Hydrophilic Interaction Liquid Chromatography Stationary Phases Research Articles

Maltose-functionalized HILIC stationary phase silica gel based on self-assembled oligopeptides and its application for the separation of polar compounds.

In this study, carbonyldiimidazole was used to bond maltose-modified oligopeptides (Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys) to the surface of silica spheres for hydrophilic interaction liquid chromatography (HILIC). Attenuated total reflectance-Fourier transform infrared spectroscopy, elemental analysis, X-ray photoelectron spectroscopy, thermogravimetric analysis, BET technique, and water contact angle measurement results confirmed the successful immobilization of the obtained material. Compared with the conventional method for preparing carbohydrate stationary phases, this method involves simpler steps and less time-consuming processes. The experimental results proved that the retention mechanism of the maltose-based HILIC column matched the typical HILIC retention mechanism. The column showed high separation efficiency and stability toward the separation of polar compounds such as amino acids, bases, nucleosides, water-soluble vitamins, and salicylic acid and its analogs. The column achieved high selectivity toward oligosaccharide separation. In addition, this efficient analysis demonstrates the applicability of the as-prepared material in the field of food inspection.

A simple generic method for analyzing water sensitive pinacol boronate compounds by hydrophilic interaction liquid chromatography

Pinacol boronates are widely used in the pharmaceutical and biomedical industries as primary building blocks for many chemical reactions, such as Suzuki coupling. However, pinacol boronate esters are often susceptible to hydrolysis and degrade into corresponding boronic acids. This causes a challenge for analyzing these boronates by reversed-phase HPLC (RPLC), where hydrolysis occurs on the column. On the other hand, boronic acids are polar compounds and difficult to be retained in RPLC. Presently, there is a lack of practical and versatile analytical methods that can accurately and simultaneously analyze a wide range of boronate esters and boronic acids. In this work, a simple generic method was developed using hydrophilic interaction liquid chromatography (HILIC) separation to overcome these challenges. More than 40 pinacol boronate compounds in a broad range of diverse structures were screened using ten different HILIC stationary phases. Among all the stationary phases studied, ZIC-cHILIC column demonstrated excellent selectivity and retention of pinacol boronate esters and boronic acids for all of the compounds studied. The high percentage of organic solvent mobile phase in HILIC mode effectively prevents the on-column hydrolysis and provides excellent solubility for boronate ester samples. The compatibility of HILIC mobile phase allows the peaks to be identified directly by mass spectrometry.

Volume and composition of semi-adsorbed stationary phases in hydrophilic interaction liquid chromatography. Comparison of water adsorption in common stationary phases and eluents

Pycnometric and homologous series retention methods are used to determine the volume and mean composition of the water-rich layers partially adsorbed on the surface of several hydrophilic interaction liquid chromatography (HILIC) column fillings with acetonitrile-water and methanol-water as eluents. The findings obtained in this work confirm earlier studies using direct methods for measuring the stationary phase water content performed by Jandera's and Irgum's research groups. Water is preferentially adsorbed on the surface of the HILIC bonded phase in hydroorganic eluents containing more than 40% acetonitrile or 70% methanol, and a gradient of several water-rich transition layers between the polar bonded phase and the poorly polar bulk mobile phase is formed. These layers of reduced mobility act as HILIC stationary phases, retaining polar solutes. The volume of these layers and concentration of adsorbed water is much larger for acetonitrile-water than for methanol-water mobile phases.In hydroorganic eluents with less than 20-30% acetonitrile or 40% methanol the amount of preferentially adsorbed water is very small, and the observed retention behavior is close to the one in reversed-phase liquid chromatography (RPLC). In eluents with intermediate acetonitrile-water or methanol-water compositions a mixed HILIC-RPLC behavior is presented.Comparison of several HILIC columns shows that the highest water enrichment in the HILIC retention region for acetonitrile-water mobile phases is observed for zwitterionic and aminopropyl bonded phases, followed in minor grade for diol and polyvinyl alcohol functionalizations. Pentafluorophenyl bonded phase, usually considered a HILIC column, does not show significant water adsorption, nor HILIC retention.

A New Definition of the Stationary Phase Volume in Mixed-Mode Chromatographic Columns in Hydrophilic Liquid Chromatography.

Polar columns used in the HILIC (Hydrophilic Interaction Liquid Chromatography) systems take up water from the mixed aqueous–organic mobile phases in excess of the water concentration in the bulk mobile phase. The adsorbed water forms a diffuse layer, which becomes a part of the HILIC stationary phase and plays dominant role in the retention of polar compounds. It is difficult to fix the exact boundary between the diffuse stationary and the bulk mobile phase, hence determining the column hold-up volume is subject to errors. Adopting a convention that presumes that the volume of the adsorbed water can be understood as the column stationary phase volume enables unambiguous determination of the volumes of the stationary and of the mobile phases in the column, which is necessary for obtaining thermodynamically correct chromatographic data in HILIC systems. The volume of the aqueous stationary phase, Vex, can be determined experimentally by frontal analysis combined with Karl Fischer titration method, yielding isotherms of water adsorbed on polar columns, which allow direct prediction of the effects of the composition of aqueous–organic mobile phase on the retention in HILIC systems, and more accurate determination of phase volumes in columns and consistent retention data for any mobile phase composition. The n phase volume ratios of 18 columns calculated according to the new phase convention strongly depend on the type of the polar column. Zwitterionic and TSK gel amide and amine columns show especially strong water adsorption.

Hydrophilic interaction liquid chromatography for removal of pesticide residues in ginseng extracts

亲水作用液相色谱法(HILIC)是一种用于改善强极性物质的保留和分离选择性的方法,广泛应用于药物分析、代谢组学、蛋白质组学等领域。该文利用农药分子与皂苷成分在HILIC上的保留行为差异,开发了一种农药残留脱除方法。以市售高纯人参提取物为例,该文评价了农药分子和人参皂苷在亲水色谱柱上的保留行为,并考察了上样量、淋洗体积、上样体积等因素对农残脱除效果的影响。实验结果证明:7种人参皂苷由于糖链上的羟基与亲水色谱固定相上的羧基形成氢键作用而具有较强保留,而农药分子由于亲水性较差且相对分子质量较小,保留很弱,从而一步实现了7种人参皂苷的富集与14种农残的脱除。在优化所得的最佳脱除工艺条件下,最终制备得到的人参总皂苷样品中,总皂苷的含量由59.87%提高到69.61%;总皂苷的回收率为94.4%;通过气相色谱-三重四极杆质谱(GC-MS/MS)对样品中的农残进行定量检测,发现原人参提取物中14种农残均得到了有效脱除,其中5种含量降至0.05 mg/kg以下,9种完全脱除。本研究是亲水色谱在中药提取物中农残脱除领域的应用,为天然产物的精制提供了一种新的技术手段,该技术对人参提取物中的农残脱除率高、人参总皂苷回收率高且安全、高效、无污染,为高品质人参提取物的研制提供了新的思路。

Performance evaluation of silica microspheres functionalized by different amine-ligands for hydrophilic interaction chromatography

In this work, for the first time five amine-ligands including mono-amine, di-amine, tri-amine, secondary and tertiary amine, were functionalized on mesoporous micro-silicas and developed as stationary phases for hydrophilic interaction liquid chromatography (HILIC). The investigations about the retention mechanisms, effects of different chromatographic conditions and stability were systematically conducted. Three kinds of polar and hydrophilic compounds (saccharides, sulfonamides, nucleosides and nucleobases) were selected as probe molecules to evaluate their separation performances. Among the five stationary phases, only aminopropyl-bonded silica has already gained wide developments and applications. Whereas, there are no related researches about the other four to be utilized as separation media. By a series of chromatographic evaluations, the results revealed the other four mesoporous micro-silica materials functionalized with di-amine, tri-amine, secondary and tertiary amine, had great potential to be explored as novel stationary phases of HILIC. Particularly, the two stationary phases functionalized with di-amine and tri-amine exhibited outstanding separation and retention abilities. This work offered some insights on the understanding of retention in HILIC mode and provided us possibility to explore other amine-based HILIC stationary phases.

Method Optimisation in Hydrophilic-Interaction Liquid Chromatography by Design of Experiments Combined with Quantitative Structure–Retention Relationships

Accurate prediction of the separation conditions for a set of target analytes with no retention data available is fundamental for routine analytical assays but remains a very challenging task. In this paper, a quality by design (QbD) optimisation workflow capable of discovering the optimal chromatographic conditions for separation of new compounds in hydrophilic-interaction liquid chromatography (HILIC) is introduced. This workflow features the application of quantitative structure−retention relationship (QSRR) methodology in conjunction with design of experiments (DoE) principles and was used to carry out a two-level full factorial DoE optimisation for a mixture of pharmaceutical analytes on zwitterionic, amide, amine, and bare silica HILIC stationary phases, with mobile phases containing varying acetonitrile content, mobile phase pH, and salt concentration. A dual-filtering approach that considers both retention time (tR) and structural similarity was used to identify the optimal set of analytes to train the QSRR in order to maximise prediction accuracy. Highly predictive retention models (average R2 of 0.98) were obtained and statistical analysis of the prediction performance of the QSRR models demonstrated their ability to predict the retention times of new compounds based solely on their molecular structures, with root-mean-square errors of prediction in the range 7.6–11.0 %. Further, the obtained retention data for pharmaceutical test compounds were used to compute their separation selectivity, which was used as input into a DoE optimiser in order to select the optimal separation conditions. Experimental separations performed under the chosen optimal working conditions showed good agreement with the theoretical predictions. To the best of our knowledge, this is the first study of a QbD optimisation workflow assisted with dual-filtering-based retention modelling to facilitate the method development process in HILIC.

HILIC-MRM-MS for Linkage-Specific Separation of Sialylated Glycopeptides to Quantify Prostate-Specific Antigen Proteoforms.

Elevated serum prostate-specific antigen (PSA) levels in body fluids may indicate prostate cancer (PCa), but it is noted that the clinical performance is rather poor. Specificity and sensitivity values of 20 and 94% at a cutoff value of 4.1 ng/mL, respectively, result in overdiagnosis and unnecessary interventions. Previous exploratory studies have indicated that the glycosylation of PSA potentially leads to improved PCa diagnosis based on qualitative analyses. However, the applied methods are not suited for a quantitative evaluation or implementation in a medical laboratory. Therefore, in this proof-of-principle study, we have evaluated the use of hydrophilic interaction liquid chromatography (HILIC) in combination with targeted quantitative mass spectrometry for the sialic acid linkage-specific analysis of PSA glyco-proteoforms based on either trypsin or ArgC peptides. The efficiency of PSA proteolysis was optimized as well as the glycopeptide separation conditions (buffer type, strength, and pH). The HILIC-based analysis of PSA glyco-proteoforms presented here has the potential for the clinical validation of patient cohorts. The method shows the feasibility of the use of a HILIC stationary phase for the separation of isomeric glycopeptides to detect specific glyco-proteoforms. This is the first step toward the development and evaluation of PSA glyco-proteoforms for use in a clinical chemistry setting aiming for improved PCa diagnosis or screening.

Chromatographic HILIC indexes to characterize the lipophilicity of zwitterions

Reverse phase high pressure liquid chromatography (RP-HPLC) is widely employed in drug discovery for lipophilicity measurements. Hydrophilic interaction liquid chromatography (HILIC) may represent a good alternative to RP-HPLC in the determination of the lipophilicity of hydrophilic compounds like zwitterions. In this paper three different HILIC stationary phases (ZIC®-HILIC, ZIC®-pHILIC and ZIC®-cHILIC) and two different mobile phases (80%ACN/20%buffer and 90%ACN/10%buffer) were combined to set-up six chromatographic systems. A computational tool named Block Relevance (BR) analysis was firstly used to deconvolute the balance of intermolecular forces governing retention in the six systems. Then the lipophilicity profiles (log k vs pH) of ten model ampholytes were determined. Results support that the lipophilicity of zwitterions at any pH can be successfully determined with a ZIC®-cHILIC stationary phase and an 80%ACN/20%buffer mobile phase. To extend the dataset and confirm results, a second series of zwitterionic drugs was also analyzed.

Accurate modelling of the retention behaviour of peptides in gradient-elution hydrophilic interaction liquid chromatography

The applicability of models to describe peptide retention in hydrophilic interaction liquid chromatography (HILIC) was investigated. A tryptic digest of bovine-serum-albumin (BSA) was used as a test sample. Several different models were considered, including adsorption, mixed-mode, exponential, quadratic and Neue–Kuss models. Gradient separations were performed on three different HILIC stationary-phases under three different mobile-phase conditions to obtain model parameters. Methods to track peaks for specific peptides across different chromatograms are shown to be essential. The optimal mobile-phase additive for the separation of BSA digest on each of the three columns was selected by considering the retention window, peak width and peak intensity with mass-spectrometric detection. The performance of the models was investigated using the Akaike information criterion (AIC) to measure the goodness-of-fit and evaluated using prediction errors. The F-test for regression was applied to support model selection. RPLC separations of the same sample were used to test the models. The adsorption model showed the best performance for all the HILIC columns investigated and the lowest prediction errors for two of the three columns. In most cases prediction errors were within 1%.

Comparison of underivatized silica and zwitterionic sulfobetaine hydrophilic interaction liquid chromatography stationary phases for global metabolomics of human plasma

To increase metabolome coverage in global LC–MS metabolomics, often both reversed-phase liquid chromatography (RPLC) and hydrophilic-interaction liquid chromatography (HILIC) are implemented in parallel. However, there is a lack of consensus in the literature on the best HILIC stationary phase to employ for global metabolomics of human biological fluids. The objective of this study was to compare in detail the performance of two commonly employed HILIC phases: zwitterionic sulfobetaine ZIC-HILIC stationary phase and an underivatized silica HILIC stationary phase. During method development, the effect of salt concentration in the mobile phase was also investigated, and 5 mM ammonium acetate was selected. The stationary phases were evaluated using a mixture of 37 polar standards covering a range of logP values (-10 to 3.73), molecular weights (59–776 Da), charges (15 anions, 11 cations, and 11 neutral) as well as 17 lipid standards to understand phospholipid behaviour on the two stationary phases. The criteria used for the comparison included the quality of the chromatographic peak shape, adequate analyte retention, peak separation capability, and metabolite coverage. The zwitterionic ZIC-HILIC column provided better chromatographic performance over the silica stationary phase with 14 standards achieving good quality peaks compared to the 7 with the silica column. Only 2 standards were undetected with the ZIC-HILIC column compared to the 14 undetected with the silica column. In human plasma, 1966 and 1650 metabolites were observed on the ZIC-HILIC column in positive and negative electrospray ionization (ESI) respectively. On the silica HILIC column, 1773 and 2028 metabolites were observed in positive and negative ESI respectively, showing comparable performance of the two phases. Next, the effect of adding 10 mM ammonium phosphate to the samples to improve the analyte peak shape and metabolite coverage was investigated for both ZIC-HILIC and silica HILIC. In contrast with recently reported results for pZIC-HILIC, there was no clear evidence that ammonium phosphate addition was beneficial for human plasma samples. In conclusion, ZIC-HILIC provided better chromatographic performance for polar plasma metabolomics than underivatized silica in terms of chromatographic peak shape and chromatographic resolution, while maintaining comparable metabolite coverage. The addition of ammonium phosphate to human plasma was not beneficial for either of the two stationary phases.

First profiling in hydrophilic interaction liquid chromatography of intact human chorionic gonadotropin isoforms

The study of glycoproteins is a rapidly growing field, which is not surprising considering that approximately 70% of human proteins are glycosylated and that numerous biological functions are associated to the glycosylation. In this work, our interest focused on the heterodimeric human Chorionic Gonadotropin (hCG) glycoprotein that is the specific hormone of the human pregnancy, consisting of an α and a β subunit, so-called hCGα and hCGβ, respectively. This protein possesses a very high structural heterogeneity, essentially due to the presence of 8 glycosylation sites, but also other types of post-translational modifications. In this study, for the first time, the potential of hydrophilic interaction liquid chromatography (HILIC) was investigated to separate the intact hCG isoforms. Three different HILIC stationary phases were tested using an hCG-based drug as standard, a recombinant hCG. For each stationary phase, the effect of the initial mobile phase composition based on ACN/H2O mixture, the slope of the gradient, the content and nature of the acidic additive (formic acid and trifluoroacetic acid (TFA)), and the addition of a volatile salt (ammonium formate) on the retention and the resolution were studied. The best HILIC separation was obtained with the amide column and a mobile phase composed of water/ACN containing 0.1% of TFA. The repeatability in terms of retention times and peak areas was then assessed. Finally, the method was applied to the analysis of a second hCG-based drug obtained from urine of pregnant women. Both drugs gave chromatograms with more than 10 peaks. However, they were significantly different, which demonstrated the potential of HILIC method for hCG isoform fingerprinting.

Hydrophilic Interaction Chromatography

Hydrophilic interaction liquid chromatography (HILIC), which includes the distribution, adsorption, ion exchange, and exclusion of analytes, has gained active development recently. The review covers the principles of the method, types of stationary and mobile phases, mechanisms of retention of highly polar compounds under HILIC conditions, implementation of 2D chromatography, and also fields of application of the method. Keywords: hydrophilic chromatography, retention mechanism, reverse phase HPLC, normal phase mode, HILIC stationary phases, zwitterionic phases, high polar analytes

Polyethyleneimine-functionalized carbon dots and their precursor co-immobilized on silica for hydrophilic interaction chromatography

A new strategy based on synergistic effect of the carbon dots and their precursor was proposed to enhance the selectivity of hydrophilic interaction liquid chromatography (HILIC). In this work, polyethyleneimine (PEI) and PEI-functionalized carbon dots (PEICDs) mix-grafted silica packing material was prepared to act as a novel HILIC stationary phase. Both inner and outer surface of the porous silica are decorated with the mixture of PEI and carbon dots. This stationary phase, namely Sil-PEI/CDs, demonstrate enhanced retention ability and selectivity toward polar analytes, with which 11 nucleosides and nucleobases and 9 ginsenosides can be nicely separated. The Sil-PEI/CDs (RSD 0.12% – 0.54%) exhibited even better stability than the traditional PEI modified stationary phase (RSD 0.39% – 0.87%) within 40 h of continuously elution. And excellent column efficiency was observed from Sil-PEI/CDs (∼65,000 plates/m for cytidine). The strategy of mixed stabilization revealed a new method to prepare good performanced carbon dots based chromatographic column.

Facile one-pot synthesized hydrothermal carbon from cyclodextrin: A stationary phase for hydrophilic interaction liquid chromatography

A new hydrothermal carbon was prepared by a “one-pot” hydrothermal transformation of β-cyclodextrin on silica microspheres (noted as silica@HTC microspheres), which was featuring as a new stationary phase for the hydrophilic interaction liquid chromatography (HILIC). The synthesized silica@HTC possessed polar groups of hydroxyl, carbonyl and carboxyl groups arising from the carbonization transformation of β-cyclodextrin. As demonstrating great hydrophilicity, the silica@HTC was applied as an excellent HILIC stationary phase for the separation of polar compounds including phenols and the endocrine disrupting chemicals (EDCs). In comparison with commercial available HILIC stationary phases, the separation performance of the silica@HTC stationary phase was superior in the separation of phenols and EDCs. The mild hydrothermal carbon transformation of the β-cyclodextrin on the silica microspheres in the “one-pot” manner would represent a new and simple approach to prepare a new class of saccharide-derived stationary phases by using saccharides as precursors.

Simultaneous determination of aminoglycosides and colistins in food

A novel method for the simultaneous identification and quantification of twelve aminoglycosides (AGs) and two colistins in meat and bovine milk has been developed. The analysis was carried out using liquid chromatography coupled to quadrupole-Orbitrap mass spectrometry (LC-Q-Orbitrap). Among the HILIC (Hydrophilic Interaction Liquid Chromatography) stationary phases tested, the bare silica Poroshell 120 provided the best results. The samples were extracted with an aqueous solution followed by an SPE clean up based on the weak cation exchange mechanism. The validation study was performed carrying out 72 experiments per matrix at six different concentrations in a range encompassing the Maximum Residue Limits. The recoveries were from 72 to 87% in meat (except colistins) and from 82 to 96% in milk. Repeatabilities and intra-lab reproducibilities were lower than 10 and 15%, respectively. Limits of detection were lower than or equal to 33 µg kg−1. Finally, test materials containing AGs prepared for interlaboratory studies were successfully analysed.

Hydrophilic interaction liquid chromatography in the speciation analysis of selenium

The hydrophilic interaction liquid chromatography (HILIC) coupled to mass spectrometry was employed to study retention behavior of selected selenium compounds using two different HILIC stationary phases: silica and zwitterionic. Two organic solvents – acetonitrile and methanol – were compared as a component of mobile phase. Separation parameters such as a content of organic modifier, the eluent pH and inorganic buffer concentration were investigated. Based on all observations, methanol seems to be beneficial for the separation of studied compounds. The optimal HILIC separation method involved silica column and eluent composed of 85% MeOH and CH3COONH4 (8 mM, pH 7) was compared to RP method in terms of time of the single run, the separation efficiency and limit of detection.

Modelling of Hydrophilic Interaction Liquid Chromatography Stationary Phases Using Chemometric Approaches.

Metabolomics is a powerful and widely used approach that aims to screen endogenous small molecules (metabolites) of different families present in biological samples. The large variety of compounds to be determined and their wide diversity of physical and chemical properties have promoted the development of different types of hydrophilic interaction liquid chromatography (HILIC) stationary phases. However, the selection of the most suitable HILIC stationary phase is not straightforward. In this work, four different HILIC stationary phases have been compared to evaluate their potential application for the analysis of a complex mixture of metabolites, a situation similar to that found in non-targeted metabolomics studies. The obtained chromatographic data were analyzed by different chemometric methods to explore the behavior of the considered stationary phases. ANOVA-simultaneous component analysis (ASCA), principal component analysis (PCA) and partial least squares regression (PLS) were used to explore the experimental factors affecting the stationary phase performance, the main similarities and differences among chromatographic conditions used (stationary phase and pH) and the molecular descriptors most useful to understand the behavior of each stationary phase.

Surface-bonded amide-functionalized imidazolium ionic liquid as stationary phase for hydrophilic interaction liquid chromatography

The amide group modified silica materials are popular stationary phases for hydrophilic interaction liquid chromatography (HILIC). Meanwhile, surface-confined imidazolium ionic liquids (ILs) have been proved to be useful HILIC stationary phases and possess many unique properties. In this study, the synthesis of an amide-functionalized imidazolium IL was conducted which was then bonded onto silica surface to obtain a novel imidazolium-embedded amide stationary phase for HILIC. The combination of the amide group and imidazolium IL moiety might bring some advantages in selectivity or retention and therefore extended its applications. After characterizing the prepared IL and the resulting modified silica materials, the chromatographic performance and separation selectivity of the packed column were evaluated and compared with a commercial amide column. Then, the retention behavior was investigated through observing the retention factors at different chromatographic conditions using a wide range of compounds. Exceptionally, the prepared amide IL column exhibited superior separation performance towards complex samples such as flavonoids mixture, soybean flavonoids and human urine. All the results indicated that the novel amide IL column possessed an anion-exchange/HILIC mixed-mode retention mechanism and could be useful in the sample analysis as a promising candidate for HILIC stationary phase.

Evaluation of hydrophilic interaction liquid chromatography stationary phases for analysis of opium alkaloids

The separation of a mixture containing five major opium alkaloids, namely morphine, codeine, thebaine, noscapine and papaverine has been investigated in hydrophilic interaction liquid chromatography (HILIC) mode using five different stationary phases: bare silica, zwitterion, aminopropyl, diol and cyanopropyl. In order to propose the appropriate column for separation and purification, retention behaviors of the five natural opioids have been studied on mentioned HILIC stationary phases. The mechanism of separation in diverse HILIC media, based on the formation of water-rich layer on surface of the HILIC stationary phases and the physicochemical properties of opium alkaloids, such as pKa (acidic pK) and the octanol-water distribution coefficient (log Do/w) are discussed. Chromatographic responses including modified limit of detection LODm, signal to noise ratio (S/N)m, and defined modified RSm have considered for suggestion of the suitable column for quantitative/qualitative and preparative purposes. According to the obtained results, diol stationary phase is best suited for analytical chromatography, whereas bare silica and zwitterionic stationary phases are appropriate for preparative applications.