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.     

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.     

Evaluation of a silicon oxynitride hydrophilic interaction liquid chromatography column in saccharide and glycoside separations

The retention characteristics of a silicon oxynitride stationary phase for carbohydrate separation were studied in hydrophilic interaction chromatography mode. Four saccharides including mono‐, di‐, and trisaccharides were employed to investigate the effects of water content and buffer concentration in the mobile phase on hydrophilic interaction liquid chromatography retention. For the tested saccharides, the silicon oxynitride column demonstrated excellent performance in terms of separation efficiency, hydrophilicity, and interesting separation selectivity for carbohydrates compared to the bare silica stationary phase. Finally, the silicon oxynitride hydrophilic interaction liquid chromatography column was employed in the separation of complex samples of fructooligosaccharides, saponins, and steviol glycoside from natural products. The resulting chromatograms demonstrated good separation efficiency and longer retention compared with silica, which further confirmed the advantages and potential application of silicon oxynitride stationary phase for hydrophilic interaction liquid chromatography separation.     

万古霉素及其杂质的亲水作用色谱分析

目前,万古霉素色谱分析方法主要为反相色谱法,该法分离万古霉素及其杂质时,存在极性选择性不足以及所使用的流动相体系与质谱兼容性差等问题。基于亲水作用色谱(HILIC)对糖肽类物质的色谱保留和极性选择性,本文选取万古霉素及其常见杂质为对象,考察了HILIC固定相、流动相组成比例、缓冲盐添加剂浓度和pH值等色谱条件,对万古霉素及其主要杂质进行了HILIC分离方法研究。确立了以Click XIon色谱柱为固定相,以甲酸铵为流动相添加剂的亲水作用色谱条件,实现了万古霉素及主要杂质的分离,为万古霉素类化合物的分离提供了新的途径。     

强极性中药组分的亲水作用色谱保留行为

采用亲水作用色谱模式,以二醇基硅胶为固定相,水-有机溶剂为流动相,通过改变流动相中有机溶剂种类及浓度、缓冲盐、有机酸种类及其浓度、柱温等条件,研究了强极性中药组分在亲水作用色谱中的保留行为。结果表明:流动相中水的比例在0～100%(V/V)变化时,溶质保留呈U型曲线,属于亲水色谱和反相色谱的混合保留机理。水含量在0～50%(V/V)范围时,亲水作用控制溶质的保留。溶质保留随流动相中缓冲盐浓度的增大而增强。对于酸性溶质,其保留随有机酸三氟乙酸、甲酸、乙酸的酸性降低而增大。     

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.     

Method development and validation for optimised separation of salicylic, acetyl salicylic and ascorbic acid in pharmaceutical formulations by hydrophilic interaction chromatography and response surface methodology

This paper introduces a design of experiments (DOE) approach for method optimisation in hydrophilic interaction chromatography (HILIC). An optimisation strategy for the separation of acetylsalicylic acid, its major impurity salicylic acid and ascorbic acid in pharmaceutical formulations by HILIC is presented, with the aid of response surface methodology (RSM) and Derringer's desirability function. A Box-Behnken experimental design was used to build the mathematical models and then to choose the significant parameters for the optimisation by simultaneously taking both resolution and retention time as the responses. The refined model had a satisfactory coefficient (R2>0.92, n=27). The four independent variables studied simultaneously were: acetonitrile content of the mobile phase, pH and concentration of buffer and column temperature each at three levels. Of these, the concentration of buffer and its cross-product with pH had a significant, positive influence on all studied responses. For the test compounds, the best separation conditions were: acetonitrile/22 mM ammonium acetate, pH 4.4 (82:18, v/v) as the mobile phase and column temperature of 28°C. The methodology also captured the interaction between variables which enabled exploration of the retention mechanism involved. It would be inferred that the retention is governed by a compromise between hydrophilic partitioning and ionic interaction. The optimised method was further validated according to the ICH guidelines with respect to linearity and range, precision, accuracy, specificity and sensitivity. The robustness of the method was also determined and confirmed by overlying counter plots of responses which were derived from the experimental design utilised for method optimisation.     

Green high‐performance liquid chromatography enantioseparation of lansoprazole using a cellulose‐based chiral stationary phase under ethanol/water mode

A simple and environmentally friendly reversed‐phase high‐performance liquid chromatography method for the separation of the enantiomers of lansoprazole has been developed. The chromatographic resolution was carried out on the cellulose‐based Chiralpak IC‐3 chiral stationary phase using a green and low‐toxicity ethanol‐aqueous mode. The effects of water content in the mobile phase and column temperature on the retention of the enantiomers of lansoprazole and its chiral and achiral related substances have been carefully investigated. A mixed‐mode hydrophilic interaction liquid chromatography and reversed‐phase retention mechanism operating on the IC‐3 chiral stationary phase allowed us to achieve simultaneous enantioselective and chemoselective separations in water‐rich conditions. The enantiomers of lansoprazole were baseline resolved with a mobile phase consisting of ethanol/water 50:50 without any interference coming from chiral and achiral impurities within 10 min.     

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.     

Retention behaviour of imidazolium ionic liquid cations on 1.7 μm ethylene bridged hybrid silica column using acetonitrile-rich and water-rich mobile phases

The potential of 1.7 μm ethylene bridged hybrid silica phase was investigated for the separation of twelve imidazolium-based ionic liquid cations. U-shaped retention profile was observed for all solutes with an increase in retention at both low and high acetonitrile content. Chromatographic behaviour of imidazolium cations in both hydrophilic interaction chromatography (HILIC) and per aqueous liquid chromatography (PALC) modes was studied by varying key parameters such as buffer concentration and pH, acid additive, organic modifier and column temperature. Experimental data provided some evidences that under PALC conditions cationic solutes are retained predominantly by mixed hydrophobic/ion-exchange interactions. In the HILIC mode, both partitioning and ion-exchange interactions are responsible for the retention of solutes. Compared to PALC, HILIC provided significantly higher efficiencies with less or even no peak tailing, better separation selectivity and greater resistance to overload. In PALC mode gradient elution was required to achieve adequate retentivity of all solutes but selectivity was not sufficient to distinguish between solutes with very similar hydrophobicity. In contrast, under HILIC conditions twelve solutes were almost completely resolved in less than 4 min by using isocratic elution. Summarizing, it could be concluded that ethylene bridged hybrid silica column providing a dual retention mechanism offers the possibility of selecting between the two retention modes with opposite separation selectivity, just by changing the composition of the mobile phase.     

Mobile phase effects on the retention on polar columns with special attention to the dual hydrophilic interaction–reversed‐phase liquid chromatography mechanism,a review

Hydrophilic interaction liquid chromatography on polar columns in aqueous–organic mobile phases has become increasingly popular for the separation of many biologically important compounds in chemical, environmental, food, toxicological, and other samples. In spite of many new applications appearing in literature, the retention mechanism is still controversial. This review addresses recent progress in understanding of the retention models in hydrophilic interaction liquid chromatography. The main attention is focused on the role of water, both adsorbed by the column and contained in the bulk mobile phase. Further, the theoretical retention models in the isocratic and gradient elution modes are discussed. The dual hydrophilic interaction liquid chromatography reversed‐phase retention mechanism on polar columns is treated in detail, especially with respect to the practical use in one‐ and two‐dimensional liquid chromatography separations.     

多羟基化合物键合亲水色谱固定相的制备及其分离性能

用硅胶与氨丙基三甲氧基硅烷反应,再与δ-葡萄糖酸内酯反应,制备了一种多羟基化合物键合的新型亲水色谱固定相。以水-有机溶剂(乙醇、乙腈、四氢呋喃)为流动相,通过改变流动相中有机溶剂的种类及浓度、缓冲盐浓度和柱温,考察了该固定相对6种强极性中药组分的保留行为和保留机理。当水的比例在0～40%(v/v)范围时,溶质的保留随着流动相中水的比例的增大而减小,属于典型的亲水色谱分离模式;而当流动相中水的比例在0～100%(v/v)范围内变化时,溶质的保留随着水的比例变化呈“U”形曲线,属于亲水色谱和反相色谱的混合保留机理。缓冲盐的浓度和pH效应说明,所选用的中药组分与所制备的固定相间还存在弱的静电作用。该固定相对6种中药组分以及丹参注射液具有良好的分离性能,表明其在强极性中药有效成分的分离及其他强极性物质的分离分析中具有一定的应用前景。     

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.     

Chromatographic behavior of new deazapurine ribonucleosides in hydrophilic interaction liquid chromatography

The chromatographic behavior of new biogenic purine nucleosides in hydrophilic interaction liquid chromatography was examined on three different stationary phases, namely bare silica, and amide‐ and cyclofructan‐based stationary phases. The effects of buffer concentration, pH and acetonitrile‐to‐aqueous‐part ratio in the mobile phase on retention and peak shape were assessed. The retention coefficients and peak symmetry values substantially differed with respect to analytes´ structures, stationary phase properties and mobile phase composition. The bare silica column was unsuitable for these compounds under the chromatographic conditions tested due to very broad and asymmetrical peaks. Furthermore, the cyclofructan‐based stationary phase provided almost Gaussian peak shapes of all deazapurine nucleosides under most conditions tested. Therefore, the cyclofructan‐based stationary phase is the most suitable choice for the chromatographic analysis of nucleosides.     

Tetrazole-Functionalized Silica for Hydrophilic Interaction Chromatography of Polar Solutes

In this work, tetrazole-functionalized stationary phase was prepared with nitrile-modified silica by an ammonium-catalyzed (3 + 2) azide-nitrile cycloaddition reaction. The prepared stationary phase was used for separation of nucleobases and nucleosides by hydrophilic interaction chromatography (HILIC) mode. A typical HILIC mechanism was observed at higher content of acetonitrile (>85%, v/v) in the mobile phase. The retention mechanism of the column was investigated by the models used for describing partitioning and surface adsorption through adjustment ratio of water in the mobile phase, and by the influence of salt concentration, buffer pH, and temperature on the retention of solutes. The results illustrated that the surface adsorption through hydrogen bonding dominated the retention behavior of nucleobases/nucleosides under HILIC mode. From the separation ability, the tetrazole-functionalized stationary phase could become a valuable alternative for the separation of the compounds concerned.     

新型咪唑嵌合氨基亲水作用色谱固定相的制备及其色谱性能

发展新型高效的亲水作用色谱分离材料对于极性化合物的分离分析具有重要的意义。本文设计合成了一种新型咪唑嵌合的氨基亲水作用色谱固定相（Sil-IEASP）,并分别采用傅里叶变换红外光谱仪、热重分析仪和元素分析仪对该固定相进行了表征,结果表明该固定相制备成功。以核苷和核酸碱基为样品,分别考察了流动相中的水含量、盐浓度和pH对其保留的影响,结果表明所发展的固定相具有良好的亲水作用特性;此外,缓冲盐浓度和pH几乎不影响上述物质的保留。进一步将该固定相应用于分离尿嘧啶、腺嘌呤、胞嘧啶、尿苷和3种位置异构体（邻三联苯、间三联苯和苯并菲）,与常用的氨基固定相相比,本文所发展的固定相具有更好的分离效果,有望在亲水作用色谱分离领域发挥潜在的应用价值。     

Preparation of a Novel Amino-Phosphate Zwitterionic Stationary Phase for Hydrophilic Interaction Chromatography

A new stationary phase which contains both negatively charged phosphate groups and positively charged amino groups was successfully synthesized by modification of amino-functionalized silica particles with trichlorophosphine oxide (POCl₃) for hydrophilic interaction chromatography (HILIC). The composition of the surface grafts was determined by Fourier transform infrared spectroscopy and elemental analysis. Various parameters, such as column temperature, water content, pH values and ionic strength of the mobile phase were investigated to study the retention mechanism. The results demonstrated that the stationary phase involved a complex retention process including surface adsorption, partitioning and electrostatic interactions. Under optimized conditions, the separation of nucleobases and nucleosides, water-soluble vitamins, organic acids on the novel stationary phase could be achieved in the HILIC mode.     

基于聚倍半硅氧烷微球的亲水/反相混合模式色谱填料的制备与评价

亲水/反相混合模式色谱应用广泛,但pH使用范围有限,不利于碱性药物的分离。该工作利用巯基-烯基点击化学合成了单分散多孔的半胱氨酸改性乙烯基功能化聚甲基倍半硅氧烷(C-V-PMSQ)微球。元素分析表明半胱氨酸成功键合在微球表面。C-V-PMSQ微球为介孔结构,单分散性好且具有优良的化学稳定性。以几种常见的核苷和核酸碱基作为测试样品,考察其色谱保留行为,溶质的保留因子随流动相中水相含量的变化呈现典型的U型曲线,表明C-V-PMSQ固定相具有亲水/反相的双重保留特征。使用该固定相可以分离苯的同系物及一系列亲水性与疏水性化合物。另外在高碱性流动相条件下利用亲水和反相模式成功分离了中药苦参中的3种主要活性成分,表明它在分离碱性药物方面具有较大的优势。     

Preparation and application of novel zwitterionic monolithic column for hydrophilic interaction chromatography

A novel zwitterionic hydrophilic porous monolithic stationary phase was prepared based on the thermal‐initiated copolymerization of N,N‐dimethyl‐N‐(3‐methacryl‐amidopropyl)‐N‐(3‐(sulfopropyl)ammonium betaine and ethylene glycol dimethacrylate. A typical hydrophilic separation mechanism was observed at a highly organic mobile phase (ACN >60%) on this optimized zwitterionic hydrophilic interaction chromatography (HILIC) monolithic stationary phase. Good permeability, stability, and column efficiency were observed on the final monolithic column. Additionally, a weak electrostatic interaction for charged analytes was confirmed in analysis of six benzoic acids by studying the influence of mobile phase pH and salt concentration on their retention behaviors on the obtained zwitterionic HILIC monolithic column. The optimized zwitterionic HILIC monolith exhibited good selectivity for a range of polar test analytes.     

糖类化合物亲水作用色谱保留行为评价

以糖类化合物为研究对象,系统评价了其在亲水模式下的色谱保留行为。分别考察了流动相、固定相和缓冲盐等对糖类化合物保留的影响,建立了糖类化合物在亲水模式下的保留方程。结果表明,糖类化合物随着流动相中乙腈比例的降低,保留时间减小;随着缓冲盐浓度的增加,保留时间增加;同时,糖类化合物的保留行为还会受到有机溶剂种类和固定相类型的影响;其保留行为可使用顶替吸附-液相相互作用模型定量描述。将该模型进一步用于实际样品中糖类化合物保留行为的预测,获得了较好的实验结果,预测保留时间与实测保留时间的相对误差小于0.3%。对糖类化合物亲水模式下的保留行为进行了系统的评价和定量描述,该研究结果将有助于糖类化合物亲水作用色谱分离方法的发展。