Silica-based Stationary Phases Research Articles

A stationary phase of tetraethylene glycol-modified silica for separation of phenolic acids

AbstractSilica, as a stationary phase, has low separation efficiency accompanied by overlapping, broadened, and tailed peaks, so it needs to be modified to improve its efficiency. This study aims to develop a silica-based stationary phase modified by tetraethylene glycol (TEG) to separate phenolic compounds. Silica was modified by a chemical bond between silanol groups on the silica surface and TEG through a 3-glycidyloxypropylmethoxysilane reaction. The modified silica was packed into a capillary column and used to separate simple phenolic compounds consisting of phenol, pyrocatechol, and pyrogallol. A sample of 0.2 µL was injected into the capillary liquid chromatography and the mobile phase employed was acetonitrile 98% with a flow rate of 3 μL min−1. Elution was also done isocratically in this process and detection was carried out at a wavelength of 254 nm. The mixture of simple phenolic compounds was successfully separated in less than 7 min. The asymmetry factor and resolution were 1.43–2.12 and 1.72–5.43 respectively. The number of the theoretical plates ranged from 213 to 7,857. Columns containing Si-TEG stationary phase also separate phenolic compounds, which consist of gallic acid, syringic acid, ferulic acid, and caffeic acid. A sample of 0.2 µL was injected into the capillary liquid chromatography and successfully separated the mixture in less than 12 min. The samples were eluted isocratically using a mixture of methanol and 50 mM phosphate buffer pH 2.5 (8:92) with a flow rate of 3 μL min−1. The phenolic acids compounds were detected at a wavelength of 280 nm. The chromatogram showed four separate peaks. The asymmetry factor and resolution were 1.53–1.63 and 1.14–1.74, respectively, but the number of the theoretical plates was low, ranging from 190 to 796.

Study of chromatographic properties of the developed hyperbranched zwitterionic silica-based stationary phase for hydrophilic interaction liquid chromatography

Study of chromatographic properties of the developed hyperbranched zwitterionic silica-based stationary phase for hydrophilic interaction liquid chromatography

The role of the cation and anion in aqueous liquid chromatography with sodium dodecyl sulphate and imidazolium-based ionic liquids as mobile phase reagents

BackgroundIn reversed-phase liquid chromatography, solute retention is primarily influenced by interactions between a nonpolar stationary phase and a moderately polar hydro-organic mobile phase, based on the solute lipophilicity. However, challenges regarding retention and peak tailing can arise due to ionic interactions between positively charged analytes and free silanols present on silica-based stationary phases. To address these challenges, incorporating surfactants and ionic liquids (ILs) into the mobile phase offers an effective solution. These additives synergistically enhance chromatographic performance through electrostatic and lipophilic interactions, which enable fine-tuning of selectivity and improved separation efficiency. ResultsThis study explores the chromatographic behaviour of several basic compounds in aqueous mixtures containing the anionic surfactant sodium dodecyl sulphate (SDS), above its critical micellar concentration, combined with various 1-alkyl-3-methylimidazolium-based ionic liquids (ILs) featuring chloride, tetrafluoroborate, and hexafluorophosphate anions, all without the addition of organic solvents. Specifically, this research investigates the influence of different anion types within the ILs and considers the impact of the IL cations. Analysis of solute peak profiles reveals narrow and symmetrical peaks. By introducing tetrafluoroborate and hexafluorophosphate IL anions into a mobile phase that contains an anionic surfactant, the study sheds light on the interactions occurring within the chromatographic column. This enhanced understanding of the combined effects of surfactants and ILs contributes to refining chromatographic methodologies. SignificanceThis research highlights the importance of carefully selecting the appropriate IL when incorporating it into a micellar mobile phase alongside SDS. This combination results in practical retention times that surpass the performance achieved with either the surfactant or IL alone in the mobile phase. The study particularly emphasises the impact of the IL anion, especially in the absence of SDS and organic solvents. This unveils interactions that are otherwise obscured in micellar and hydro-organic media, providing new insights into chromatographic dynamics.

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.

Per-aqueous liquid chromatography stationary phase with poly(ethylene oxide-co-dimethylsiloxane) immobilized on mesoporous aluminized silica support

This work proposes an alternative to searching for HPLC stationary phases to separate polar compounds. The method involves preparing an aluminized silica support and thermally immobilizing a thin layer of pre-synthesized poly(ethylene oxide-co-dimethylsiloxane) to create a chemically stable and polar stationary phase. The polymer immobilized aluminized silica-based stationary phase has a mesoporous structure (10.5 nm of mean size pore), high surface area (374 m2 g−1), and highly regular spheres. The presence of alumina (1.6% Al) ensures high chemical stability of the material under phosphate mobile phases at high pH and temperature. Using highly aqueous mobile phases and retention mechanisms of per-aqueous liquid chromatography mode, separations of polar and non-polar compounds using the proposed stationary phase are fast and efficient (N/m > 70,000 plates m−1). The aluminized silica-based stationary phase provides a viable and effective alternative to commercial reversed phases designed for polar compounds.

Polybutylene terephthalate-based stationary phase for ion-pair-free reversed-phase liquid chromatography of small interfering RNA. Part 1: Direct coupling with mass spectrometry

Nowadays, ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the dominating generic method for the analysis of nucleic acid related compounds, such as antisense-oligonucleotides (ASO), small-interfering ribonucleic acid (siRNA) or other DNA or RNA type molecules and their conjugates. Despite of its effective performance, the usage of a high concentration of ion-pairing reagent in the eluent in IP-RPLC is unfavorable for the hyphenation with mass spectrometry (MS) which is required for a detailed structural characterization of the analytes and their structurally related impurities. In this work, we tested a polybutylene terephthalate (PBT)-bonded silica-based stationary phase for the separation of generically synthesized Patisiran as siRNA (antisense and sense single strands as well as their annealed double strand) giving some unexpected selectivity without any presence of ion-pairing reagents. Important chromatographic conditions affecting the separation have been investigated and evaluated. Furthermore, MS and tandem MS (MS/MS) characterization was possible without contamination of the MS system with ion-pair agent and related problems.

Mechanism and inhibition of abnormal chromatographic behavior of serotype type a inactivated foot and mouth disease virus in high-performance size-exclusion chromatography

High-performance size-exclusion chromatography (HPSEC) has been developed for the rapid and quantitative analysis of inactivated foot and mouth disease virus (FMDV) and adopted by regulatory agencies and vaccine manufacturers. However, strong non-specific adsorption of type A/AKT III FMDV was found on some batches of TSK G4000 SWXL column, which significantly affected the analysis accuracy. The adsorption mechanism was studied by investigating the charge and hydrophobicity of A/AKT III FMDV and another serotype O/Mya 98, as well as several model proteins, by zeta potential and hydrophobic interaction chromatography analysis. Adsorption was related to both the FMDV strain and column lots. Some specific amino acids residues on the A/AKT III FMDV surface may strongly interact with the column if the silica-based stationary phase was not completely diol-modified. Several amino acids and chaotropic salts were screened as additives in the mobile phase to suppress the non-specific adsorption of AKT III FMDV in HPSEC analysis. Results showed that adding 0.4 M of arginine (Arg), lysine (Lys), NaClO4, or NaSCN achieved 100% FMDV recovery and normal retention time. Suppression of interaction between FMDV and the backbone of the silica matrix through competitive binding with residues of FMDV or the matrix is considered as the main mechanism by which these four additives act as suppressors. The addition of Arg, NaClO4, or NaSCN led to an apparent decrease in the thermal dissociation temperature Tm of FMDV, whereas Lys slightly increased viral stability. Finally, the mobile phase comprising 0.4 M Lys was screened as optimum that allowed accurate quantification of both two serotypes of FMDV according to method validation; particularly, a relative standard deviation (RSD) < 5% was achieved for AKT III FMDV using three different lots of columns.

N-Vinyl pyrrolidone and undecylenic acid copolymerized on silica surface as mixed-mode stationary phases for reversed-phase and hydrophilic interaction chromatography

In this work, three new mixed-mode stationary phases were prepared, based on different ratio of N-vinyl pyrrolidone (NVP) copolymerized together with undecylenic acid (UA) on silica microspheres surface without silanization, which named Sil@NVPUA series. The combination of NVP and UA rendered the Sil@NVPUA suitable for reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC), and shown excellent methyl, planar, isomers and ion selectivity. Five types of model analytes including eight polycyclic aromatic hydrocarbons, six alkylbenzenes, eight nucleosides and nucleobases, seven ginsenosides and five oxazolidinones can be well separated on this stationary phase. The preparation method of NVP and UA modified silica-based stationary phase is simple, and it also provides a new idea for the design of synthetic polymers to develop mixed-mode chromatography.

Non-conjugated flexible network for the functional design of silica-based stationary phase for mixed-mode liquid chromatography

The functionalized non-conjugated organic network modified silica microspheres are proposed as the stationary phase of liquid chromatography, which can effectively avoid some defects of organic framework materials in liquid chromatography. Due to the co-existing of pyridine ring, carbonyl group, amide group and triazine ring in the non-conjugated flexible organic network on the silica surface, the developed mixed-mode stationary phase exhibits multiple interactions between the stationary phase and the analytes during the separation process. A variety of nucleoside bases, organic acids, antibiotics, pesticides, alkylbenzenes, polycyclic aromatic hydrocarbons and sulfonamides achieved ideal resolution and flexible selectivity in separation. Compared with the commercial chromatographic columns under their optimized chromatographic conditions, it shows better performance for the separation of complex analytes. The influence of chromatographic conditions on retention behavior indicates that the column's multiple retention mechanisms make it suitable for mixed-mode liquid chromatography. The stationary phase prepared by the new design strategy also has excellent chromatographic reproducibility, repeatability and stability with the intraday RSD of 0.09%–0.12% (n = 10) and the interday RSD of 0.37%–1.64% (n = 5) for the retention time. The separation results of actual samples also prove its potential in the analysis of complex samples. In short, we designed and prepared the non-conjugated flexible network modified silica stationary phase material for liquid chromatography that is different from organic framework materials. Its excellent separation ability shows that we have successfully reported a new kind of liquid chromatography packing with functional design and facile preparation method.

LC Troubleshooting Odds and Ends from 2020

Sometimes our approach to troubleshooting specific problems has to change in response to changes in high performance liquid chromatography (HPLC) technology over time. In this installment, we discuss changes in technologies for mobile-phase degassing, silica-based stationary phases, and models for reversed-phase selectivity.

Hydrophilic interaction liquid chromatography method for eremomycin determination in pre-clinical study

A complex of hydrophilic interaction liquid chromatography (HILIC) methods for simple and efficient determination of eremomycin (ERM) as an active pharmaceutical ingredient (API) of a novel drug is proposed for preclinical study, which includes the dissolution test and pharmacokinetic study on the animals.A home-made HILIC silica-based stationary phase (SP) containing diol functionalities and positively charged nitrogen atoms in its structure was synthesized for this research and applied for the first time for performing the first step of preclinical study (dissolution test) of the novel ERM-containing drug. HILIC method developed using novel home-made SP allowed us to avoid any interferences from polyethylene glycol (PEG) contained in the drug matrix thus providing a unique advantage of the proposed approach over RP HPLC. The home-made SP demonstrated better chromatographic performance as compared to the tested commercially available columns with various functionalities. Different retention behaviour and mechanisms with various electrostatic impact were demonstrated for two glycopeptide antibiotics, namely, ERM and its analogue vancomycin (VAN), on the home-made SP. For the second step of the preclinical study HILIC-MS/MS method for ERM determination in rabbit plasma was developed and validated in accordance with the EMA requirements and successfully applied to the preclinical study on rabbits after intravenous and intraperitoneal drug administration. The results of dissolution test and pharmacokinetic study revealed similar in vitro solubility of ERM and VAN and low ERM bioavailability, which proved the potential safety and efficiency of the novel drug.

Application of mobile phases containing ionic liquid for the analysis of selected psychotropic drugs by HPLC–DAD and HPLC–MS

In reversed-phase liquid chromatography, in case of the absence of additives, cationic basic compounds give rise to asymmetrical and broad peaks as a result of interactions of analyte cations with residual free silanols on silica-based stationary phases. Ionic liquids, added to the mobile phase, have been suggested as alternatives to amines to block the activity of free silanols. The different parameters affecting the retention behavior, symmetry of peak, system efficiency, and separation selectivity of selected psychotropic drugs, especially the effect of concentration of ionic liquid, kind and concentration of organic modifiers of mobile phases, and kind of stationary phases were investigated. The most selective and efficient systems are used for separations of psychotropic drug standards' mixture and for determination of selected psychotropic drugs in human serum.

Ultrasensitive high‐performance liquid chromatography determination of tetracycline antibiotics and their 4‐epimer derivatives based on dual effect of methanesulfonic acid

In reversed-phase liquid chromatography, tetracycline antibiotics yield broad and asymmetrical peaks, as a result of their ionic interaction with the anionic free silanol groups and metal ion present in the silica-based stationary phases (commonly derivatized with C18 groups). These frequently encountered difficulties were absolutely eliminated based on dual effect when methanesulfonic acid was employed as an eluent additive. The study revealed that the performance of methanesulfonic acid to minimize the "silanol effect" is mainly explained by both direct neutralization of the anionic silanol sites and electrostatic attraction with analytes. Based on these dual action mechanisms, an ultrasensitive method has been successfully developed for the simultaneous determination of tetracycline antibiotics and their derivatives (minocycline, oxytetracycline, tetracycline, chlortetracycline, metacycline, doxycycline, 4-epitetracycline, and 4-epichlortetracycline) in bovine milk with convenient ultraviolet detection within 15 min. Under the optimal conditions, the calibration curves showed good linearity (r2 >0.999) for all analytes in the range of 1∼200ng/mL with the instrument limits of detection as low as 0.3ng/mL. The study sheds new light on suitable additives to analyze basic compounds with the advantage of good compatibility with MS detection.

Modelling sub-micron particle slip flow in liquid chromatography

The profoundly fast flow rates, observed at unexpectedly low column back-pressures, of capillaries packed with colloidal silica-based stationary phases is explained using slip flow theory applied to the particles themselves rather than the capillary walls. An equation is proposed that leverages nanoscale thermodynamics, geometrical considerations and Hagen-Poiseuille flow without the traditional no-slip boundary condition. It is found that the proposed model successfully fits/predicts real-world data taken from the literature, presented in a plot of the observed flow (enhancement) as a function of the (diminishing) mean particle size, so long as the particles do not exceed ~1 μm. This finding lends support to the continued use of colloidal silica packings in liquid chromatographic applications to achieve efficient separations of molecules on the smallest scale.

Influence of silica functionalization by amino acids and peptides on the stationary phases zeta potential

In this study, the zeta potentials of silica-based stationary phases with chemically bonded amino acids and peptides of different types (glycine, alanine, phenylalanine, leucine, and aspartic acid) and chain lengths (amino acid, di- and tripeptides) have been measured in water/acetonitrile and water/methanol solutions. The zeta potential changes with ionic strength of the mobile phase were tested using phosphate buffer. A series of home-made stationary phases were also studied in different pH conditions. In order to compare how the modifications based on the amino acids vary from the conventional packings, pure silica, silica modified with aminopropyl ligands, and octadecyl groups were also studied. The results show that the increase of the amino acid polarity and sequence length causes higher zeta potential values. The changing conditions from acidic to basic result in shifting the zeta potential values from positive to negative. Depending on the environment, different ionization states of the chemically bonded functionalities provide different charge distribution on the surface of the particles.

Low-Bleed Silica-Based Stationary Phase for Hydrophilic Interaction Liquid Chromatography.

Bleeding of hydrophilic interaction liquid chromatographic (HILIC) phases is a major problem. A hydrophobic silica surface exhibits low bleeding but does not behave as a HILIC phase. A hydrophilic coating, such as that of poly(vinyl alcohol) (PVA) on a hydrophobic particle should result in a low-bleed hydrophilic phase. However, a silica particle functionalized in a hydrophobic manner is not wetted by PVA and cannot be coated with it. Coating with PVA becomes possible if one region of the hydrophobic functionality is polar. We describe a low-bleed HILIC stationary phase, PVA-coated benzylthioethyl-silica. The benzyl groups shield the silica from water erosion, while the thiol group provides sufficient local polarity for PVA to wet the material. The new stationary phase demonstrated good chromatographic performance and typical HILIC retention behavior. The measured silica concentration in the effluent was ∼80-fold lower than that from a bare silica column. The new stationary phase exhibited a lower background level, lower background noise, and lower background drift under gradient conditions than benchmark commercial columns.

N-Propyl-N′-2-pyridylurea-modified silica as mixed-mode stationary phase with moderate weak anion exchange capacity and pH-dependent surface charge reversal

Herein, we present a novel silica-based stationary phase modified with N-propyl-N′-2-pyridylurea selector. Due to the weakly basic properties of the pyridine selector and the presence of residual silanols after selector immobilization, a zwitterionic surface with a pI observed at approximately pH 5.5 was measured by electrophoretic light scattering in pH-dependent ζ-potential determinations. The capability of the new N-propyl-N′-2-pyridylurea-modified silica to serve as mixed-mode stationary phase was investigated. For this purpose, it was characterized under RP and HILIC conditions using test mixtures. Subsequent classification of this stationary phase in comparison to in-house and commercial benchmarks was carried by principal component analysis of resultant retention factors from chromatographic tests. The results show a relatively unique mixed-mode character amongst the tested stationary phases. The chromatographic retention characteristics of acidic compounds matched well the ζ-potential determinations. The application of anion-exchange at low pH values (e.g. pH 5) and ion exclusion chromatography at pH 7 for the separation of uridine 5′-mono-, di- and triphosphate demonstrated a pH-dependent umpolung of the stationary phase surface. The combination of these separation principles in a pH gradient from 5 to 7 gave rise to weak anion-exchange selectivity with a charge-inducted elution due to repulsive interactions at higher pH and resulted in a significant faster separation with improved peak shape under mild elution conditions.

Protein separation using a novel silica-based RPLC/IEC mixed-mode stationary phase modified with N-methylimidazolium ionic liquid

Ionic liquids (ILs) immobilized on silica as a novel high-performance liquid chromatography (HPLC) stationary phase have attracted considerable attentions. However, it has not been applied to protein separation. In this paper, N-methylimidazolium IL-modified silica-based stationary phase (SilprMim) was prepared and investigated as a novel multi-interaction stationary phase with positive charges for protein separation. The results indicate that all of the basic proteins tested cannot be adsorbed on this novel stationary phase, whereas all of the acidic proteins tested can be retained, and the baseline separation of eight kinds of acidic protein standards can be achieved when being performed under reversed phase/ion-exchange chromatography (RPLC/IEC) mode. Compared with commonly used commercial C4 column, the novel stationary phase can show good selectivity and resolution to acidic proteins. The effects of acetonitrile and salt concentration, pH as well as the ligand structure on protein separation were investigated in detail. In addition, the mix-mode retention mechanism of proteins on the SilprMim column was also discussed using stoichiometric displacement theory for retention in LC (SDT-R). The result shows that the protein retention can be controlled mainly by the electrostatic and hydrophobic interactions between the proteins and the stationary phase. As a result, with such characteristics of multi-interaction mechanism and multi-modal separation, not only the selectivity to the acidic proteins can be enhanced, but also a better resolution can be achieved. The result demonstrates that the SilprMim mixed-mode chromatography (MMC) column has a promising application in the separation and analysis of acidic proteins from the complex samples.

Selective enrichment of N-linked glycopeptides and glycans by using a dextran-modified hydrophilic material.

Glycosylation analysis of proteins from biological sources utilizing mass spectrometry based approaches is challenging due to the relatively low abundance of glycopeptides, the structural diversity of glycans, and the coexisting matrices. In this study, a customized dextran-bonded silica-based stationary phase was introduced for selective enrichment of glycopeptides and glycans from complex biological samples. This material has exhibited superior selectivity and broader glycosylation site coverage over commercial Sepharose in glycoproteomic evaluation. Additionally, the glycomic analysis of fetuin, α1 -acid glycoprotein, and human serum N-glycome also indicated the relatively higher sensitivity, selectivity, and glycoform coverage of dextran-bonded silica than that of Sepharose and porous graphitized carbon. Therefore, the dextran-bonded silica is expected to make contributions in the fields of glycoproteomics and glycomics.

Suitability of 1-hexyl-3-methylimidazolium ionic liquids for the analysis of pharmaceutical formulations containing tricyclic antidepressants

The reversed-phase chromatographic behaviour of six tricyclic antidepressants (amitryptiline, clomipramine, doxepin, imipramine, nortryptiline and maprotiline) was examined in this work with acetonitrile-water mobile phases, in the absence and presence of the ionic liquids 1-hexyl-3-methylimidazolium chloride and 1-hexyl-3-methylimidazolium tetrafluoroborate, which have interesting features for the separation of basic compounds, in terms of peak shape combined with reduced retention. Tricyclic antidepressants are low polarity drugs that strongly associate to the alkyl chains of conventional stationary phases. They are also positively charged in the usual working pH range (2–8) in reversed-phase liquid chromatography, due to their strong basic character. In consequence, they may interact with the residual ionised silanols present in conventional silica-based stationary phases, which is translated in stronger retention, and tailed and broad peaks. A simple chromatographic procedure for the control of tricyclic antidepressants in pharmaceutical formulations was developed using a C8 column and a mobile phase containing 30% acetonitrile/10 mM 1-hexyl-3-methylimidazolium chloride at pH 3, with UV detection. Intra- and inter-day precisions were usually below +1.0%, and intra- and inter-day bias (trueness) ranged between ‒2.1% and +2.4%, and between ‒3.0% and +2.3%, respectively. Sample preparation was simple and only required solubilisation and filtration previous to injection.