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。     

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。     

亲水作用色谱及其在环境分析中的应用进展

亲水作用色谱(HILIC)是一种分离极性和亲水性化合物的液相色谱模式,其作为反相液相色谱(RPLC)的重要补充,近年来越来越受到各个领域的关注和重视。这不只是因为强极性化合物的分离问题在各个领域引起了重视,而且因为与RPLC比较,HILIC具有流动相组成黏度低、色谱柱渗透性好、与质谱联用的灵敏度高及反压较低等优势。本文简要概述了HILIC的发展历程、特点及保留机理,重点介绍了HILIC用于环境分析的最新进展,评述了HILIC及RPLC用于污染物分析的优缺点,并指出了HILIC用于环境分析的未来发展趋势。     

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。     

Determination of catecholamines in urine using hydrophilic interaction chromatography with electrochemical detection

The determination of catecholamines in urine was investigated using hydrophilic interaction chromatography (HILIC) as an alternative to the commonly used reversed-phase (RP) method. A number of different approaches were explored, including per-aqueous liquid chromatography (PALC), and HILIC using bare silica, bonded amide and zwitterionic phases. The bonded phases gave superior results in terms of both peak shape and selectivity. The mechanism of the HILIC separation was investigated particularly with respect to the contribution of ion exchange to retention. The electrochemical detection of catecholamines was studied and optimised in typical HILIC mobile phases that contain high concentrations of acetonitrile. HILIC offered a number of advantages over the conventional RP approach, giving good retention of the solutes without use of ion pair reagents, the absence of which also would facilitate detection by mass spectrometry. HILIC used in conjunction with solid phase extraction based on RP also gives orthogonal separation mechanisms in the cleanup and analysis steps. Furthermore, good recoveries from the cleanup stage were obtained, as high concentrations of acetonitrile can be used as eluting solvent that are fully compatible with HILIC, and lipophilic impurities are eluted close to the void volume of the HILIC column.     

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。     

低共熔溶剂——新型亲水作用色谱流动相改性剂

低共熔溶剂被用作亲水作用色谱流动相的新型改性剂。选用硅胶柱(150 mm×4.6 mm, 3 μm),以乙腈与低共熔溶剂(氯化胆碱-乙二醇(摩尔比为1:3))的混合溶液为流动相,考察了6个碱基与核苷的色谱分离效果,并讨论了低共熔溶剂在流动相中的比例及温度条件对分离的影响。结果表明,与传统的水相流动相条件相比,在加入低共熔溶剂改性后的流动相条件下,碱基与核苷分离效果得到明显的改善,尤其是胞嘧啶与胞苷能达到完全分离;同时,随着低共熔溶剂在乙腈中浓度的增加,6个碱基与核苷在色谱柱上的保留均有不同程度的减小,其中胞苷的保留减小最为显著;随着柱温的升高,碱基与核苷的保留同样有所减小。本文验证了低共熔溶剂作为亲水作用色谱流动相改性剂的可行性,可在一定程度上解决传统亲水作用色谱分离的困难。     

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。     

Retention and selectivity of stationary phases for hydrophilic interaction chromatography

More and more polar stationary phases have become available for the separation of small polar compounds in the past decade as hydrophilic interaction chromatography (HILIC) continues to find applications in new fields (e.g., metabolomics and proteomics). Bare silica phases remain popular, especially in the bio-analytical area. A wide range of functional groups (e.g., amino, amide, diol, sulfobetaine, and triazole) have been employed as polar stationary phases for HILIC separation. This review provides a survey of the popular stationary phases commercially available and discusses the retention and selectivity characteristics of the polar stationary phases in HILIC. The purpose of the review is not to provide a comprehensive overview of literature reports, but rather focuses on findings that demonstrate retention and selectivity of the polar stationary phases in HILIC.     

Determination of selectivity of HPLC systems by correspondence factor analysis

Correspondence factor analysis （CFA） was employed to study the selectivity of 14 HPLC systems. The tested LC systems were classified as reversed-phase （RP）, ion-exchange （IE） and hydrophilic interaction chromatography （HILIC） modes. It was found that the retentions of the hydrophilic solutes on HILIC column were significantly influenced by the second-order effects besides their hydrophilic properties. Organic modifiers and residue silanol groups on silica surface both participated in retention. HypersilTM amino column performed separation in the HILIC mode at appropriate conditions, and its retention mechanism was more similar to that of HILIC silica column than that of HILIC column coating poly（aspartamide） groups.     

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.     

亲水作用色谱固定相及其在中药分离中的应用

亲水作用色谱(HILIC)作为一种分离极性化合物的液相色谱模式,近年来越来越受到关注和重视。一方面是因为强极性化合物的分离问题引起了各个研究领域的重视,如药物分析、代谢组学、蛋白质组学等研究领域都不同程度地涉及强极性化合物的分离问题;另一方面是由于HILIC具有流动相组成简单、分离效率较高、与质谱兼容以及反压较低等优势。固定相是HILIC发展和应用的基础,本文主要从固定相分子结构的角度对HILIC固定相的结构特征、保留特性以及应用概况等进行了综述。对传统正相色谱固定相用于HILIC以及专门设计的HILIC固定相进行了介绍,评述了各自的优缺点和应用概况;对近年来HILIC固定相在中药分离中的应用进行了介绍;并对HILIC固定相的发展进行了展望。     

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.     

Hydrophilic interaction chromatography of seized drugs and related compounds with sub 2 μm particle columns

The use of hydrophilic interaction chromatography (HILIC) with sub 2 μm particle columns for the analysis of drugs and related compounds of forensic interest is described. This technique uses a high organic/low aqueous buffered mobile phase with a polar stationary phase, and is excellent for the separation of many of the charged solutes that are found in forensic drug exhibits. In this study, HILIC is investigated for 11 solutes of forensic interest, including weak bases, weak acids, and a neutral solute. In addition, for columns containing either ethylene bridged hybrid particles with or without an amide bonded phase, the effects of acetonitrile concentration, buffer type, buffer concentration, linear velocity, and sample concentration were studied. Based on these studies, HILIC with sub 2 μm particle columns can offer highly efficient, selective, and rapid isocratic separations of drugs and related compounds of forensic interest, with excellent peak shapes and low back pressures. This is in contrast to reverse phase chromatography (RPLC), where gradient elution is usually required, which can result in extensive overlap between acidic, neutral, and basic solutes. In addition, since HILIC exhibits a much greater loading capacity than RPLC, it could be a preferred technique for drug profiling. Furthermore, because high organic content mobile phases are highly amenable to mass spectrometric detection, the use of HILIC with tandem mass spectrometric detection for the analysis of seized drugs is described.     

Click novel glycosyl amino acid hydrophilic interaction chromatography stationary phase and its application in enrichment of glycopeptides

A novel glycosyl amino acid hydrophilic interaction chromatography (HILIC) stationary phase was prepared via click chemistry. The key intermediate N₃-glycosyl d-phenylglycine was prepared by a three steps procedure, including selective condensation of amino glucose with N-succinimidyl ester of Boc-d-phenylglycine, deprotection and transformation of amino group to azido group. The structure of all the intermediates and functionalized silica beads were confirmed by ¹H NMR, IR, elemental analysis and ¹³C CP-MAS. The chromatography test showed that this new type of separation material possessed good HILIC properties and glycopeptide enrichment characteristics. Nucleosides and bases could be separated in a simple eluent composition (only acetonitrile in combined with water), and with the same condition, these model compounds could not be separated on the commercial HILIC column (Atlantis). Click glycosyl amino acid thus prepared also showed longer retention and better separation ability in the separation of polar organic acids.     

Simple and Rapid Determination of Denaturants in Alcohol Formulations by Hydrophilic Interaction Chromatography

A hydrophilic interaction chromatography (HILIC) technique has been developed and validated for determination of common denaturants (denatonium benzoate, crystal violet and methylene blue) in denaturated alcohol formulations. Among the three different polar stationary phases (i.e., aminopropyl, cyanoethyl and silica) studied the cyanoethyl phase provided much stronger retention for the organic cations. It was shown that high efficiencies were reached only with anionic ion-pairing reagent that reduces the interactions with the silanol groups. The anion ion-pairing strength under HILIC conditions was: acetate < formate < trifluoroacetate < perchlorate. This study also investigated the effect of various experimental factors on the retention of the cyanoethyl stationary phase, such as acetonitrile content, pH and ion-pairing anion concentration in the mobile phase. The separation of three denaturants was achieved in about 8 min with a mobile phase containing 60% (v/v) acetonitrile and 10 mmol L⁻¹ HClO4. The proposed method was validated and applied to the determination of danaturating agents in various Lithuanian denaturated alcohol formulations.     

Direct determination of hydrazine,methylhydrazine, and 1,1-dimethylhydrazine by zwitterionic hydrophilic interaction liquid chromatography with amperometric detection

A promising alternative to ion-chromatographic methods currently used for the direct determination of hydrazines is provided by hydrophilic interaction liquid chromatography (HILIC). In this work, we propose a method for the simultaneous determination of hydrazine, methylhydrazine and 1,1-dimethylhydrazine in natural waters and soils based on a combination of chromatographic separation on a zwitterionic sulfobetaine stationary phase (Nucleodur HILIC) in the HILIC mode with amperometric detection.

Effects of different factors on the retention of analytes were studied and the optimum conditions of analysis were found. We recommend a mixture of acetonitrile with an aqueous phosphate buffer solution of pH 2.5 (78:22 v/v) with an ionic strength of 20 mM as a mobile phase. Detection in the direct current mode was performed at a working electrode potential of 1.1 V.

The advantages of the method are the high efficiency of separation, rapidity, high sensitivity and a wide dynamic range of analyte concentrations, covering four orders of magnitude. The attained LOD values for analytes lie in the range 0.07–0.13 μg L^–1, which is two orders of magnitude lower than those in currently used methods of ion chromatography with electrochemical and mass spectrometric detection.

The method was validated on samples of natural waters of different origin using the added–found technique. It was found that the error of analysis did not exceed 10% for river and ground waters and increased to 20–30% for peat bog surface waters.

The possibility of application of the developed method to the analysis of soils was shown on samples of peat bog soils selected at places of impact of the first steps of carrier rockets and polluted by rocket fuel based on 1,1-dimethylhydrazine.     

Separation of phospholipid classes by hydrophilic interaction chromatography detected by electrospray ionization mass spectrometry

A new isocratic separation method was developed for separation of phospholipid (PL) classes based on a silica hydrophilic interaction liquid chromatography (HILIC) column with electrospray ionization (ESI) mass spectrometric detection. Although HILIC is typically used for polar compounds, also amphiphilic molecules like phospholipids can be separated very well. Compared to normal-phase (NP) chromatography, which is usually used for PL class separation, HILIC has the advantage to use on-line ESI-MS detection because its eluents are ESI compatible. Furthermore, this HILIC method is isocratic and hence less time consuming than most (gradient) NP HPLC methods. A chromatographic baseline separation of a standard mixture containing phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingomyelin (SM) and lysophosphatidylcholine (LPC) was achieved within a total run time of 17 min using a mobile phase consisting of acetonitrile, methanol and ammonium acetate 10 mM. The new method was subsequently tested on phospholipid fractions of a body fluid (human blood plasma) and a tissue extract (swine brain) whereby it achieved nearly the same baseline separation of the PL classes. The detected classes in both cases were PE, PC, SM and LPC.     

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).     

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.