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.     

Retention behavior of a homologous series and positional isomers of aliphatic amino acids in hydrophilic interaction chromatography

The retention behavior of several series of free α‐ and ω‐amino acids and positional isomers of amino pentanoic acid in the hydrophilic interaction chromatography mode (HILIC) was studied. The study was carried out on three stationary phases followed by post‐column derivatization with fluorescence detection in order to describe the retention mechanism of the tested amino acids. The effect of chromatographic conditions including acetonitrile content in the mobile phase, mobile phase pH (ranging from 3.5 to 6.5) and concentration of buffer in the mobile phase was investigated. The effect of the number of carbon atoms (n_C) in aliphatic chains of the individual homologue of α‐ and ω‐amino acids and the logarithm of the partition coefficient (logD) on retention was also a part of the presented study. A good correlation (r > 0.98) between the logk and logD values of amino acids or n_C, respectively, was observed. The described linear relationships were subsequently applied to predict the retention behavior of individual members of the homologous series of amino acids and to optimize the mobile phase composition in HILIC. The obtained results confirmed that the retention mechanism of α‐amino acids, ω‐amino acids and positional isomers of amino acids was based on the logD values and the number of carbon atoms in the aliphatic chains of amino acids. The elution order of ω‐amino acids and positional isomers of amino pentanoic acid was strongly dependent on the mobile phase pH in the investigated range whereas the retention factors of all α‐amino acids remained essentially unchanged on all tested stationary phases.     

Retention behavior of ginsenosides on a poly(vinyl alcohol)-bonded stationary phase in hydrophilic interaction chromatography

The influences of the organic component of the mobile phase and the column temperature on the retention of ginsenosides on a poly(vinyl alcohol) (PVA) bonded stationary phase operated under hydrophilic interaction chromatographic mode were investigated. The retention of the ginsenosides was found to increase with increasing amount of acetonitrile (MeCN) in the mobile phase, which is typical of hydrophilic interaction chromatographic behavior. It was also found that the retention of the analytes was highly affected by the type of the organic modifier used. Aqueous MeCN (75–90%) gave the most satisfactory retention and separation of ginsenosides Rf, Rg1, Rd, Re, Rc, Rb2 and Rb1 compared with aqueous methanol, isopropyl alcohol or tetrahydrofuran at the same composition levels. The effects of the different types of organic modifiers on the retention of the analytes were attributed to their solvent strength and hydrogen-bond accepting/donating properties. The effect of temperature on the retention of ginsenoside on the PVA-bonded phase was assessed by constructing van’t Hoff plots for two temperature ranges: subambient (273–293 K) and ambient-elevated (298–333 K) temperatures. van’t Hoff plots for all analytes were linear at the two temperature intervals; however, the slopes of the lines corresponding to ginsenosides Rg1 and Re were completely different from those for the rest of the analytes especially in the subambient temperature range. Enthalpy-entropy compensation (EEC) studies were conducted to verify the difference in thermodynamics observed for ginsenosides Rg1 and Re compared with the other analytes. EEC plots showed that Rf, Rd, Rc, Rb2 and Rb1 were possibly retained by the same retention mechanism, which was completely different from that of Rg1 and Re at subambient temperatures. Retention prediction models were derived using multiple linear regression to identify solute attributes that affected the retention of the analytes on the PVA-bonded phase. The mathematical models derived revealed that the number of hydrogen-bond donors and the ovality of the molecules are important molecular properties that govern the retention of the compounds on the chromatographic system.     

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.     

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。     

Separation efficiencies in hydrophilic interaction chromatography

Hydrophilic interaction chromatography (HILIC) is important for the separation of highly polar substances including biologically active compounds, such as pharmaceutical drugs, neurotransmitters, nucleosides, nucleotides, amino acids, peptides, proteins, oligosaccharides, carbohydrates, etc. In the HILIC mode separation, aqueous organic solvents are used as mobile phases on more polar stationary phases that consist of bare silica, and silica phases modified with amino, amide, zwitterionic functional group, polyols including saccharides and other polar groups. This review discusses the column efficiency of HILIC materials in relation to solute and stationary phase structures, as well as comparisons between particle-packed and monolithic columns. In addition, a literature review consisting of 2006-2007 data is included, as a follow up to the excellent review by Hemstr?m and Irgum.     

Probing the interaction mode in hydrophilic interaction chromatography

This work aims at characterizing interactions between a select set of probes and 22 hydrophilic and polar commercial stationary phases, to develop an understanding of the relationship between the chemical properties of those phases and their interplay with the eluent and solutes in hydrophilic interaction chromatography. "Hydrophilic interaction" is a somewhat inexact term, and an attempt was therefore made to characterize the interactions involved in HILIC as hydrophilic, hydrophobic, electrostatic, hydrogen bonding, dipole-dipole, π-π interaction, and shape-selectivity. Each specific interaction was quantified from the separation factors of a pair of similar substances of which one had properties promoting the interaction mode being probed while the other did not. The effects of particle size and pore size of the phases on retention and selectivity were also studied. The phases investigated covered a wide range of surface functional groups including zwitterionic (sulfobetaine and phosphocholine), neutral (amide and hydroxyl), cationic (amine), and anionic (sulfonic acid and silanol). Principal component analysis of the data showed that partitioning was a dominating mechanism for uncharged solutes in HILIC. However, correlations between functional groups and interactions were also observed, which confirms that the HILIC retention mechanism is partly contributed by adsorption mechanisms involving electrostatic interaction and multipoint hydrogen bonding. Phases with smaller pore diameters yielded longer retention of solutes, but did not significantly change the column selectivities. The particle diameter had no significant effect, neither on retention, nor on the selectivities. An increased water content in the eluent reduced the multipoint hydrogen bonding interactions, while an increased electrolyte concentration lowered the selectivities of the tested columns and made their interaction patterns more similar.     

Retention prediction of a set of amino acids under gradient elution conditions in hydrophilic interaction liquid chromatography

The analysis of amino acids presents significant challenges to contemporary analytical separations. The present paper investigates the possibility of retention prediction in hydrophilic interaction chromatography (HILIC) gradient elution based on the analytical solution of the fundamental equation of the multilinear gradient elution derived for reversed‐phase systems. A simple linear dependence of the logarithm of the solute retention (ln k) upon the volume fraction of organic modifier (φ) in a binary aqueous‐organic mobile is adopted. Utility of the developed methodology was tested on the separation of a mixture of 21 amino acids carried out with 14 different gradient elution programs (from simple linear to multilinear and curved shaped) using ternary eluents in which a mixture of methanol and water (1:1, v/v) was the strong eluting member and acetonitrile was the weak solvent. Starting from at least two gradient runs, the prediction of solute retention obtained under all the rest gradients was excellent, even when curved gradient profiles were used. Development of such methodologies can be of great interest for a wide range of applications.     

Chromatographic behavior of epirubicin and its analogues on high-purity silica in hydrophilic interaction chromatography

Hydrophilic interaction chromatography has been applied for the separation of epirubicin and its analogues using high-purity silica column with aqueous-organic mobile phase. Parameters affecting the chromatographic behavior of the solutes such as organic modifier, buffer pH, ionic strength and sample size, have been investigated. Of utmost importance for successful separation of these analogues is the choice of organic modifier, since it impacts both the solvent selectivity and the ionization of silica silanols as well as buffer solution, and consequently the retention behavior of solutes. Acetonitrile was shown to offer superior separation of these analogues to methanol, isopropanol or tetrahydrofuran. Results of the effects of organic modifier, buffer pH and ion strength indicate that the retention mechanism is a mixed-mode of adsorption and ion exchange. In addition, an irreversible adsorption of these compounds was found on silica in the weakly acidic or neutral mobile phases, and the effect of various factors on irreversible adsorption was also preliminarily discussed. More significantly, these basic compounds have exhibited peaks with a slanted front and a sharp tail, a typical overloading peak profile belonging to the behavior of competitive anti-Langmuir isotherm by increasing the sample size at the experimental conditions.     

Adsorption equilibria of proline in hydrophilic interaction chromatography

The adsorption behavior of proline under hydrophilic interaction chromatography conditions was investigated from six aqueous solutions of acetonitrile. Proline adsorption isotherms were recorded at each mobile phase composition by frontal analysis and inverse method. The BET model was found to be the best choice to describe the nonlinear behavior of proline adsorption under hydrophilic interaction chromatography conditions. The adsorption isotherm parameters were derived from two independent parameter estimation methods. The parameters derived from regression analysis of the frontal analysis data and from overloaded elution bands were found to be in good agreement with the excess isotherm of water. The mobile phase composition at which the maximum excess adsorption of water was observed corresponded to the maximum saturation capacity measured for proline.     

Retention behavior of peptides in hydrophilic-interaction chromatography

Selected hydrophilic interaction chromatography (HILIC) columns packed with bare silica, bridge-ethyl hybrid silica, or an amide sorbent chemistry were utilized for an investigation of chromatographic behavior and separation selectivity of tryptic peptides. Retention model was proposed allowing for retention prediction of peptides with correlation coefficient R(2)~0.92-0.97 for various columns. The values of optimized amino acid retention coefficients were compared to those obtained for reversed-phase liquid chromatography (Gilar et al., Anal. Chem. 2010, 82, 265-275) and used to elucidate the impact of different amino acid on peptide HILIC retention. In contrast to reversed-phase chromatography, where presence of Phe, Trp, Ile, and Leu amino acid residues in sequence strongly promoted, and presence of hydrophilic His, Lys and Arg residues strongly reduced peptide retention, the effects of these amino acid residues in HILIC were opposite (His, Lys and Arg promote, Phe, Trp, Ile and Leu demote peptide retention in HILIC). Retention coefficient optimized for pH experiments illustrated the impact of silanols on HILIC retention.     

Antioxidant oligomeric proanthocyanidins from Cistus salvifolius

The purified proanthocyanidin oligomers of Cistus salvifolius herb extract accounted for 78% of the total proanthocyanidins and 73% of the total antioxidant activity of this extract. To elucidate the structure of the oligomer, it was depolymerized by acid catalysis in the presence of phloroglucinol. The structures of the resulting flavan-3-ols and phloroglucinol adducts were determined on the basis of 1D- and reverse 2D-NMR (HSQC, HMBC) experiments of their peracetylated derivatives, MALDI-TOF-MS and CD spectroscopy. These observations resulting from the degradation with phloroglucinol were confirmed by 13C NMR spectroscopy of the oligomer. The mean molecular weight of the higher oligomeric fraction was estimated to be 5-6 flavan-3-ol-units.     

他汀类药物在微乳液相色谱体系中的保留行为

以普伐他汀钠、阿托伐他汀钙、辛伐他汀和洛伐他汀为研究对象,考察了微乳液流动相中表面活性剂的浓度、油相浓度、助表面活性剂浓度以及流动相的pH值等对他汀类药物在微乳液相色谱体系中保留行为的影响。实验结果表明,微乳流动相中表面活性剂、助表面活性剂、亲脂性溶剂的浓度对他汀类药物保留行为的影响与理论模型一致;流动相的pH值对酸性他汀类药物保留行为的影响与理论模型基本一致,对中性他汀类药物保留行为的影响存在着隐函数关系。所建立的保留模型能较好地反映微乳液组成对他汀类药物保留行为的影响。     

Organic monoliths for hydrophilic interaction electrochromatography/chromatography and immunoaffinity chromatography

This article is aimed at providing a review of the progress made over the past decade in the preparation of polar monoliths for hydrophilic interaction LC (HILIC)/capillary electrochromatography (HI-CEC) and in the design of immuno-monoliths for immunoaffinity chromatography that are based on some of the polar monolith precursors used in HILIC/HI-CEC. In addition, this review article discusses some of the applications of polar monoliths by HILIC and HI-CEC, and the applications of immuno-monoliths. This article is by no means an exhaustive review of the literature; it is rather a survey of the recent progress made in the field with 83 references published in the past decade on the topics of HILIC and immunoaffinity chromatography monoliths.     

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.

Evaluation of hydrophilic interaction chromatography versus reversed-phase chromatography in a plant metabolomics perspective

The metabolomics goal, the unbiased relative quantification of all metabolites in a biological system, still lacks a universal analytical approach. In the LC-MS line of approach, one of the major problems encountered is the polar nature of a large group of (plant) metabolites. Here, we investigate the potential of hydrophilic interaction chromatography (HILIC) and compare its qualities with extended polarity RP chromatography. Two opposite LC phase compositions (Atlantis dC18 vs. TSKgel Amide-80) are compared in a plant metabolomics setting. Both performed equally well with regard to retentive capacities, but variation in peak area was about 5% higher for the HILIC approach. Focussing on matrix effects (ME) on the other hand, it was observed that this well-known problem in RP LC-MS metabolomics was not reduced on using hydrophilic interaction chromatography.     

基于亲水作用色谱的寡糖色谱分离新进展

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,可以作为反相液相色谱的重要补充,解决极性化合物的分离问题。近几年,HILIC研究发展十分迅速,应用领域也不断拓展,其中寡糖分离是HILIC最典型的应用领域。HILIC适合分离强极性和亲水性样品,是寡糖分离的理想色谱模式。基于HILIC的寡糖分离技术发展迅速,本文以近期发表在主要国际刊物的几个工作为典型案例,介绍HILIC用于寡糖色谱分离的若干进展。