Phase For Hydrophilic Interaction Chromatography Research Articles

A lysine and amide functionalized polymer-based polar stationary phase for hydrophilic interaction chromatography

A novel polymer-based polar stationary phase for hydrophilic interaction chromatography (HILIC) is described. It was obtained by grafting lysine and acrylamide onto poly (glycidyl methacrylate-divinylbenzene) (GMA-DVB) microspheres via ring-opening reaction of epoxy groups and free radical polymerization with pendant double bonds of the microspheres. Multiple types of polar groups including zwitterionic (carboxylate and amine), amide and diol onto the microspheres make them highly hydrophilic. It showed typical HILIC character and good separation performance towards model polar analytes. Negligible bleed level under gradient elution mode (up to 50% fraction of water) was observed. It also exhibited specific separation selectivity to ionic analytes and simultaneous separation of anions and cations could be achieved in ideal electrostatic selectivity elution order, e.g. I−< NO3− <Br− <Cl− or K+< Na+ < Li+.

Tetraethylenepentamine-derived carbon dots and tetraethylenepentamine co-immobilized silica stationary phase for hydrophilic interaction chromatography

In this work, tetraethylenepentamine (TEPA) was used as precursor and reaction medium to prepare tetraethylenepentamine-functionalized carbon dots (TEPACDs), the resultant mixture was subsequently silanized and then grafted on the surface of bare silica. The obtained tetraethylenepentamine-functionalized carbon dots and tetraethylenepentamine co-modified silica stationary phase (Sil-TEPA/CDs) was characterized by multiple ways, such as Fourier transformed infrared spectroscopy (FTIR), elemental analysis and transmission electron microscope, which revealed the successful preparation of the mixed stationary phase and higher density of functional groups on co-modified stationary phase than precursor single-modified stationary phase. The synergistic effect of TEPACDs and TEPA was proved by comparing the separation performance of Sil-TEPA/CDs and Sil-TEPA toward amino acids, nucleosides, and nucleobases, which distinctly enhanced the selectivity of Sil-TEPA/CDs. Thus, 12 nucleosides and nucleobases and 11 amino acids was nicely separated on Sil-TEPA/CDs. By study the influences of the changes of mobile phase composition, mobile phase buffer concentration and buffer pH on the retention behaviors of Sil-TEPA and Sil-TEPA/CDs, it was found that both hydrophilic partitioning and adsorption of analytes on Sil-TEPA/CDs were enhanced benefit from the co-existence of TEPA and TEPACDs, which provided the analytes better separation performance. By comparing the column quality of Sil-TEPA/CDs with four commercially available columns, Sil-TEPA/CDs exhibited the best peak asymmetry of 0.98, and second best column efficiency of 43895 m−1 using guanosine as analyte. The RSD (n = 9) of the retention times of five selected analytes on Sil-TEPA/CDs were within 0.30–0.61% during 40 h of continuously elution, which implied excellent stability of prepared packing material.

Studies on the retention mechanism of solutes in hydrophilic interaction chromatography using stoichiometric displacement theory II. HILIC/RPLC dual-retention mechanism of solutes in hydrophilic interaction chromatography over the entire range of water concentration in mobile phase

This paper is a continuation of research into the retention behavior and mechanism of solutes in hydrophilic interaction chromatography (HILIC) using stoichiometric displacement theory (SDT). A HILIC/reversed-phase liquid chromatography (RPLC) dual-retention mechanism was studied in detail using a β-CD HILIC column. The retention behaviors of three groups of solutes with varying polarities were studied over the entire range of water concentrations in the mobile phase on the β-CD column, resulting in the formation of “U-shape” curves when lgk’ was plotted against lg[H2O]. Additionally, the effect of hydrophobic distribution coefficient lgPO/W on the retention behaviors of solutes in HILIC and RPLC modes was also examined. A four-parameter equation derived from the SDT-R was found to accurately describe the “U-shaped” curves of solutes with RPLC/HILIC dual-retention mechanisms on β-CD column. The theoretical lgk’ values of solutes calculated using the equation were found to be in agreement with their experimental values, with correlation coefficients greater than 0.99. This indicates that the four-parameter equation derived from SDT-R can effectively describe the retention behaviors of solutes over the entire range of water concentrations in the mobile phase in HILIC. As such, SDT can be used as a theoretical guide for the development of HILIC, including the exploration of new dual-function stationary phases to enhance separation efficiency.

A detailed evaluation of the advantages and limitations of online RP-LC×HILIC compared to HILIC×RP-LC for phenolic analysis

The combination of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RP-LC) has proved effective in the LC × LC analysis of polyphenols due to the high degree of orthogonality associated with these separation modes for various classes of phenolic compounds. However, despite the growing number of such applications, HILIC is almost exclusively used as the first dimension (1D) separation mode, and RP-LC in the second dimension (2D). This is somewhat surprising in light of the potential advantages of swapping these separation modes. In this contribution, we present a detailed evaluation of the potential of online RP-LC × HILIC-MS for the analysis of phenolic compounds, comparing the performance of this system to the more established HILIC × RP-LC-MS configuration.Method development was performed using a predictive optimisation program, and fixed solvent modulation was employed to combat the solvent incompatibility between HILIC and RP-LC mobile phases. Red wine, rooibos tea, Protea and chestnut phenolic extracts containing a large diversity of phenolic compound classes were analysed by both HILIC × RP-LC- and RP-LC × HILIC-MS in order to compare the separation performance.Overall, the kinetic performance of HILIC × RP-LC was found to be clearly superior, with higher peak capacities and better resolution obtained for the majority of samples compared to RP-LC × HILIC analyses using similar column dimensions. Dilution of the 1D solvent combined with large volume injections proved insufficient to focus especially phenolic acids in the 2D HILIC separation, which resulted in severe 2D peak distortion for these compounds, and negatively impacted on method performance. On the other hand, a noteworthy improvement in the sensitivity of RP-LC × HILIC-MS analyses was observed due to higher ESI-MS response for the 2D HILIC mobile phase and greater sample loading capacity of the 1D RP-LC column, brought on by the high solubility of phenolic samples in aqueous solutions. As a result, a significantly higher number of compounds were detected in the RP-LC × HILIC-MS separations. These findings point to the potential advantage of RP-LC × HILIC as a complementary configuration to HILIC × RP-LC for phenolic analysis.

A survey of polar stationary phases for hydrophilic interaction chromatography and recent progress in understanding retention and selectivity.

Various polar stationary phases are available for hydrophilic interaction chromatography (HILIC) and help drive continuous applications in biomedical, environmental, and pharmaceutical areas in the past decade. Although the stationary phases for HILIC have been reviewed previously, it is an appropriate time to take another look at the progresses made during the past 5years. The current review provides an overview of the polar stationary phases commercially available for HILIC applications in an effort to assist scientists in selecting suitable columns. New types of stationary phases that were published in literature in the past 5years are summarized and discussed. The trend in stationary phase research and development is also highlighted. Of particular interest is the experimental evidence for direct interactions of polar analytes with the ligands of the stationary phases under HILIC conditions. In addition, two different approaches have been developed to delineate the relative significance of the partitioning and adsorption mechanisms in HILIC, representing an important advancement in our understanding of the retention mechanisms in HILIC.

A hydrolytically stable amide polar stationary phase for hydrophilic interaction chromatography

A novel amide functionalized polar stationary phase has been described by grafting acrylamide polymer coating onto hydrolysed poly (glycidyl methacrylate-divinylbenzene) (GMA-DVB) microsphere. The grafting of acrylamide coating is performed via free radical polymerization of acrylamide with pendant double bonds of hydrolysed GMA-DVB microsphere. The obtained stationary phase (G-pAM) possesses favorable hydrophilicity, as proved by strong retention and good separation ability towards several types of model polar analytes (e.g. 71913 plates/m plate count of sucrose), and excellent hydrolytically stability, as indicated by extremely low bleed level (much superior to commercial ones, ~23.7-fold–~77.4-fold lower). Negligible baseline drift under gradient elution (water fraction even up to 50%) was observed. It also exhibits good selectivity in the separation of isomers and homologue sugars.

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.

Nitrogen-doping to enhance the separation selectivity of glucose-based carbon dots-modified silica stationary phase for hydrophilic interaction chromatography

Doping is often used to alter or improve the properties of nanomaterials. In this work, a novel glucose-based nitrogen-doped carbon dots (Glc-NCDs)-bonded silica stationary phase (Sil-Glc-NCDs) was synthesized and characterized carefully. Glc-NCDs were prepared using glucose and aspartic acid as carbon and nitrogen sources, and then grafted on silica surface via isocyanatopropyl as linker using deep eutectic solvents as reaction medium. The synthesis was confirmed by elemental analysis, Fourier transform infrared spectroscopy and microscopic imaging techniques. And then Sil-Glc-NCDs were packed in the stainless columns for hydrophilic interaction chromatography (HILIC). Due to the synergistic effect between the functional groups of doped carbon dots, Sil-Glc-NCDs column showed enhanced separation selectivity compared with previous non-doped Sil-Glc-CDs column. Sil-Glc-NCDs column can be used for separation of base, nucleosides and antibiotics etc. This column was successfully applied to determine the content of roxithromycin in the capsule, which was found that the concentration was 2.45 mg/mL.

A HILIC stationary phase fuctionalized with glutathione by thiol-ene chemistry on monodisperse-porous polymer microparticles

Monodisperse-porous microparticles functionalized a with a zwitterionic ligand were synthesized as a new stationary phase for hydrophilic interaction chromatography. Monodisperse-porous poly(3-trimethoxysilylpropyl methacrylate-co-ethylene dimethacrylate), poly(TMSPM-co-EDMA) microspheres (6 μm in size) were obtained with different seed latex/monomer ratios and diluent compositions by multistage microsuspension copolymerization. The zwitterionic chromatographic ligand containing thiol moiety (i.e. glutathione) were covalently attached onto the TMSPM attached-poly(TMSPM-co-EDMA) microparticles. The selected starting material allowed the direct attachment of zwitterionic ligand onto the support material using thiol-ene chemistry. The derivatized microparticles were slurry packed into the microbore columns with 2 mm i.d. and used as stationary phase for the separation of nucleosides in hydrophilic interaction chromatography with the plate numbers up to 54.000 plates/m. The results showed the usability of tailored poly (TMSPM-co-EDMA) microparticles as a stationary phase and thiol-ene chemistry in the manufacture of a chromatographic stationary phase with high efficiency in hydrophilic liquid chromatography applications.

Preparation and application of quinine-functionalized poly(1-vinylimidazole)-modified silica stationary phase in hydrophilic interaction chromatography

A quinine-functionalized poly(1-vinylimidazole)-modified hydrophilic silica stationary phase (Sil-PIm-Qn) was prepared by surface radical chain transfer and nucleophilic substitution reactions. Sil-PIm-Qn was characterized by elemental analysis and FT-IR. The chromatographic characteristics of the stationary phase were investigated in the hydrophilic interaction chromatographic (HILIC) mode. The results showed that five amino acids, nine sulfonamides, and ten nucleosides/nucleobases could be successfully separated on this stationary phase, respectively. The retention behavior of the compounds on the column was evaluated under different chromatographic conditions, i. e., for different salt concentrations and acetonitrile contents in the mobile phases. The stability of this column was also investigated, and the relative standard deviation (RSD, n=10) of the retention time of Sil-PIm-Qn was 0.08%-2.30%. Given its simple and green preparation as well as its unique selectivity and high separation efficiency, Sil-PIm-Qn is expected to find widespread application in the separation of hydrophilic compounds, including sulfonamides and nucleosides/nucleobases, in real samples.

A polymer-based zwitterionic stationary phase for hydrophilic interaction chromatography

We describe a novel polymer-based zwitterionic stationary phase for hydrophilic interaction chromatography (HILIC). It is prepared by chemical modification of poly (glycidyl methacrylate-divinylbenzene) (GMA-DVB) by converting epoxide groups of microsphere surface to diol groups via hydrolysis, then clicking cysteine onto the microsphere with pendant double bonds of microsphere surface via “thiol-ene” click chemistry. The phase has been characterized by scanning electron micrograph, elemental analysis and zeta potential measurement. Diol and zwitterionic group (carboxylate and amine group associated with cysteine) onto the surface of GMA-DVB microspheres make them possess good hydrophilicity, as supported by effective separation towards common polar analytes. It shows good stability at alkaline solution (e.g. pH 10) and negligible bleed (e.g. only 1.7-fold blank and ~55-fold lower than a commercial silica-based polar phase tested with the minimal bleed level). Such phase exhibits specific separation selectivity to ionic analytes and simultaneous separation of anions and cations is achieved in the retention order of I− < NO3− < Choline < Br− < Cl− < K+< Na+.

Glucose-based carbon dots-modified silica stationary phase for hydrophilic interaction chromatography

A new stationary phase based on glucose-derived carbon dots-modified on silica (Sil-Glc-CDs) was prepared and applied in hydrophilic interaction chromatography (HILIC). The Sil-Glc-CDs column showed better retention ability and separation selectivity toward polar analytes, including amino acids, saccharides, ginsenosides, antibiotics, nucleosides, and nucleobases when compared with a commercial HILIC column and a home-made glucose-modified silica (Sil-Glc) column. The effect of the organic phase ratio, salt concentration, pH and column temperature on chromatographic retention behavior was investigated. In addition, the column exhibited good stability and column-to-column reproducibility (RSDs 0.80–1.97%, n = 3). Particularly, this column was successfully applied for the determination of the concentration of glucose (2.2 mg mL−1) and fructose (3.4 mg mL−1) in the goji berry solution.

A phenylenediamine-based carbon dot-modified silica stationary phase for hydrophilic interaction chromatography.

Red emitting carbon dots derived from p-phenylenediamine (PPDCDs) were successfully prepared and grafted onto the surface of silica spheres, which served as a new stationary phase (Sil-PPDCDs) for liquid chromatography with excellent hydrophilic selectivity. Various hydrophilic analytes including nucleosides and bases, amino acids, saccharides and ginsenosides can be well separated on this stationary phase. Compared with the precursor-modified silica, i.e. p-phenylenediamine functionalized silica stationary phase (Sil-PPD), Sil-PPDCDs possessed enhanced selectivity for the separation of tested polar compounds. Moreover, compared with two commercially available hydrophilic columns, the selective behavior of the stationary phase was also considerable. The successful application of the Sil-PPDCDs stationary phase again indicated the great potential of carbon dots as a new modifier of the stationary phase in high performance liquid chromatography, and this kind of new stationary phase is very promising to be used for the separation of hydrophilic compounds in biochemical and pharmaceutical analysis.

Imidazolium ionic liquids-derived carbon dots-modified silica stationary phase for hydrophilic interaction chromatography

In this study, imidazolium ionic liquids-derived carbon dots (ImCDs) were successfully synthesized and bonded on the surface of spherical porous silica as a new stationary phase (Sil-ImCDs) for hydrophilic interaction chromatography (HILIC). First, the ImCDs were synthesized by the reaction of amino ionic liquids with citric acid, and then they were grafted onto the surface of aminopropyl-modified silica. A series of characterizations including infrared spectra, elemental analysis and laser confocal microscope proved that the preparation was successful. It was found that the Sil-ImCDs had good separation efficiency in the separation of bases, nucleosides, amino acids, saccharides and ginsenosides. It is interesting that we found that Sil-ImCDs has better hydrophilic chromatographic performance compared with methylimidazole-modified silica and aminopropyl-modified silica. It may be due to the rich functional group which is available after the formation of carbon dots from imidazolium ionic liquids and citric acid.

A polar stationary phase obtained by surface-initiated polymerization of hyperbranched polyglycerol onto silica

A novel polar stationary phase for hydrophilic interaction chromatography (HILIC) is prepared by surface-initiated polymerization of hyperbranched polyglycerol onto silica. Such polymerization offers an easy way to tune the cabon load. The stationary phase showed obvious hydrogen bonding interaction and typical HILIC character, exhibiting nealy-universal separation ability towards polar analytes. Good peak shape and high separation efficiency was observed for model polar analytes, including nucleosides and nucleic bases, aromatic acids, organic bases, vitamins, saccharides, amino acids, e.g. 35,000–58,000 plate counts per meter for five water-soluble vitamins.

Retention characteristics of poly(N-(1H-tetrazole-5-yl)-methacrylamide)-bonded stationary phase in hydrophilic interaction chromatography

A Poly(N-(1H-tetrazole-5-yl)methacrylamide)-bonded (PTZ) stationary phase has recently gained increased attention in hydrophilic liquid chromatography (HILIC) mode for separation of polar compounds and is now commercially available as DCpak PTZ. It is chromatographically characterized in this work. The property of the new column was proven to be the most hydrophilic and acidic by separation of mixtures of purines and pyrimidines (theophylline, theobromine, uridine and 2′-deoxyuridine) in comparison to other two commercial columns Luna HILIC and LiChrospher Diol column. The retention mechanism of the new column was investigated by design-of-experiment (DoE) approach using factorial design models. Water/acetonitrile ratio in the mobile phase, buffer salt concentration and buffer pH were considered factors employing the purine/pyrimidine mixture and glucose derivatives as test samples. The resultant retention model coefficients and contour plots documented the complementary retention and selectivity profiles of the new PTZ column as compared to Diol and Luna HILIC. Moreover, it became clearly evident that the PTZ column exhibits its best HILIC performance at eluent pH ≥ 5 because the NH group in tetrazolyl moiety is dissociated under these conditions. The applicability and great potential of the new HILIC column was proven by the chromatographic separation of complex mixtures of very hydrophilic glucose and glucose derivatives (sucrose, glucosamine, glucuronic acid, glucose-1-phosphate and trehalose, glucose, maltose, glucosamine-6-phosphate, glucose-6-phosphate and gluconic acid δ-lactone) as well as monosaccharides found in N-glycans. It is concluded that the new DCpak PTZ HILIC column could have good prospects for the separation of polar compounds e.g. in metabolomics and glycomics.

Evaluating Relative Retention of Polar Stationary Phases in Hydrophilic Interaction Chromatography

A large number of polar stationary phases with diverse chemistry have been developed for various applications in hydrophilic interaction chromatography (HILIC). However, column manufacturers employ different testing procedures to evaluate retention of the polar stationary phases. This renders the retention data impossible for comparison and makes it difficult for the users to select the right stationary phase based on retention. We have evaluated 25 polar stationary phases using cytosine and uracil as the model compounds in various mobile phase conditions. These stationary phases show a wide range of retention characteristics for the model compounds. The ranking of the stationary phases does not change drastically with the acetonitrile level in the mobile phase.

Evaluating the Adsorbed Water Layer on Polar Stationary Phases for Hydrophilic Interaction Chromatography (HILIC)

The water-rich liquid layer immobilized on the surface of the polar stationary phases is critical to the retention of polar compounds in hydrophilic interaction chromatography (HILIC). Although the presence of the adsorbed water layer has been investigated and confirmed by multiple techniques, there is a lack of quantitative measures that can be easily determined and linked to chromatographic parameters. This study proposes a simple measure termed volume ratio (the ratio of the adsorbed water layer volume and the mobile phase volume) that can be easily determined using toluene elution volume. The volume ratio values measured using the proposed method indicate that the volume of the adsorbed water layer varies in a wide range in the stationary phases commonly used in HILIC separation. It was observed that the volume ratio increases with the acetonitrile content and ammonium acetate concentration in the mobile phase. In addition, increasing the column temperature had the effect of reducing the volume ratio and diminishing the adsorbed water layer.

L‐Lysine‐derived highly selective stationary phases for hydrophilic interaction chromatography: Effect of chain length on selectivity, efficiency, resolution, and asymmetry

AbstractStationary phase development for hydrophilic interaction chromatography is rapidly gaining attention due to its versatile application in the separation of diversified solutes. Considering the hydrogen bonding property of urea, we designed and synthesized double‐alkylated L‐lysine derived urea containing short and long chain systems for use as stationary phases in hydrophilic interaction chromatography after immobilizing onto silica. The compounds were characterized by elemental analysis, NMR, and Fourier transform infrared spectroscopy. The compounds were further investigated by thermogravimetric analysis and diffuse reflectance infrared Fourier transform spectroscopy after immobilization onto silica. The effect of chain length in hydrophilic interaction chromatography, the phases were used for the separation of nucleic acid bases, nucleosides, and sulfa drugs. Enhanced selectivity was observed for both phases. However, the L‐lysine‐derived urea‐containing phase with short alkyl chains showed better separation ability than did the phase with longer alkyl chains. The former also showed better performance in terms of other chromatographic parameters such as efficiency, resolution, and asymmetry as compared to the latter.

Interaction of toluene with polar stationary phases under conditions typical of hydrophilic interaction chromatography probed by saturation transfer difference nuclear magnetic resonance spectroscopy

Toluene has been used as void volume (zero retention) marker since the inception of hydrophilic interaction chromatography (HILIC), based on the assumption that its hydrophobicity should prevent it from interacting with stationary phases envisioned to be covered by relatively thick layers of water. Recent work has shown that the void volumes of partly water-swollen HILIC phases are not identical to the volumes probed by toluene, yet the compound is still ubiquitously used as void volume marker. As part of our investigations of the retention mechanisms in HILIC, we probed the extent to which toluene is capable of penetrating into the water-enriched layer and to interact with the functional groups of three commercially available hydrophilic and polar stationary phases with different charge properties and water-retaining abilities, using saturation transfer difference 1H nuclear magnetic resonance (STD-NMR) spectroscopy at high resolution magic angle spinning (HR-MAS) conditions. The test solutions were 1000 ppm of toluene in deuterated acetonitrile and water mixtures, with and without addition of ammonium acetate, in order to mimic a set of conditions typically encountered in HILIC separations. Interactions between toluene and the functional groups on the stationary phases were probed by equilibrating the phases with these eluent mimics and measuring the transfer of magnetization from stationary phase protons to the protons of toluene. Our results show that toluene is indeed capable of traversing the water-enriched layers of all the three tested phases and of interacting with protons that are tightly associated with the stationary phases.