Interaction Chromatography Stationary Phase Research Articles

Preparation of poly(N-vinylpyrrolidone-co-pentaerythritol triacrylate) monolithic column for hydrophilic interaction chromatography.

A method for the preparation of poly(N-vinylpyrrolidone-co-pentaerythritol triacrylate copolymerization)-based monolithic capillary column was reported for the separation of polar small molecular weight compounds with nano-liquid chromatography in hydrophilic interaction chromatography mode. The monolithic columns were prepared by in situ copolymerization of N-vinylpyrrolidone and a cross-linker pentaerythritol triacrylate in a binary porogenic agent consisting of methanol and water. The composition of the polymerization solution was systematically optimized in terms of column permeability, theoretical plate number, asymmetric factor, and retention factor. A typical hydrophilic chromatography retention mechanism was observed with a mobile phase composed of a high content of organic solvent. The preparation method is simple and robust, the precursor N-vinylpyrrolidone is chemically stable, cheap, and easily available. The N-vinylpyrrolidone-based hydrophilic interaction chromatography stationary phase displays satisfactory separation selectivity for a range of polar test analytes, including benzoic acid derivatives, nucleosides, and phenols.

Characterization of a highly stable zwitterionic hydrophilic interaction chromatography stationary phase based on hybrid organic-inorganic particles.

We have characterized a sulfobetaine stationary phase based on 1.7 μm ethylene‐bridged hybrid organic–inorganic particles, which is intended for use in hydrophilic interaction chromatography. The efficiency of a column packed with this material was determined as a function of flow rate, demonstrating a minimum reduced plate height of 2.4. The batch‐to‐batch reproducibility was assessed using the separation of a mixture of acids, bases, and neutrals. We compared the retention and selectivity of the hybrid sulfobetaine stationary phase to that of several benchmark materials. The hybrid sulfobetaine material gave strong retention for polar neutrals and high selectivity for methyl groups, hydroxy groups, and configurational isomers. Large differences in cation and anion retention were observed among the columns. We characterized the acid and base stability of the hybrid sulfobetaine stationary phase, using accelerated tests at pH 1.3 and 11.0, both at 70°C. The results support a recommended pH range of 2–10. We also investigated the performance of columns packed with this material for metal‐sensitive analytes, comparing conventional stainless steel column hardware to hardware that incorporates hybrid surface technology to mitigate interactions with metal surfaces. Compared to the conventional columns, the hybrid surface technology columns showed a greatly improved peak shape.

Preparation and chromatographic evaluation of the hydrophilic interaction chromatography stationary phase based on nucleosides or nucleotides

In this work, a series of novel hydrophilic interaction chromatography (HILIC) stationary phases were prepared by grafting nucleosides or nucleotides on the surface of silica gel. Firstly, the silica was modified with 3-glycidoxypropyltrimethoxysilane (GPTMS). And then nucleosides or nucleotides were bonded on the surface of GPTMS-modified silica through the epoxy-amine ring-opening reaction to provide four HILIC materials. These obtained stationary phases were successfully characterized by Fourier transform-infrared spectroscopy (FT-IR) and elemental analysis (EA), respectively. Effects of column temperature, water content of the mobile phase, pH and buffer concentration on the retention behavior of these HILIC materials and the corresponding separation mechanism were evaluated using various nucleosides and nucleobases, respectively. In addition, polar and hydrophilic compounds such as amino acids and water-soluble vitamins were successfully separated using the corresponding columns, showing application potential for the separation of bioactive substances.

Managing the column equilibration time in hydrophilic interaction chromatography

For a wide variety of hydrophilic interaction chromatography stationary phases, a repeatable partial equilibration was demonstrated in gradient elution after purging with as little as 12 column volumes of mobile phase. Relative standard deviations of retention time of on average ~0.15% could be obtained after 1 or 2 conditioning (blank) runs. The equilibration period must be kept strictly constant, otherwise selectivity changes occur, but this is not problematic on modern instruments. Partial equilibration was largely independent of stationary phase or gradient slope. Alternatively, full column equilibration is favoured for stationary phases that do not trap extensive water layers, and for materials with a wider pore size that have a lower surface area. Temperatures somewhat above ambient also shorten the equilibration time. Some stationary phases under optimum conditions can achieve full column equilibration using purging with ~12 column volumes, which is useful for rapid set-up of isocratic separations or for conventional gradient analysis.

Hydrophilic polymeric monoliths containing choline phosphate for separation science applications

In this research, a hydrophilic polymeric monolith containing choline phosphate (CP) was fabricated through the thermally initiated free-radical polymerization of 2-{2-(methacryloyloxy) ethyldimethylammonium}ethyl n-butyl phosphate (MBP) with ethylene dimethacrylate (EDMA) using isopropanol and tetrahydrofuran as the porogenic system. Excellent mechanical strength, permeability, stability and reproducibility were obtained on the optimized poly(MBP-co-EDMA) monolith. Column efficiency as high as 107,500 plates/m was achieved for the analysis of thiourea at a flow velocity of 0.2 mm/s on the monolith. Both hydrophilic and electrostatic interactions were observed for the retention of charged analytes on the poly(MBP-co-EDMA) monolith. It is worth noting that the resulting monolith exhibits higher selectivity and efficiency than the classical 2-(methacryloyloxy)ethyl phosphorylcholine functionalized polymeric monolith for the chromatographic separation of polar analytes. Also, the novel monolithic column was successfully applied to enrich N-glycopeptides from tryptic digest of human IgG. In a word, the versatile MBP functionalized polymeric monolith not only opens up interesting possibilities for reverse zwitterionic CP derivative-based polymeric materials in separation science, but also represents a promising hydrophilic interaction chromatography stationary phase.

Preparation and evaluation of chromatographic properties of sulfobetaine zwitterionic hydrophilic interaction chromatography stationary phase

Preparation and evaluation of chromatographic properties of sulfobetaine zwitterionic hydrophilic interaction chromatography stationary phase

Preparation of a silica-based high-performance hydrophobic interaction chromatography stationary phase for protein separation and renaturation.

In this work, based on the structural characteristics of bio-membrane molecules, a novel type of high-performance hydrophobic interaction chromatography stationary phase was prepared using cholesterol as a ligand. Investigating the separation performance of this stationary phase, the effect of pH and salt concentration of the mobile phase on the retention time, the absorption capacity, and the hydrophobic ability revealed that this stationary phase had a high loading capacity and moderate hydrophobic interactions compared with four different hydrophobic interaction chromatography stationary phase ligands. Five types of standard proteins could be baseline separated with a great selection for protein separation. When 3.0 M urea was added to the mobile phase, it could be refolded with simultaneous purification of denatured lysozyme by one-step chromatography. The mass recovery of lysozyme reached 89.5%, and the active recovery was 96.8%. Compared with traditional hydrophobic interaction chromatography, this new stationary phase has a good hydrophobic ability and a significant refolding efficiency.

A novel dual-function weak cation exchange/hydrophobic interaction chromatography stationary phase with aminocaproic acid as the ligand for protein separation

A novel dual-function weak cation exchange/hydrophobic interaction chromatography stationary phase with aminocaproic acid as the ligand for protein separation

Hydrophilic interaction chromatography: A promising alternative to reversed-phase liquid chromatography systems for the purification of small protonated bases.

The overloaded band profiles of the protonated species of propranolol and amitriptyline were recorded under acidic conditions on four classes of stationary phases including a conventional silica/organic hybrid material in reversed-phase liquid chromatography mode (BEH-C18), an electrostatic repulsion reversed-phase liquid chromatography C18 column (BEH-C18+), a poly(styrene-divinylbenzene) monolithic column, and a hydrophilic interaction chromatography stationary phase (underivatized BEH). The same amounts of protonated bases per unit volume of stationary phase were injected in each column (16, 47, and 141 μg/cm(3)). The performance of the propranolol/amitriptyline purification was assessed on the basis of the asymmetry of the recorded band profiles and on the selectivity factor achieved. The results show that the separation performed under reversed-phase liquid chromatography like conditions (with BEH-C18, BEH-C18+, and polymer monolith materials) provide the largest selectivity factors due to the difference in the hydrophobic character of the two compounds. However, they also provide the most distorted overloaded band profiles due to a too small loading capacity. Remarkably, symmetric band profiles were observed with the hydrophilic interaction chromatography column. The larger loading capacity of the hydrophilic interaction chromatography column is due to the accumulation of the protonated bases into the diffuse water layer formed at the surface of the polar adsorbent. This work encourages purifying ionizable compounds on hydrophilic interaction chromatography columns rather than on reversed-phase liquid chromatography columns.

Applications of hydrophilic interaction chromatography to amino acids, peptides, and proteins.

This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity. Hydrophilic interaction chromatography is also recognized as a powerful technique for peptide analysis, and there are a lot of papers showing its applicability for proteomic applications (peptide mapping). It is expected that its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed-phase liquid chromatography, in a two-dimensional setup, to reach very high resolving power. Finally, the interest in hydrophilic interaction chromatography for intact proteins analysis is less evident due to possible solubility issues and a lack of suitable hydrophilic interaction chromatography stationary phases. To date, it has been successfully employed only for the characterization of membrane proteins, histones, and the separation of glycosylated isoforms of an intact glycoprotein. From our point of view, the number of hydrophilic interaction chromatography columns compatible with intact proteins (higher upper temperature limit, large pore size, etc.) is still too limited.

Novel imidazolium-embedded amine stationary phase: preparation and its performance for hydrophilic interaction chromatography

The development of the novel and highly efficient hydrophilic interaction chromatography stationary phase is important for the separation of polar compounds. In the present work, a novel imidazolium-embedded amine stationary phase (Sil-IEASP) was designed and prepared for hydrophilic interaction chromatographic separation. The prepared material was respectively characterized by Fourier transform infrared spectrometer, thermogravimetric analyzer and organic element analyzer. The results indicated that the novel stationary phase was successfully prepared with the present used method. The effects of the contents of water, salt concentration and pH on the retention of nucleosides and nucleic acid bases were respectively investigated, and the results indicated that Sil-IEASP is of hydrophilic interaction chromatographic nature. Furthermore, no obvious effect of buffer pH and salt concentration on the retention of these compounds were found. The developed material was further used for the separation of uracil, adenine, cytosine, uridine and positional isomers (o-terphenyl, m-terphenyl and triphenylene). Compared with the commonly used amino stationary phase, improved separation efficiency was achieved with the developed material, indicating the potential merit of the developed stationary phase for highly efficient hydrophilic interaction chromatographic separation. We anticipate that the novel stationary phase is promising for the hydrophilic interaction chromatography of polar compounds in the future research.

Preparation of a novel dual-function strong cation exchange/hydrophobic interaction chromatography stationary phase for protein separation

We have explored a novel dual-function stationary phase which combines both strong cation exchange (SCX) and hydrophobic interaction chromatography (HIC) characteristics. The novel dual-function stationary phase is based on porous and spherical silica gel functionalized with ligand containing sulfonic and benzyl groups capable of electrostatic and hydrophobic interaction functionalities, which displays HIC character in a high salt concentration, and IEC character in a low salt concentration in mobile phase employed. As a result, it can be employed to separate proteins with SCX and HIC modes, respectively. The resolution and selectivity of the dual-function stationary phase were evaluated under both HIC and SCX modes with standard proteins and can be comparable to that of conventional IEC and HIC columns. More than 96% of mass and bioactivity recoveries of proteins can be achieved in both HIC and SCX modes, respectively. The results indicated that the novel dual-function column could replace two individual SCX and HIC columns for protein separation. Mixed retention mechanism of proteins on this dual-function column based on stoichiometric displacement theory (SDT) in LC was investigated to find the optimal balance of the magnitude of electrostatic and hydrophobic interactions between protein and the ligand on the silica surface in order to obtain high resolution and selectivity for protein separation. In addition, the effects of the hydrophobicity of the ligand of the dual-function packings and pH of the mobile phase used on protein separation were also investigated in detail. The results show that the ligand with suitable hydrophobicity to match the electrostatic interaction is very important to prepare the dual-function stationary phase, and a better resolution and selectivity can be obtained at pH 6.5 in SCX mode. Therefore, the dual-function column can replace two individual SCX and HIC columns for protein separation and be used to set up two-dimensional liquid chromatography with a single column (2DLC-1C), which can also be employed to separate three kinds of active proteins completely, such as lysozyme, ovotransferrin and ovalbumin from egg white. The result is very important not only to the development of new 2DLC technology with a single column for proteomics, but also to recombinant protein drug production for saving column expense and simplifying the process in biotechnology.

Click novel glycosyl amino acid hydrophilic interaction chromatography stationary phase and its application in enrichment of glycopeptides

A novel glycosyl amino acid hydrophilic interaction chromatography (HILIC) stationary phase was prepared via click chemistry. The key intermediate N3-glycosyl d-phenylglycine was prepared by a three steps procedure, including selective condensation of amino glucose with N-succinimidyl ester of Boc-d-phenylglycine, deprotection and transformation of amino group to azido group. The structure of all the intermediates and functionalized silica beads were confirmed by 1H NMR, IR, elemental analysis and 13C CP-MAS. The chromatography test showed that this new type of separation material possessed good HILIC properties and glycopeptide enrichment characteristics. Nucleosides and bases could be separated in a simple eluent composition (only acetonitrile in combined with water), and with the same condition, these model compounds could not be separated on the commercial HILIC column (Atlantis). Click glycosyl amino acid thus prepared also showed longer retention and better separation ability in the separation of polar organic acids.

Comparison of different liquid chromatography stationary phases in LC‐HRMS metabolomics for the detection of recombinant growth hormone doping control

Growth hormone (GH) is a polypeptide suspected of being used in horse racing to speed up physical performances. Despite scientific advances in the recent years, the control of its administration remains difficult. In order to improve it, a metabolomics study through LC-high resolution mass spectrometry measurements was recently initiated to assess the metabolic perturbations caused by recombinant equine growth hormone administration. Few tens of ions not identified structurally were highlighted as compounds responsible for the modification of metabolic profiling observed in treated animals. This previous work was based on the use of Uptisphere Strategy NEC as the chromatographic column. In parallel, more and more metabolomics studies showed the interest of the use of new chromatographic supports such as hydrophilic interaction chromatography for the analysis of polar compounds. It is in this context that an investigation was conducted on Uptisphere HDO and Luna hydrophilic interaction chromatography stationary phases to generate and process urinary metabolomics fingerprints, which could allow to establish a comparison with Uptisphere Strategy NEC. The chromatographic column the most adapted for the detection of new biomarkers of GH administration has been used to set up a relevant statistical model based on the analysis of more than hundred biological samples.

Evaluation of hybrid hydrophilic interaction chromatography stationary phases for ultra‐HPLC in analysis of polar pteridines

Retention characteristics of ultra-HPLC (UHPLC) hybrid stationary phases (bridged ethyl hybrid (BEH) and BEH Amide) were studied in hydrophilic interaction chromatography system in the group of polar basic pteridines (neopterin, biopterin, dihydroneopterin and dihydrobiopterin). The effect of mobile phase composition, buffer type, pH and concentration on retention of pteridines were examined in detail under UHPLC-fluorescence detection and UHPLC-MS conditions. The selectivity, retention properties and column performance were examined. BEH HILIC did not provide sufficient retention and selectivity for the separation of four pteridines under any tested conditions. BEH Amide provided strong retention for all pteridines especially at high pH values such as 9.8. However, at pH 9.8 the selectivity of separation for the pairs neopterin-dihydroneopterin and biopterin-dihydrobiopterin substantially decreased and resulted in very long analysis time. The best separation of four pteridine derivatives was obtained in the pH range 4.8-7.8 within a reasonable analysis time up to 8 min for UHPLC-fluorescence detection using higher concentrations of ammonium acetate buffer and up to 4 min for UHPLC-MS using lower concentrations of ammonium acetate.

Retention behavior of estrogen metabolites on hydrophilic interaction chromatography stationary phases

Estrogens and estrogen metabolites are important biological mediators of the endocrine system. They have also been implicated in detrimental carcinogenesis and beneficial neuroprotective processes. The retention behavior of estrogen metabolites was investigated on five polar stationary phases, used for hydrophilic interaction chromatography, and coupled with ESI-MS. Data were fit to partitioning and surface adsorption models. Retention of the compounds, especially estrogen glucuronides, on the amide- and diol-bonded stationary phases, could be best described by the surface adsorption model; however, mixed modes of retention were observed on most stationary phases. Retention time increased while the peak efficiency decreased proportional to the number of hydroxyl groups in the analytes. The effects of salt concentration and salt type were also investigated. The presence of solvated salt ions, which interact with the stationary phase and the analyte, enhanced retention of the analytes. This was believed to be due to two effects. The increased ionic strength reduced the contribution of secondary electrostatic interactions (mixed-mode effects). It also enhanced hydrogen-bonding and partitioning (hydrophilic interaction) between the analyte and the stationary phase, likely facilitated by the associated solvated salt ions.

Nylon-6 capillary-channeled polymer (C-CP) fibers as a hydrophobic interaction chromatography stationary phase for the separation of proteins

Nylon-6 capillary-channeled polymer (C-CP) fibers are used as the stationary phase for the hydrophobic interaction chromatography (HIC) separation of a synthetic protein mixture composed of ribonuclease A, lysozyme, and holotransferrin. Nylon is a useful polymer phase for HIC as it has an alkyl backbone, while the amide functionality is hydrophilic (in fact ionic) in nature. The combination of a nonporous polymer surface of the fiber phases and high column permeability yields very efficient mass transfer characteristics, as exhibited by narrowing of peak widths with increases in mobile phase linear velocity. Retention factors and resolution were evaluated at flow rates ranging from 0.5 to 9 mL/min (linear velocities of ca. 2 to 15 mm/s) and at gradient slopes between 3.3 and 20 %B/min. Optimum resolution was achieved by employing fast flow rates (9 mL/min) and slow gradients (3 %B/min), also resulting in the highest peak capacities.