Hydrophilic Interaction Liquid Chromatography Retention Research Articles

Silica-Based Stationary Phase with Surface Bound N-Acetyl-glucosamine for Hydrophilic Interaction Liquid Chromatography.

A hydrophilic silica-based stationary phase with surface bound N-acetylglucosamine (GlcNAc-silica) was prepared in house and characterized physically via Fourier transform infrared (FTIR) analysis and thermogravimetric analysis (TGA) and chromatographically over a wide range of mobile phase compositions. While both FTIR and TGA confirmed the attachment of the GlcNAc ligands to the silica surface, the chromatographic evaluation of GlcNAc-silica with polar and slightly polar standard solutes (e.g., sugars, nucleic acid fragments, phenolic, and benzoic acid derivatives) yielded the typical hydrophilic interaction liquid chromatography (HILIC) behaviors in the sense that retention increased with increases in solute polarity and the organic content (i.e., acetonitrile) of the hydro-organic mobile phase (i.e., ACN-rich mobile phase). Sugars derivatized with 1-naphthylamine (1-NA) and 2-aminoanthrcene (2-AA) such as xylose, glucose, and short chains maltooligosaccharides constituted the most polar species for HILIC retention evaluation, and in addition, the maltooligosaccharides offered a polar homologous series for gauging the hydrophilicity of GlcNAc-silica in analogy with alkylbenzene homologous series and other nonpolar homologues for evaluating the hydrophobicity of non-polar stationary phases. On the other hand, the benzoic acid and phenolic acid derivatives were the probe solutes for evaluating the HILIC retention dependence of ionizable solutes on the pH of the mobile phase. Similarly, the nucleobase and nucleoside weak basic solutes as well as some typical cyclic nucleotide acidic solutes allowed for the examination of the dependence of solute retention on the pH of the mobile as well as the polarity of the species.

Exploration of the effects of chloride ions on the analysis of polar compounds at low concentrations by hydrophilic interaction liquid chromatography coupled to a charged aerosol detector: Application to tromethamine.

In this study, we discuss the origin of the slightly increased response of the charged aerosol detector when low-concentration polar drugs formulated with sodium chloride are analyzed by hydrophilic interaction liquid chromatography coupled to the charged aerosol detector. In the case of tromethamine mixed with saline solutions, we investigated several levels including the mobile phase, sample matrix, and detection. We show that the analysis of the rich-salted sample results in both interactions with the mobile phase modifiers and the stationary phase during the run time. With 150mM NaCl as a compounding solution, a slight increase in the tromethamine peak area was observed(<5.5%). Our study suggests that chloride ions in excess sequentially interact firstly with the counterions from the organic modifiers and secondly with the analyte via the stationary phase and the contribution of hydrophilic interaction liquid chromatography retention mechanisms. Because of these effects, the hydrophilic interaction liquid chromatography-charged aerosol detector analysis of drugs in saline solutions requires particular attention, and a correction factor for quantitative purposes that accounts for formulation ions remains appropriate.

Separation of minor steviol glycosides using hydrophilic interaction liquid chromatography (HILIC) and off-line two-dimensional reversed-phase liquid chromatography/HILIC methods

Steviol glycosides (SGs) are increasingly used as noncaloric sweeteners. In this study, hydrophilic interaction liquid chromatography (HILIC) methods and an off-line environmentally friendly two-dimensional reversed-phase liquid chromatography/HILIC (2D-RPLC/HILIC) for the separation of SGs were developed. The retention behavior on the polyacrylamide-based silica (PA) column was investigated by separating twelve SGs and comparing with three HILIC columns (Diol, XAmide and Unitary NH2 columns). Ethanol instead of acetonitrile as the weak eluent in HILIC mode was investigated. The PA column exhibited superior hydrophilicity, higher selectivity and better resolution. Two HILIC-UV methods for separation of SGs on a PA column with water/acetonitrile (LOD, 0.0049–0.013 mg/mL; RSD <3.73%; recovery, 95.02–102.5%) or water/ethanol (LOD, 0.010–0.016 mg/mL; RSD <4.52%; recovery, 91.90–97.76%) as elution system were developed. The preparative separation of minor SGs from stevia extracts was investigated by 2D-RPLC/HILIC using a preparative C18 column with the methanol/water mobile phase in the first dimension and a preparative PA column with the ethanol/water mobile phase in the second dimension. Two SGs were isolated and identified. In summary, safer and cheaper ethanol/water binary eluent system increased the diversity of HILIC retention and improved the solubility of SGs, which is greatly beneficial at a preparative scale in the food industry.

Solute–Solvent Interactions in Hydrophilic Interaction Liquid Chromatography: Characterization of the Retention in a Silica Column by the Abraham Linear Free Energy Relationship Model

The Abraham linear free energy relationship model has been used to characterize a hydrophilic interaction liquid chromatography (HILIC) silica column with acetonitrile/water and methanol/water mobile phases. Analysis by the model for acetonitrile/water mobile phases points to solute volume and hydrogen bond basicity as the main properties affecting retention, whereas solute hydrogen bond acidity, dipolarity and polarizability practically do not affect it. Formation of a cavity is easier in acetonitrile-rich mobile phases than in the aqueous stationary phase, and hence increase of solute volume decreases retention. Conversely, hydrogen bond acidity is stronger in the aqueous stationary phase than in the acetonitrile-rich mobile phase and thus an increase of solute hydrogen bond basicity increases retention. Results are similar for methanol/water mobile phases with the difference that solute hydrogen bond acidity is significant too. Increase in hydrogen bond acidity of the solute decreases retention showing that methanol mobile phases must be better hydrogen bond acceptors than acetonitrile ones, and even than water-rich stationary phases. The results are like the ones obtained in zwitterionic HILIC columns bonded to silica or polymer supports for acetonitrile/water mobile phases, but different for solute hydrogen bond acidity for a polymer bonded zwitterionic column with methanol/water mobile phases, indicating that bonding support plays an important role in HILIC retention. Comparison to RPLC characterized systems confirms the complementarity of HILIC systems to RPLC ones because the main properties affecting retention are the same but with reversed coefficients. The least retained solutes in RPLC are the most retained in HILIC.

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.

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.

Surface-confined guanidinium ionic liquid as a new type of stationary phase for hydrophilic interaction liquid chromatography.

Guanidinium-based ionic liquids possess lower toxicity and greater designability than commonly used species and have presented good performances in liquid-phase extraction and stationary phase for capillary gas chromatography. In the present work, a novel type of surface-confined guanidinium ionic liquid stationary phase was developed by bonding a hexaalkylguanidinium ionic liquid N,N,N',N'-tetramethyl-N",N"-diallylguanidinium bromide onto the surface of 3-mercaptopropyl modified silica. The obtained surface-confined guanidinium ionic liquid silica materials were characterized by elemental analysis, infrared spectroscopy and thermogravimetric analysis, and then packed as a high-performance liquid chromatography column for the evaluation of chromatographic retention behavior. Typical polar compounds were used to evaluate the separation performances, and the changes of retention with water content in mobile phase further suggested the hydrophilic interaction liquid chromatography retention mechanism. Moreover, the effect of different chromatographic factors (salt concentration, mobile phase pH, and column temperature) on retention was investigated with a series of compounds as test solutes to gain insights into the retention mechanism. The results indicated that the surface-confined guanidinium ionic liquid stationary phase exhibited a hydrophilic interaction liquid chromatography/anion-exchange mixed-mode retention behavior and possessed promising potential in separating a wide range of compounds as an alternative stationary phase for high-performance liquid chromatography.

Separation of multiphosphorylated cyclopeptides and their positional isomers by hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI-MS)

Peptides are efficient models used in different fields such as toxicology to study the interactions of several contaminants at the molecular scale, requiring the development of bio-analytical strategies. In this context, Hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI-MS) was used to separate synthetic multiphosphorylated cyclopeptides and their positional isomers at physiological pH. We assessed (i) the selectivity of eleven HILIC columns, from different manufacturers and packed with diverse polar sorbents, and (ii) the effect of mobile phase composition on the separation selectivity. The best selectivity and baseline resolution were achieved with the columns grafted by neutral sorbents amide and diol. Furthermore, we investigated the HILIC retention mechanism of these peptides by examining the effect of the number of phosphorylated residues in the peptide scaffold on their retention. The peptide behavior followed the classical hydrophilic partitioning mechanism exclusively on amide and diol columns. This trend was not fully respected on bare and hybrid silica due to the attractive/repulsive interactions of the deprotonated surface silanol groups with the Arginine or Glutamate residues in the peptide scaffold according to the peptide sequence. The position of the phosphorylated amino acid in the peptide backbone also showed to have an impact on the retention, making possible the separation of positional isomers of these multiphosphorylated cyclic peptides using HILIC.

One-pot hydrothermal cross-linking preparation of poly(vinylpyrrolidone) immobilized silica stationary phase for hydrophilic interaction chromatography

Hydrothermally cross-linked polyvinylpyrrolidone (PVP) immobilized SiO2 stationary phase (CPVP-Sil) was prepared via a green and facile one-pot method which was demonstrated for hydrophilic interaction liquid chromatography (HILIC) as well as reverse phase chromatography(RP). A water or organic solvent-insoluble permanent CPVP immobilizing on the silica particle surface can be formed simply by dipping silica particles into PVP solution and low temperature hydrothermal treatment. The cross-linked PVP network coating on SiO2 endow it ring lactam functional groups which exhibited excellent separation ability of polar compounds by a typical HILIC retention mechanism at higher organic solvent contents (>55% ACN) and additionally polyvinyl groups for separation of alkylbenzenes in RP mode(<25% ACN). A high column efficiency of about 7 × 104 plates per meter was obtained for the test catechol compound. Remarkably, the CPVP-Sil packing materials showed good stability in acid (at pH 3.5) or basic (at pH 9.5) conditions, with 5400-fold column volumes and 3500-fold column volumes respectively.

Insight into the hydrophilic interaction liquid chromatographic retention behaviors of hydrophilic compounds on different stationary phases

In this work, the correlations between retention behavior and lipophilicity of a large set of hydrophilic neutral and ionic analytes were studied based on three hydrophilic interaction liquid chromatography (HILIC) stationary phases, including zwitterionic, crosslinked diol and triazole stationary phases. It was found that HILIC, due to the diversity of retention mechanism, is a more complex chromatography separation mode than reversed-phase liquid chromatography (RPLC) which has been widely accepted for lipophilicity assessment. Because electrostatic interactions contributed to the overall retention of the charged solutes on all three stationary phases, ion-strength of the mobile phase kept the same during the whole experiment. After the correlations between retention factor log k and water volume fraction Φ were investigated, the mixed retention model was revealed to be more suitable for HILIC retention behavior than other single models including partitioning and adsorption model. Moreover, in order to bridge the relationship between HILIC log k and lipophilicity parameter log D, net charge ne and Abraham solvation parameter were introduced in the quantitative structure-retention relationship (QSRR) model. Although the correlation coefficients between log D and log k were still moderate, the significant improvement in correlation has made HILIC a potential choice as the complement of RPLC for log D measurement.

Retention of stevioside polar compounds on a sulfonic acid-functionalized stationary phase

To explore the retention and separation of stevioside polar compounds on a sulfonic acid-functionalized cation exchange column, the effects of different organic solvent-water mobile phases on the retention behavior of polar rebaudioside A (RA) and its analogues on the column were investigated over a wide range of organic solvent contents. The obtained U-shape curves hinted that the retention of the compounds on the same column transitioned from a reversed-phase liquid chromatography (RPLC) mode to a hydrophilic interaction liquid chromatography (HILIC) mode when the water-rich state in the mobile phases changed to an organic solvent-rich state. Under the RPLC mode, no separation of RA from its analogues was observed. The HILIC mode was beneficial to the retention and separation of RA and its analogues. Compared with polar protic solvents, aprotic solvents were more conducive to the retention and separation of the polar compounds based on the HILIC mode in organic solvent-rich mobile phases. Three models were used to evaluate and discuss the HILIC retention and separation of the compounds on the column. In the aprotic solvent-rich mobile phase, the HILIC retention of RA and its analogues was effectively described by a mixed-mode model; in the polar proton solvent-rich mobile phase, the retention of analytes was best described by an linear solvation strength (LSS) model. The content and composition of the organic solvent in the mobile phase were determined to be important influencing factors that regulated the retention time for the RA and its analogues, and even the separation mechanism for HILIC. The present work provides a theoretical basis for guiding one to prepare high-purity RA from its analogues by predicting the retention time.

Characterization of hydrophilic interaction liquid chromatography retention by a linear free energy relationship. Comparison to reversed- and normal-phase retentions

The Abraham solvation parameter model, a linear free energy relationship (LFER) approach, has been used to characterize a polymeric zwitterionic (sulfobetaine) column in HILIC mode. When acetonitrile (MeCN) is used in the preparation of mobile phases the main solute characteristics affecting the chromatographic behavior of analytes are the molecular size and the hydrogen-bonding (both acidity and basicity) interactions. The former property is more favorable in the acetonitrile-rich mobile phase, reducing thus the retention, but the latter reveals a higher affinity for the water layer adsorbed on the stationary phase, enhancing retention. However, if the aprotic acetonitrile is replaced by methanol, a hydrogen-bond acidic solvent, solute hydrogen-bond basicity does not contribute any more to retention, quite the opposite. Thus, a slightly different selectivity is observed in methanol/water than in acetonitrile/water. Normal-phase mode and HILIC-MeCN share the same main factors affecting retention. For reversed-phase and immobilized artificial membrane (IAM) chromatography, the solute molecular size increase retention because of the lower amount of energy required in the formation of a cavity in the solvated stationary phase. On the contrary, the analyte hydrogen-bond basicity favors interactions with the hydroorganic mobile phase and reduces retention. The determined parameters justify the reversed selectivity commonly observed in HILIC in reference to reversed-phase. In most instances, the least retained solutes in reversed-phase are the most retained in HILIC.

Adduct formation in electrospray ionisation-mass spectrometry with hydrophilic interaction liquid chromatography is strongly affected by the inorganic ion concentration of the samples

Hydrophilic interaction liquid chromatography (HILIC)/ electrospray ionisation-mass spectrometry (ESI-MS) has gained interest for the analysis of polar analytes in bioanalytical applications in recent years. However, ESI-MS is prone to adduct formation of analytes. In contrast to reversed phase chromatography, small inorganic ions have retention in HILIC, i.e. analytes and inorganic ions may co-elute, which could influence the adduct formation. In the present paper, it was demonstrated that the co-elution of sodium ions or potassium ions and analytes in HILIC/ESI-MS affect the adduct formation and that different concentrations of sodium ions and potassium ions in biological samples could have an impact on the quantitative response of the respective adducts as well as the quantitative response of the protonated adduct. The co-elution also lead to cluster formation of analytes and sodium formate or potassium formate, causing extremely complicated spectra. In analytical applications using HILIC/ESI-MS where internal standards are rarely used or not properly matched, great care needs to be taken to ensure minimal variation of inorganic ion concentration between samples. Moreover, the use of alkali metal ion adducts as quantitative target ions in relative quantitative applications should be made with caution if proper internal standards are not used.

Predicting the HILIC Retention Behavior of the N-Linked Glycopeptides Produced by Trypsin Digestion of Immunoglobulin Gs (IgGs).

The prediction of the retention behavior/time would facilitate the identification and characterization of glycoproteins, particularly the analytical challenges, such as the characterization of low-abundance glycoforms. This task is essential in the biotherapeutics industry, where the type and amount of glycosylation on recombinant IgG alter the efficacy, function, and immunogenicity. Models exist for the prediction of the hydrophilic interaction liquid chromatography retention of peptides and glycans. Here, we have devised a unified model to predict the retention behavior of glycopeptides from human IgGs and applied this to the analysis of glycopeptides from rabbit IgGs. The combined model is capable of accurately predicting the retention of native IgG glycopeptides on 2 completely different liquid chromatography-mass spectrometry systems.

Investigation and prediction of retention characteristics of imidazoline and serotonin receptor ligands and their related compounds on mixed-mode stationary phase

Investigation of the retention behavior of a wide range of analytes, 43 nitrogen containing heterocyclic and guanidine derivatives such, as imidazoline and serotonin receptor ligands or their related compounds, was performed on mixed-mode stationary phase in the combined reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) modes.Suitability of the linear retention modelling in the HILIC and RP modes was tested including separate contributions from adsorption and partition. For the HILIC retention, the partition model was found to provide better description compared with the adsorption model. In a wider range of the aqueous eluent volume fractions, φ(aq), retention was described as a function of volume fractions and total polarity of mobile phase using the mixed-mode retention modelling. The obtained results revealed that the shift of the chromatographic mode can be calculated from the change of total polarity of mobile phase in a multi-modal relation, logarithm of retention factor vs. total polarity, with the minimum value representing the turning point between the HILIC and the RP mode.Molecular properties of the investigated compounds that influence the retention behavior and the turning point were selected using Multiple Linear Regression (MLR) and Support Vector Machine (SVM). Slightly better statistical results were found for the logkwRP(aq)/MLR, logkwHILIC(org)/MLR, logkbHILIC(aq)/MLR, and φmin (aq)/SVM (RBF) QSRR models than for the logkwRP(aq)/SVM, logkwHILIC(org)/SVM, logkbHILIC(aq)/SVM, and φmin(aq)/MLR modelling. With this insight, it is possible to precisely define and predict the retention characteristics based on physico-chemical properties of imidazoline and piperazine related compounds.

High performance liquid chromatography as a molecular probe in quantitative structure-retention relationships studies of selected lipid classes on polar-embedded stationary phases

Studies on the retention mechanism of lipid classes (phospholipids, sphingomyelin) were performed using three polar-embedded stationary phases for which diol, phosphate, amino, and amide moieties were incorporated into the alkyl chains of the stationary phases. Their structural descriptors were determined using the quantum-mechanical method. The retention behavior of the analytes was investigated as a function of different binary hydro-organic mobile phases containing (90%/10% acetonitrile (or methanol)/0.1% formic acid). It was found that the elution order on the tested stationary phases was governed chiefly by the hydrophilicity of the analyte and indicated the existence of a hydrophilic interaction liquid chromatography retention mechanism. Quantitative structure-retention relationships studies were performed to further elucidate the retention mechanism. These studies showed that the dominant analyte descriptor influencing retention on the alkyl-amine stationary phase was the logarithm of the octanol-water partition coefficient. For the phospho-diol and alkyl-amide stationary phases, the dominant analyte descriptor influencing retention was the molar volume and solvent accessible area of the analyte, respectively.

Modeling of HILIC retention behavior with theoretical models and new spline interpolation technique

When it is taken into account that hydrophilic interaction liquid chromatography (HILIC) as an analytical method is relatively young compared with the other techniques, retention modeling could still bring scientifically valuable data to the field. Therefore, in this paper, olanzapine and its 8 impurities were selected as a test mixture, considering that they have never been analyzed in HILIC before. Their investigation on 4 different HILIC columns (bare silica, cyanopropyl, diol and zwitterionic) has been performed. The mixture of 9 structurally similar substances allows the examination of complex HILIC retention behavior depending on the chemical properties of the analytes, as well as of the stationary phase. To describe the nature of the relationship between the retention and the stronger eluent content in the mobile phase, we fitted experimentally obtained data to several theoretical (localized adsorption, nonlocalized partition, quadratic, and mixed) models. Results show that the best fit is the quadratic model with the highest R2 and cross‐validated coefficient of determination (Q2) values, but its usage has some drawbacks. With the aim to improve the possibility to predict retention behavior in HILIC, a new empirical model was proposed. For that purpose, a spline interpolation technique was performed, by dividing the experimental range into several subdivisions. This type of interpolation was performed for the first time in the chromatographic field. The estimation of the polynomial equations was performed using Q2 values. Obtained Q2 values pointed out the goodness of fit of the model, as well as its good predictive capabilities. In the end, the prediction capabilities were experimentally verified, under randomly chosen conditions from the experimental range. The errors in prediction were all under 10%, which is satisfying for HILIC.

A rapid hydrophilic interaction liquid chromatographic determination of glimepiride in pharmaceutical formulations

Glimepiride is one of the most widely prescribed antidiabetic drugs and contains both hydrophobic and hydrophilic functional groups in its molecules, and thus could be analyzed by either reversed-phase high performance liquid chromatography (HPLC) or hydrophilic interaction liquid chromatography (HILIC). In the literature, however, only reversed-phase HPLC has been reported. In this study, a simple, rapid and accurate hydrophilic interaction liquid chromatographic method was developed for the determination of glimepiride in pharmaceutical formulations. The analytical method comprised a fast ultrasound-assisted extraction with acetonitrile as a solvent followed by HILIC separation and quantification using a Waters Spherisorb S5NH2 hydrophilic column with a mobile phase consisting of acetonitrile and aqueous acetate buffer (5.0mM). The retention time of glimepiride increased slightly with decrease of mobile phase pH value from 6.8 to 5.8 and of acetonitrile content from 60% to 40%, indicating that both hydrophilic, ionic, and hydrophobic interactions were involved in the HILIC retention and elution mechanisms. Quantitation was carried out with a mobile phase of 40% acetonitrile and 60% aqueous acetate buffer (5.0mM) at pH 6.3, by relating the peak area of glimepiride to that of the internal standard, with a detection limit of 15.0μg/L. UV light absorption responses at 228nm were linear over a wide concentration range from 50.0μg/L to 6.00mg/L. The recoveries of the standard added to pharmaceutical tablet samples were 99.4–103.0% for glimepiride, and the relative standard deviation for the analyte was less than 1.0%. This method has been successfully applied to determine the glimepiride contents in pharmaceutical formulations.

Evaluation of the dual retention properties of stationary phases based on silica hydride: Perfluorinated bonded material.

The synthesis of a new perfluorinated stationary phase based on silica hydride using a hydrosilation reaction was investigated. The material was characterized by elemental analysis, diffuse reflectance infrared Fourier transform spectroscopy and (13) C cross-polarization magic-angle spinning NMR spectroscopy. The retention properties of this new material were tested in the reversed-phase and normal-phase mode. Variable buffer strength experiments at two pH conditions for selected polar compounds were used to compare the new phase to hydrophilic interaction liquid chromatography retention. These results and previous data reported in the literature were used to postulate differences in the retention mechanism between hydrophilic interaction liquid chromatography and silica hydride-based stationary phases.

Preparation and evaluation of surface-bonded tricationic ionic liquid silica as stationary phases for high-performance liquid chromatography

Two tricationic ionic liquids were prepared and then bonded onto the surface of supporting silica materials through “thiol-ene” click chemistry as new stationary phases for high-performance liquid chromatography. The obtained columns of tricationic ionic liquids were evaluated respectively in the reversed-phase liquid chromatography (RPLC) mode and hydrophilic interaction liquid chromatography (HILIC) mode, and possess ideal column efficiency of 80,000 plates/m in the RPLC mode with naphthalene as the test solute. The tricationic ionic liquid stationary phases exhibit good hydrophobic and shape selectivity to hydrophobic compounds, and RPLC retention behavior with multiple interactions. In the HILIC mode, the retention and selectivity were evaluated through the efficient separation of nucleosides and bases as well as flavonoids, and the typical HILIC retention behavior was demonstrated by investigating retention changes of hydrophilic solutes with water volume fraction in mobile phase. The results show that the tricationic ionic liquid columns possess great prospect for applications in analysis of hydrophobic and hydrophilic samples.