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.

     

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。     

Zwitterionic stationary phase with covalently bonded phosphorylcholine type polymer grafts and its applicability to separation of peptides in the hydrophilic interaction liquid chromatography mode

A novel phosphorylcholine type zwitterionic stationary phase was synthesized by graft polymerization of 2-methacryloyloxyethyl phosphorylcholine onto the surface of porous silica particles. The resulting material possesses both negatively charged phosphoric acid and positively charged quaternary ammonium groups, which renders it a low net charge over a wide pH range. The composition of the surface grafts were determined by elemental analysis and solid state NMR, and the surface charge (zeta-potential) in different buffer solutions were measured using photon correlation spectroscopy. Separation of several peptides was investigated on packed columns in the hydrophilic interaction liquid chromatography (HILIC) separation mode. It was shown that small peptides can be separated based on hydrophilic interaction and ionic interaction between the stationary phase and analyte. The organic solvent composition, the pH and the salt concentration of the eluent have strong effects on the retention time. Compared to native silica before grafting, the newly synthesized zwitterionic material gave more stable retention times for basic peptides over pH range 3-7 due to elimination of the dissociation of silanol groups.     

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.     

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。     

Impact of column temperature and mobile phase components on selectivity of hydrophilic interaction chromatography (HILIC)

The retention mechanism and chromatographic behavior for different polar analytes under hydrophilic interaction chromatography (HILIC) conditions have been studied by application of different mobile phases and stationary phases to various analytes at different temperatures. In addition to the commonly accepted mechanism of analyte liquid-liquid partitioning between mobile phase and water-enriched solvent layer which is partially immobilized onto the surface of the stationary phase, hydrogen-bonding, hydrophobic interaction, and ion-exchange interactions may also be involved. The predominant retention mechanism in HILIC separation is not always easily predictable. It can depend not only on the characteristics of the analytes but also on the selection of mobile and stationary phase compositions. The objective of this review is to evaluate the potential application of column temperature and mobile phase composition toward improving HILIC selectivity. The functional groups from analyte structures, stationary phase materials and organic mobile phase solvents will be highlighted.     

一种可控离子比例的两性亲水色谱固定相的制备及性能研究

将不同比例的氨基和巯基的硅烷偶联剂键合到硅胶表面，再利用巯基与乙烯基膦酸之间的点击化学反应将膦酸基团引入到硅胶表面，制备了一种可调节正负离子比例的两性亲水色谱固定相。通过测定固定相中C、H、N、P元素的含量，证明了氨基与膦酸基团已成功键合到固定相的表面，同时通过N元素与P元素的质量分数确定固定相表面氨基与膦酸基团的比例。制备了3种不同电荷比例的氨基膦酸固定相，将其作为亲水模式下的固定相填料填装在150 mm×4.6 mm不锈钢色谱柱中。以一系列经典的极性小分子作为探针，研究了流动相中乙腈含量、缓冲盐pH值及缓冲盐浓度等因素对探针分子在3种色谱柱上的保留的影响，结果表明，分析物在固定相上是多重保留机理。最后通过比较核苷、水溶性维生素、碱性化合物、苯甲酸这几类标准物质在3种色谱柱上的保留行为来对比3种不同电荷比例的固定相的分离选择性与色谱性能。结果表明，对于不同的分析物，3种固定相表现出完全不同的分离选择性和色谱行为。可以根据分析物的特征选取不同电荷比例的固定相，表明此种固定相在极性化合物的分离上具有良好的应用前景。     

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

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

Development and evaluation of new zwitterionic hydrophilic interaction liquid chromatography stationary phases based on 3-P,P-diphenylphosphonium-propylsulfonate

New zwitterionic stationary phases were synthesized by covalently bonding 3-P,P-diphenylphosphonium-propylsulfonate to silica gel. The resulting materials possess both a negatively charged sulfonate group and a positively charged quaternary phosphonium group, which means that there is no net charge over a wide pH range. The retention mechanism and chromatographic behavior of polar solutes under HILIC conditions were studied on these zwitterionic phases. Compared to the commercial ZIC-HILIC column and a bare silica gel stationary phase, the newly synthesized zwitterionic stationary phases provided greater retention, higher peak efficiency and better peak symmetry in the HILIC mode. The analytes examined included: β-blockers, nucleic acid bases and nucleosides, salicylic acid and its analogues, and water soluble vitamins. Factors, such as the type of organic modifiers, solvent composition, pH and the buffer concentration of the mobile phase, have been considered as potential variables for controlling the chromatographic retention of polar analytes.     

Underivatized amylose and cellulose as new stationary phases for hydrophilic interaction chromatography

Two polysaccharide stationary phases have been newly suggested for application in hydrophilic interaction chromatography (HILIC). Both columns (amylose‐silica, 250 × 4.6 mm, 5 μm and cellulose‐silica, 250 × 4.6 mm, 5 μm) demonstrated a satisfactory retention of polar compounds. The influence of the mobile‐phase composition (acetonitrile content, pH, salt concentration) on the retention was in agreement with the HILIC concept. The phases showed a very similar behavior, typical efficiency of about 50 000 plates/m, cellulose retained test compounds somewhat more strongly. Under the experimental conditions, electrostatic (non‐HILIC‐type) interactions due to the dissociation of silanol groups on the silica surface did not influence the retention, noticeably. The applicability of polysaccharide stationary phases for the chromatography of polar compounds was proven by the separation of mixtures of sugars (fructose, glucose, saccharose, maltose, trehalose) or vitamins (nicotinamide, pyridoxine, riboflavin, thiamine, nicotinic acid, ascorbic acid).     

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.     

Separation of nucleotides by hydrophilic interaction chromatography using the FRULIC-N column

A stationary phase composed of silica-bonded cyclofructan 6 (FRULIC-N) was evaluated for the separation of four cyclic nucleotides, six nucleoside monophosphates, four nucleoside diphosphates, and five nucleoside triphosphates via hydrophilic interaction chromatography (HILIC) in both isocratic and gradient conditions. The gradient conditions gave significantly better separations by narrowing peak widths. Sixteen out of nineteen nucleotides were baseline separated on the FRULIC-N column in one run. Unlike other known HILIC stationary phases, there can be dual-retention mechanisms unique to this media. Traditional hydrogen bonding/dipolar interactions can be supplemented by dynamic ion interaction effects for anionic analytes. This occurs because the FRULIC-N stationary phase is able to bind certain buffer cations. The extent of the ion interaction is tunable, in comparison to stationary phases with embedded charged groups, where the inherent ionic properties are fixed. The best mobile phase conditions were determined by varying the organic modifier (acetonitrile) content, as well as salt type/concentration and electrolyte pH. The thermodynamic characteristic of the FRULIC-N column was investigated by evaluating the column temperature effect on retention and utilizing van’t Hoff plots. This study shows that there is a greater entropic contribution for the retention of nucleotide di and triphosphates, whereas there is a greater enthalphic contribution for the cyclic nucleotides with the FRULIC-N column.     

Preparation of imidazole-functionalized silica by surface-initiated atom transfer radical polymerization and its application for hydrophilic interaction chromatography

A novel imidazole-functionalized stationary phase for hydrophilic interaction chromatography (HILIC) was prepared via surface-initiated atom transfer radical polymerization (SI-ATRP). 1-Vinylimidazole as a monomer was polymerized on the surface of initiator-immobilized silica by SI-ATRP using CuCl and 2,2'-bipyridyl as a catalyst. The graft chain length and polymer grafting density were controlled by varying the ratio of monomer to initiator. The resulting materials were characterized by elemental analysis and thermogravimetric analysis. Then, high-performance liquid chromatography separation of eight nucleobases/nucleosides was performed on the imidazole-functionalized chromatographic column in HILIC mode. The effects of mobile phase composition, buffer pH, and column temperature on the separation of nucleobases/nucleosides were investigated, and the retention mechanisms were studied. Chromatographic parameters were calculated, and the results showed that surface adsorption through hydrogen bonding and electrostatic interaction dominated the retention behavior of the solutes in HILIC mode. Lastly, the stationary phase was successfully used to determine the nucleobases and nucleosides from Cordyceps militaris.     

Preparation and Evaluation of Poly-l-Lysine Stationary Phase for Hydrophilic Interaction/Reversed-Phase Mixed-Mode Chromatography

In this work, a poly-l-lysine-grafted stationary phase was synthesized by polymerization of N-carboxyanhydride of l-lysine initiated by 3-aminopropylated silica. The resulting material was characterized by FT-IR spectra, elemental analysis and thermogravimetric analysis, which clearly indicated that the new phase had been prepared successfully. The retention of polar solutes depending on acetonitrile content in mobile phase exhibited ??U-shaped?? curves, which was an indication of hydrophilic interaction liquid chromatography (HILIC)/reversed-phase liquid chromatography (RPLC) mixed-mode retention behavior. The retention mechanisms in HILIC and RPLC modes also were investigated. Phenol compounds, aniline compounds and hydrophilic compounds were separated in RPLC or HILIC mode on the new stationary phase, respectively. This result shows that the new phase could be used for both RPLC and HILIC applications, providing greater flexibility for real sample analysis.     

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.     

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。     

羧基甜菜碱型亲水作用色谱柱的制备及性能研究

以甲基丙烯酰氧乙基二甲基乙酸铵(CBMA)为功能单体,利用表面引发原子转移自由基聚合(Surface-initiated atom transfer radical polymerization, SI-ATRP)技术,将CBMA接枝到硅胶表面,得到接枝聚合物CBMA的亲水作用色谱固定相(Silica-CBMA).通过改变SI-ATRP反应体系中单体的浓度,制备了3种不同接枝量的亲水作用色谱固定相.考察了Silica-CBMA固定相对有机酸类化合物的分离性能以及流动相中pH值、盐浓度、水含量等因素对溶质保留行为的影响.结果表明,在亲水作用色谱模式下,Silica-CBMA固定相对有机酸类化合物的分离是离子交换作用与亲水作用的混合色谱模式.流动相中盐浓度增大,溶质保留减弱,符合离子交换作用特征;固定相和溶质的离子化程度受流动相pH值影响较大,pH值增大,溶质保留增强;而溶质的保留时间随流动相水含量增加而降低则是典型的亲水作用色谱特征.使用自制Silica-CBMA柱,建立了芦丁片中维生素C、芦丁含量的亲水作用色谱测定方法,操作方法简单,为强极性样品的分离测定提供了新方法.     

Investigating the Effect of Chromatographic Conditions on Retention of Organic Acids in Hydrophilic Interaction Chromatography Using a Design of Experiment

Small organic acids have shown significant retention on various stationary phases, such as amide, amino, aspartamide, silica and sulfobetaine phase commonly used in hydrophilic interaction chromatography (HILIC). This study investigated the effect of chromatographic conditions on the retention behavior of organic acids in HILIC using the tool of design of experiment (DOE). The results of the DOE study indicated that both the content of organic solvent (i.e., acetonitrile) and salt concentration in the mobile phase had significant effects on the retention of organic acids. Higher content of organic solvent in the mobile phase led to a significant increase in retention on all types of stationary phases. Increasing salt concentration also resulted in a moderate increase in retention; however, the effect of salt concentration varied with the type of stationary phase. The study also revealed that column temperature had less impact on retention than organic solvent content and salt concentration in HILIC.     

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。     

Stationary phases for hydrophilic interaction chromatography, their characterization and implementation into multidimensional chromatography concepts

Hydrophilic interaction chromatography (HILIC) is becoming increasingly popular for separation of polar samples on polar columns in aqueous-organic mobile phases rich in organic solvents (usually ACN). Silica gel with decreased surface concentration of silanol groups, or with chemically bonded amino-, amido-, cyano-, carbamate-, diol-, polyol-, or zwitterionic sulfobetaine ligands are used as the stationary phases for HILIC separations, in addition to the original poly(2-sulphoethyl aspartamide) strong cation-exchange HILIC material. The type of the stationary and the composition of the mobile phase play important roles in the mixed-mode HILIC retention mechanism and can be flexibly tuned to suit specific separation problems. Because of excellent mobile phase compatibility and complementary selectivity to RP chromatography, HILIC is ideally suited for highly orthogonal 2-D LC-LC separations of complex samples containing polar compounds, such as peptides, proteins, oligosaccharides, drugs, metabolites and natural compounds. This review attempts to present an overview of the HILIC separation systems, possibilities for their characterization and emerging HILIC applications in 2-D off-line and on-line LC-LC separations of various samples, in combination with RP and other separation modes.