This allows an exceptionally flexible adjustment of retention and selectivity by tuning the experimental conditions. The distinct separation mechanisms were outlined by selected examples of chromatographic separations in the different modes. In reversed‐phase liquid chromatography, this new stationary phase presented specific chromatographic performance when evaluated using a Tanaka test mixture. Seven dinitro aromatic isomers, four steroids, and seven flavonoids were separated successfully in simple reversed‐phase mode. This stationary phase can also be used in hydrophilic interaction chromatography because of the existing polar functional groups; for this, nucleosides and their bases were used as a test mixture. Interestingly, the same nucleosides and bases can also be separated in per aqueous liquid chromatography using the same stationary phase. Three ginsenosides including Rg1, Re, and Rb1 were successfully separated in hydrophilic mode. There is the potential for more applications to benefit from this useful column.     

Silica modified with a thiourea derivative as a new stationary phase for hydrophilic interaction liquid chromatography

Nowadays there are limited types of commercially available stationary phases for hydrophilic interaction liquid chromatography and therefore new ones with unique selectivity are urgently in demand to meet the need of separations of various polar and hydrophilic analytes. The present study describes the preparation and evaluation of a new stationary phase based on thiourea derivative modified silica for hydrophilic interaction liquid chromatography. Thiourea derivative was bonded onto the surface of silica particles via a mild addition reaction between –NH₂ and –SCN, and the result of elemental analysis together with infrared analysis and solid‐state NMR spectroscopy proved that the synthesis method was feasible. The new stationary phase succeeded in fast separations of a wide range of polar and hydrophilic analytes and exhibited excellent separation performance, especially unique selectivity. Furthermore, the effects of water content, buffer pH, and salt concentration on retention indicated that a complicated separation mechanism rather than partitioning was involved in the stationary phase and hydrogen bonding interaction between analytes and thiourea functional group could play a very important role in its selectivity. For sure, the new stationary phase is of a great potential as a new type of hydrophilic interaction liquid chromatographic stationary phase.     

纳米氧化锆沉积硅胶色谱固定相的制备及亲水作用色谱行为

采用液相沉积法(LPD)制备了纳米氧化锆沉积硅胶色谱固定相(ZrO₂/SiO₂), 并将其应用于亲水作用色谱分离中. 考察并比较了ZrO₂/SiO₂、 硅胶(SiO₂)和氧化锆(ZrO₂) 3种色谱固定相在不同有机调节剂比例、 不同pH值及不同盐浓度的流动相条件下的色谱行为. 结果表明, 制备的ZrO₂/SiO₂色谱柱不仅具有SiO₂色谱柱高柱效的优点, 表面沉积的纳米氧化锆还能有效屏蔽硅羟基, 有利于碱性物质的保留和分离, 表现出良好的亲水作用色谱性能. 将ZrO₂/SiO₂色谱柱用于4种脱氧核苷和5种碱性化合物的分离, 均得到了较好的效果, 展现出其作为色谱固定相良好的应用前景.     

硅胶色谱柱的亲水作用保留机理及其影响因素

亲水作用色谱（HILIC）是替代反相色谱（RPLC）分离强极性及亲水性化合物的另一色谱模式,其分离机理与RPLC有很大不同,具有和RPLC互补的选择性。在HILIC模式中,采用正相色谱（NPLC）中的极性固定相及含高浓度有机溶剂（通常为乙腈）的水溶液为流动相。硅胶是开发最早、研究最为深入及应用最为广泛的HILIC固定相,本文介绍了硅胶色谱柱的HILIC保留机理,详细概述了操作条件如硅胶柱类型、流动相组成及柱温对HILIC分离的影响,并对硅胶填料色谱柱的HILIC模式的发展方向与应用前景进行了展望。     

Novel reversed-phase high-performance liquid chromatography stationary phase with oligo(ethylene glycol) "click" to silica

Oligo(ethylene glycol) (OEG) covalently bonded silica was prepared by using click chemistry and employed as a stationary phase for reversed-phase high-performance liquid chromatography. The column efficiency and effect of organic modifier content on retention were investigated. The separation selectivity was also studied with phenyl compounds and an actual sample of natural products. The results indicated that the stationary phase possessed good separation efficiency and separation selectivity in RP-HPLC mode. Moreover, the stationary phase showed good complementary separation selectivity to the C18 stationary phase. The OEG stationary phase had "clustering" function for "homologous component" in the separation of natural products.     

Hydrophilic interaction liquid chromatography for the separation of acidic agricultural compounds

Organic acids with very low pK_a require extremely low pH conditions to achieve adequate retention in reversed‐phase liquid chromatography, but an extremely low pH mobile phase can cause instrument reliability problems and limit the choice of columns. Hydrophilic interaction chromatography is a potential alternative to reversed‐phase liquid chromatography for the separation of organic acids using more moderate conditions. However, the hydrophilic interaction chromatography separation mechanism is known to be very complex and involves multiple competing mechanisms. In the present study, a hydrophilic interaction chromatography column packed with bare silica core–shell particles was used as the separation column and six agricultural organic acids were used as model analytes to evaluate the effects of buffer concentration, buffer pH, and temperature on sample loading capacity, selectivity, retention, and repeatability. It was found that using a higher concentration of buffer can lead to a significant improvement in the overall performance and reproducibility of the separation. Investigation of column equilibration time revealed that a very long equilibration time is needed when changing mobile phase conditions in between runs. This limitation needs to be acknowledged in hydrophilic interaction chromatography method development and sufficient equilibration time needs to be allowed in method scouting.     

Imidazolium‐embedded iodoacetamide‐functionalized silica‐based stationary phase for hydrophilic interaction/reversed‐phase mixed‐mode chromatography

A novel imidazolium‐embedded iodoacetamide‐functionalized silica‐based stationary phase has been prepared by surface radical chain‐transfer polymerization. The stationary phase was characterized by Fourier transform infrared spectrometry, thermogravimetric analysis, and element analysis. Fast and efficient separations of polar analytes, such as nucleosides and nucleic acid bases, water‐soluble vitamins and saponins, were well achieved in hydrophilic interaction chromatography mode. Additionally, a mixed mode of hydrophilic interaction and reversed‐phase could be also obtained in the analysis of polar and nonpolar compounds, including weak acidic phenols, basic anilines and positional isomers, with high resolution and molecular‐planarity selectivity, outperforming the commercially available amino column. Moreover, simultaneous separation of polar and nonpolar compounds was also achieved. In conclusion, the multimodal retention capabilities of the imidazolium‐embedded iodoacetamide‐functionalized silica‐based column could offer a wide range of retention behavior and flexible selectivity toward hydrophilic and hydrophobic compounds.     

Hydrophilic interaction chromatography: A promising alternative to reversed‐phase liquid chromatography systems for the purification of small protonated bases

The overloaded band profiles of the protonated species of propranolol and amitriptyline were recorded under acidic conditions on four classes of stationary phases including a conventional silica/organic hybrid material in reversed‐phase liquid chromatography mode (BEH‐C₁₈), an electrostatic repulsion reversed‐phase liquid chromatography C₁₈ column (BEH‐C₁₈+), a poly(styrene‐divinylbenzene) monolithic column, and a hydrophilic interaction chromatography stationary phase (underivatized BEH). The same amounts of protonated bases per unit volume of stationary phase were injected in each column (16, 47, and 141 μg/cm³). The performance of the propranolol/amitriptyline purification was assessed on the basis of the asymmetry of the recorded band profiles and on the selectivity factor achieved. The results show that the separation performed under reversed‐phase liquid chromatography like conditions (with BEH‐C₁₈, BEH‐C₁₈+, and polymer monolith materials) provide the largest selectivity factors due to the difference in the hydrophobic character of the two compounds. However, they also provide the most distorted overloaded band profiles due to a too small loading capacity. Remarkably, symmetric band profiles were observed with the hydrophilic interaction chromatography column. The larger loading capacity of the hydrophilic interaction chromatography column is due to the accumulation of the protonated bases into the diffuse water layer formed at the surface of the polar adsorbent. This work encourages purifying ionizable compounds on hydrophilic interaction chromatography columns rather than on reversed‐phase liquid chromatography columns.     

Evaluation of the dual retention properties of stationary phases based on silica hydride: Perfluorinated bonded material

The synthesis of a new perfluorinated stationary phase based on silica hydride using a hydrosilation reaction was investigated. The material was characterized by elemental analysis, diffuse reflectance infrared Fourier transform spectroscopy and ¹³C cross‐polarization magic‐angle spinning NMR spectroscopy. The retention properties of this new material were tested in the reversed‐phase and normal‐phase mode. Variable buffer strength experiments at two pH conditions for selected polar compounds were used to compare the new phase to hydrophilic interaction liquid chromatography retention. These results and previous data reported in the literature were used to postulate differences in the retention mechanism between hydrophilic interaction liquid chromatography and silica hydride‐based stationary phases.     

木糖醇和麦芽糖醇型亲水作用色谱固定相的制备及其分离性能的评价

利用-NCO和-OH的加成反应,通过简单的两步反应将木糖醇和麦芽糖醇成功地键合于硅胶表面,制备了两种新型糖醇类亲水作用色谱固定相。流动相中乙腈含量对保留的影响曲线表明,这两种糖醇固定相具有典型的亲水作用色谱固定相性质,对极性和亲水性化合物有很强的保留作用。利用这两种固定相成功分离了水溶性维生素、水杨酸及其类似物、碱基及其相应的核苷和淫羊藿苷类似物等模型混合物,同时糖醇固定相展现了新颖的选择性,特别是相对于线形的木糖醇键合固定相,非线形的麦芽糖醇键合固定相表现出了对糖基的独特保留能力。此外,缓冲盐的pH和浓度对保留的影响表明静电作用在这两种糖醇固定相的保留机理中也发挥着一定的作用。本文所发展的糖醇类固定相具有良好的分离性能,有望在亲水作用色谱分离领域发挥潜在的应用价值。