Separation properties of novel and commercial polar stationary phases in hydrophilic interaction and reversed-phase liquid chromatography mode

Four novel nonionic polar stationary phases were synthesised by anchoring first 2-mercaptoethanol and 1-thioglycerol, respectively, onto vinylised silica (ME and TG packings) followed by an on-phase oxidation with excess hydrogen peroxide in aqueous medium which yielded sulphoxide analogues of the embedded sulphide groups, i. e. oxidised 2-mercaptoethanol (MEO) and oxidised 1-thioglycerol (TGO) packings. Chromatographic characteristics of these stationary phases were evaluated comparatively to three commercial so-called 'diol' columns. U-shaped response curves of retention factors of adenosine and guanosine with hydro-organic eluents containing 5-95% v/v ACN as well as noticeable CH(2)-increment selectivity demonstrated multimodal separation capabilities of the developed amphiphilic materials, i. e. columns can be operated both in hydrophilic interaction chromatography (HILIC) and in RP mode. Although the selector ligands were physico-chemically related, considerably differing retention and selectivity patterns were observed in the HILIC mode. Thereby the introduction of additional hydroxyl groups in the chromatographic ligand resulted in selectivity increments that were different from those obtained by sulphur oxidation. For example, a set of five vitamins delivered five different elution orders with the overall seven columns. A close examination of HILIC separations of nucleobases and nucleosides on the developed packings revealed that (i) the amount of ACN in the eluent adopts a pivotal role in adjusting retention, (ii) the linearity of the relationship log (retention factor) versus log (volume fraction of water in the eluent) increases with phase polarity in the range of 5-40% v/v water, (iii) the slopes are higher with solutes having more polar interactive sites, (iv) the van't Hoff plots are linear (range 15-45 degrees C) with negative retention enthalpy values DeltaH (-4.5 to -14.5 kJ/mol) and (v) the -DeltaH values tend to be higher with more polar phases and more polar analytes. Based on these data the HILIC retention mechanism is described to be composed of both partitioning and adsorption processes. Distinct types of polar interactive sites in the chromatographic ligands may generate mixed-mode HILIC separation conditions that may additionally be superimposed by surface silanol contributions.     

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.     

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.     

Hydrophilic interaction chromatography in nonaqueous elution mode for separation of hydrophilic analytes on silica-based packings with noncharged polar bondings

Chromatographic effects of dedicated stationary and mobile phase variations in hydrophilic interaction chromatography (HILIC) were investigated using a set of nucleobases, nucleosides and deoxynucleosides as polar test solutes. Retention and selectivity profiles were comparatively mapped on four in-house developed silica materials modified with short alkyl chains (C4, C5) which carry hydroxyl functionalities (including diol motifs) as well as embedded sulphide or sulphoxide groups. These data were complemented by results obtained with two commercially available diol-type phases and a bare silica column. Besides elucidation of packing-related aspects this work concentrated specifically on extending aqueous HILIC (AQ-HILIC) to nonaqueous polar-organic elution conditions herein termed NA-HILIC. The exchange of the polar modifier water by various alcohols in ACN-rich mobile phases containing 5 mM ammonium acetate decreased the eluotropic strength of the resulting eluents. The gain in retention largely followed the order ethanol (EtOH)>methanol (MeOH)>1,2-ethanediol (Et(OH)2) and was accompanied by distinct effects on chromatographic selectivity. For example, on the most polar home-made packing the purine nucleoside selectivity guanosine/adenosine increased from 2.25 in the AQ-HILIC (kguanosine=8.3) to 7.33 (kguanosine=59) in the NA-HILIC mode when EtOH was employed as NA modifier while this value was 5.84 and 2.93 with MeOH and Et(OH)2, respectively (eluent: 5 mM ammonium acetate in ACN/modifier 90:10 v/v). Besides the type of protic modifier its percentage as well the retention and selectivity effects upon varying the ammonium acetate concentration and column temperature, respectively, were also investigated. Notable inter-column differences were found for all of these elution parameters. A mixed-mode retention model composed of partitioning and adsorption is proposed for both AQ- and NA-HILIC retention processes. The potential of (i) the implementation of novel polar bondings (such as ones containing sulphoxide functionalities) and (ii) the comprehensive exploitation of elution variables (type of protic modifiers, salt, etc.) for providing new selectivity increments to the separation of polar analytes in HILIC is emphasised.     

Retention Mechanism Studies of Selected Amino Acids and Vitamin B6 on HILIC Columns with Evaporative Light Scattering Detection

The goal of the study was to investigate separation mechanism of selected “essential” amino acids (leucine, isoleucine, threonine, tryptophan, proline, and glycine) and vitamin B6 in hydrophilic interaction liquid chromatography (HILIC) with the evaporative light scattering detection. Chromatographic measurements were made on three different HILIC columns: amide-silica (TSK-gel Amide-80), amino-silica (TSK-gel NH₂-100), and cross-linked diol (Luna HILIC). The retention behaviour of the analytes was investigated as a function of different binary hydro-organic mobile phases containing 10–90 % (v/v) acetonitrile. The compounds studied were separated under isocratic and gradient conditions. The best results of tested biologically active compounds separation were obtained on the TSK-gel NH₂-100 column. TSK-gel NH₂ column showed mixed HILIC–ion-exchange mechanism, the highest separation efficiency and better selectivity and resolution for tested analytes than the other studied column, especially at concentration of water in mobile phase lower than 30 % (v/v). Special attention was dedicated to the study of interactions among the stationary phase, mobile phase and the analytes.     

Retention Mechanism Studies of Selected Amino Acids and Vitamin B6 on HILIC Columns with Evaporative Light Scattering Detection

The goal of the study was to investigate separation mechanism of selected “essential” amino acids (leucine, isoleucine, threonine, tryptophan, proline, and glycine) and vitamin B6 in hydrophilic interaction liquid chromatography (HILIC) with the evaporative light scattering detection. Chromatographic measurements were made on three different HILIC columns: amide-silica (TSK-gel Amide-80), amino-silica (TSK-gel NH₂-100), and cross-linked diol (Luna HILIC). The retention behaviour of the analytes was investigated as a function of different binary hydro-organic mobile phases containing 10–90 % (v/v) acetonitrile. The compounds studied were separated under isocratic and gradient conditions. The best results of tested biologically active compounds separation were obtained on the TSK-gel NH₂-100 column. TSK-gel NH₂ column showed mixed HILIC–ion-exchange mechanism, the highest separation efficiency and better selectivity and resolution for tested analytes than the other studied column, especially at concentration of water in mobile phase lower than 30 % (v/v). Special attention was dedicated to the study of interactions among the stationary phase, mobile phase and the analytes.

     

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。     

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。     

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.     

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.

     

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.     

Characterization of enhanced-fluidity liquid hydrophilic interaction chromatography for the separation of nucleosides and nucleotides

Hydrophilic interaction chromatography (HILIC) is a liquid chromatographic separation mechanism commonly used for polar biological molecules. The use of enhanced-fluidity liquid chromatography (EFLC) with mixtures of methanol/water/carbon dioxide is compared to acetonitrile/water mobile phases for the separation of nucleosides and nucleotides under HILIC conditions. Enhanced-fluidity liquid chromatography involves using common mobile phases with the addition of substantial proportions of a dissolved gas which provides greater mobile phase diffusivity and lower viscosity. The impact of varying several experimental parameters, including temperature, addition of base, salt, and CO₂ was studied to provide optimized HILIC separations. Each of these parameters plays a key role in the retention of the analytes, which demonstrates the complexity of the retention mechanism in HILIC. The tailing of phosphorylated compounds was overcome with the use of phosphate salts and the addition of a strong base; efficiency and peak asymmetry were compared with the addition of either triethylamine (TEA), 1,4-diazabicyclo [2.2.2] octane (DABCO) or 1,5-diazabicyclo [4.3.0] non-5-ene (DBN). DBN and DABCO both led to increased efficiency and lower peak asymmetry; DBN provided the best results. Sodium chloride and carbon dioxide were added to enhance the selectivity between the analytes, giving a successful isocratic separation of nucleosides and nucleotides within 8 min. The retention mechanism involved in EFL-HILIC was explored by varying the temperature and the mole fraction of CO₂. These studies showed that partitioning was the dominant mechanism. The thermodynamics study confirmed that the solvent strength is maintained in EFLC and that a change in entropy was mainly responsible for the improved selectivity. The selectivity using methanol/water/carbon dioxide varied greatly compared to that obtained with acetonitrile/water. Finally while this study highlights the optimization of EFL-HILIC for the separation of nucleosides and nucleotides under isocratic conditions, this is also an example of the broad range of polarities of compounds that EFL-HILIC can separate.     

Approach to hydrophilic interaction chromatography column selection: Application to neurotransmitters analysis

This paper presents the comparison between 12 hydrophilic interaction liquid chromatography columns that are commercially available. The main factors influencing the retention and selectivity toward 12 neurotransmitters for these different HILIC systems have been studied. For additional information, the retention data have been analyzed statistically by factor analysis. Principal component analyses (PCA) were calculated to evidence different separation behaviour between the stationary phases, based on the retention data measured for three compound classes: anionic acidic compounds, cationic basic compounds and zwitterionic amino acids. Finally, a generic procedure is suggested for optimization of HILIC analyses, depending on the ionization state of the analytes.     

Retention mechanism assessment and method development for the analysis of iohexol and its related compounds in hydrophilic interaction liquid chromatography

Hydrophilic interaction liquid chromatography (HILIC) has emerged in recent years as a valuable alternative to reversed-phase liquid chromatography in the analysis of polar compounds. Research in HILIC is divided into two directions: the assessment of the retention mechanism and retention behavior, and the development of HILIC methods. In this work, four polar neutral analytes (iohexol and its related compounds A, B, and C) were analyzed on two silica and two diol columns in HILIC mode with the aim to investigate thoroughly the retention mechanisms and retention behavior of polar neutral compounds on these four columns. The adsorption and partition contribution to the overall HILIC retention mechanism was investigated by fitting the retention data to linear (adsorption and partition) and nonlinear (mixed-retention and quadratic) theoretical models. On the other hand, the establishment of empirical second-order polynomial retention models on the basis of D-optimal design made possible the estimation of the simultaneous influence of several mobile-phase-related factors. Furthermore, these models were also used as the basis for the application of indirect modeling of the selectivity factor and a grid point search approach in order to achieve the optimal separation of analytes. After the optimization goals had been set, the grids were searched and the optimal conditions were identified. Finally, the optimized method was subjected to validation.

有机酸在阴离子交换树脂上的保留行为及其机理

研究了有机酸在阴离子交换树脂上的保留行为。首次报道了有机酸在阴离子交换色谱过程中的“多峰现象”,即一种有机酸有两个或两个以上的色谱峰流出色谱柱。对这种“多峰现象”形成的机理进行了初步探讨,提出了有机酸离解后的不同价数阴离子在阴离子交换树脂上同时保留的机理。研究中还发现离解很弱的有机酸的保留行为不完全符合离子交换机理。     

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.     

Hydrophilic interaction chromatography using amino and silica columns for the determination of polar pharmaceuticals and impurities

Hydrophilic interaction chromatography (HILIC) is described as a useful alternative to reversed-phase chromatography for applications involving polar compounds. In the HILIC mode, an aqueous-organic mobile phase is used with a polar stationary phase to provide normal-phase retention behavior. Silica and amino columns with aqueous-acetonitrile mobile phases offer potential for use in the HILIC mode. An examination of the retention and separation of several pyrimidines, purines, and amides on silica and amino columns from three manufacturers revealed that mobile phases should contain a buffer or acid for pH control to achieve similar and reproducible results among columns from different sources. Amino columns may also be used in an anion-exchange mode, which provides an advantage for some applications. In some cases, silica can provide different selectivity and better separation than an amino column. Example applications include: low-molecular-mass organic acids and amides as impurities in non-polar drug substances, 5-fluorouracil in 5-fluorocytosine, guanine in acyclovir, and different selectivity for polar basic compounds compared to an ion-pairing system.     

Hydrophilic interaction liquid chromatography for separation and quantification of selected room-temperature ionic liquids

Hydrophilic interaction liquid chromatography (HILIC) is an alternative technique to ion pairing-reversed-phase liquid chromatography (IP-RPLC) and classical RPLC for separation of alkylimidazolium room-temperature ionic liquids (RTILs). Particularly, HILIC offers better retention and selectivity for short-chains RTILs imidazolium compounds. HILIC mechanisms were investigated by studying the influence of organic modifier content and salt concentration in the mobile phase. HILIC method was validated by quantifying 1-butyl-3-methylimidazolium cation (BMIM) degradation under gamma radiation at 2.5MGy. Development of separative reproducible analytical methods, including for low concentration, applicable to RTILs are today mandatory to improve RTILs chemistry.     

Stationary and mobile phases in hydrophilic interaction chromatography: a review

Hydrophilic interaction chromatography (HILIC) is valuable alternative to reversed-phase liquid chromatography separations of polar, weakly acidic or basic samples. In principle, this separation mode can be characterized as normal-phase chromatography on polar columns in aqueous-organic mobile phases rich in organic solvents (usually acetonitrile). Highly organic HILIC mobile phases usually enhance ionization in the electrospray ion source of a mass spectrometer, in comparison to mobile phases with higher concentrations of water generally used in reversed-phase (RP) LC separations of polar or ionic compounds, which is another reason for increasing popularity of this technique. Various columns can be used in the HILIC mode for separations of peptides, proteins, oligosaccharides, drugs, metabolites and various natural compounds: bare silica gel, silica-based amino-, amido-, cyano-, carbamate-, diol-, polyol-, zwitterionic sulfobetaine, or poly(2-sulphoethyl aspartamide) and other polar stationary phases chemically bonded on silica gel support, but also ion exchangers or zwitterionic materials showing combined HILIC-ion interaction retention mechanism. Some stationary phases are designed to enhance the mixed-mode retention character. Many polar columns show some contributions of reversed phase (hydrophobic) separation mechanism, depending on the composition of the mobile phase, which can be tuned to suit specific separation problems. Because the separation selectivity in the HILIC mode is complementary to that in reversed-phase and other modes, combinations of the HILIC, RP and other systems are attractive for two-dimensional applications. This review deals with recent advances in the development of HILIC phase separation systems with special attention to the properties of stationary phases. The effects of the mobile phase, of sample structure and of temperature on separation are addressed, too.     

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.