Investigation of the novel mixed-mode stationary phase for capillary electrochromatography. II. Separation of amino acids and peptides on sulfonated naphthalimido-modified silyl silica gel

The potential of 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP) as a mixed-mode stationary phase for capillary electrochromatography (CEC) was investigated for the separation of charged analytes, taking four amino acids (tyrosine, phenylalanine, tryptophan, histidine) as model analytes. The elution process of these charged analytes in CEC with SNAIP was dominated by a combination of both electrophoretic process and chromatographic process involving hydrophobic as well as electrostatic interactions. In order to study the retention mechanism, the CEC retention factor k* and the velocity factor ke* were measured for the amino acids, which allowed the assessment of the respective contribution from the differential processes underlying the separation. Migration and retention could be mediated by changing various mobile phase compositions, including buffer pH, buffer concentration, and concentration of organic solvent. Based on the results obtained by separation of the amino acids, the separation of eight peptides (Gly-Val, Gly-Phe, Gly-Ile, Gly-His, Gly-Lys, Lys-Lys, Gly-Gly-Gly, Gly-Gly-His) was attempted. A good separation was achieved under an isocratic elution with a mobile phase consisting of 35 mM phosphate buffer (pH 3.8) and 40% methanol.     

Comparison of the chromatographic behavior of monolithic capillary columns in capillary electrochromatography and nano-high-performance liquid chromatography

Porous monoliths based on N,N-dimethylacrylamide (DMAA) or methacrylamide (MAA) were prepared inside fused silica capillaries as stationary phases for nano-chromatography. The columns were characterized in terms of flow rate and backpressure and showed, e.g. differences as a function of the salt concentration added to the polymerization mixture. When the columns were investigated for the separation of uncharged (polar hydroxylated aromatic compounds) and charged (amino acids) analytes under pressure driven conditions (pLC), differences to the previously observed behavior under voltage driven conditions (CEC) were observed. Whereas the non-charged analytes showed similar behavior in both cases--thus, corroborating the previous assumption of a mainly chromatographic separation mode driven by hydrophilic interactions in CEC--the charged amino acids did not. Assuming that the separation was governed by chromatographic phenomena in the pLC mode and by both chromatographic and electrophoretic effects in the CEC mode, the experiments allowed deconvoluting the two contributions. In particular, the charged amino acids appeared to interact with the stationary phases mainly by electrostatic interactions modified by some hydrophilic effects.     

Simultaneous separation of hydrophobic and hydrophilic peptides with a silica hydride stationary phase using aqueous normal phase conditions

The application of a silica hydride modified stationary phase with low organic loading has been investigated as a new type of chromatographic material suitable for the separation and analysis of peptides with electrospray ionization mass spectrometric detection. Retention maps were established to delineate the chromatographic characteristics of a series of peptides with physical properties ranging from strongly hydrophobic to very hydrophilic and encompassing a broad range of pI values (pI 5.5-9.4). The effects of low concentrations of two additives (formic acid and acetic acid) in the mobile phase were also investigated with respect to their contribution to separation selectivity and retention under comparable conditions. Significantly, strong retention of both the hydrophobic and the hydrophilic peptides was observed when high-organic low-aqueous mobile phases were employed, thus providing a new avenue to achieve high resolution peptide separations. For example, simultaneous separation of hydrophobic and hydrophilic peptides was achieved under aqueous normal phase (ANP) chromatographic conditions with linear gradient elution procedures in a single run, whilst further gradient optimization enabled improved peak efficiencies of the more strongly retained hydrophobic and hydrophilic peptides.     

Investigation of the ion-exchange properties of methacrylate-based mixed-mode monolithic stationary phases employed as stationary phases in capillary electrochromatography

The potential of methacrylate-based mixed-mode monolithic stationary phases bearing sulfonic acid groups for the separation of positively charged analytes (alkylanilines, amino acids, and peptides) by capillary electrochromatography (CEC) is investigated. The retention mechanism of protonated alkylanilines as positively charged model solutes on these negatively charged mixed-mode stationary phases is investigated by studying the influence of mobile phase and stationary phase parameters on the corrected retention factor which was calculated by taking the electrophoretic mobility of the solutes into consideration. It is shown that both solvophobic and ion-exchange interactions contribute to the retention of these analytes. The dependence of the corrected retention factor on (1) the concentration of the counter ion ammonium and (2) the number of methylene groups in the alkyl chain of the model analytes investigated shows clearly that a one-site model (solvophobic and ion-exchange interactions take place simultaneously at a single type of site) has to be taken to describe the retention behaviour observed. Comparison of the CEC separation of these charged analytes with electrophoretic mobilities determined by open-tubular capillary electrophoresis shows that mainly chromatographic interactions (solvophobic and ion-exchange interactions) are responsible for the selectivity observed in CEC, while the electrophoretic migration of these analytes plays only a minor role.     

Histidine‐modified organic‐silica hybrid monolithic column for mixed‐mode per aqueous and ion‐exchange capillary electrochromatography

A novel organic‐silica hybrid monolith was prepared through the binding of histidine onto the surface of monolithic matrix for mixed‐mode per aqueous and ion‐exchange capillary electrochromatography. The imidazolium and amino groups on the surface of the monolithic stationary phase were used to generate an anodic electro‐osmotic flow as well as to provide electrostatic interaction sites for the charged compounds at low pH. Typical per aqueous chromatographic behavior was observed in water‐rich mobile phases. Various polar and hydrophilic analytes were selected to evaluate the characteristics and chromatographic performance of the obtained monolith. Under per aqueous conditions, the mixed‐mode mechanism of hydrophobic and ion‐exchange interactions was observed and the resultant monolithic column proved to be very versatile for the efficient separations of these polar and hydrophilic compounds (including amides, nucleosides and nucleotide bases, benzoic acid derivatives, and amino acids) in highly aqueous mobile phases. The successful applications suggested that the histidine‐modified organic‐silica hybrid monolithic column could offer a wide range of retention behaviors and flexible selectivities toward polar and hydrophilic compounds.     

一种可控离子比例的两性亲水色谱固定相的制备及性能研究

将不同比例的氨基和巯基的硅烷偶联剂键合到硅胶表面，再利用巯基与乙烯基膦酸之间的点击化学反应将膦酸基团引入到硅胶表面，制备了一种可调节正负离子比例的两性亲水色谱固定相。通过测定固定相中C、H、N、P元素的含量，证明了氨基与膦酸基团已成功键合到固定相的表面，同时通过N元素与P元素的质量分数确定固定相表面氨基与膦酸基团的比例。制备了3种不同电荷比例的氨基膦酸固定相，将其作为亲水模式下的固定相填料填装在150 mm×4.6 mm不锈钢色谱柱中。以一系列经典的极性小分子作为探针，研究了流动相中乙腈含量、缓冲盐pH值及缓冲盐浓度等因素对探针分子在3种色谱柱上的保留的影响，结果表明，分析物在固定相上是多重保留机理。最后通过比较核苷、水溶性维生素、碱性化合物、苯甲酸这几类标准物质在3种色谱柱上的保留行为来对比3种不同电荷比例的固定相的分离选择性与色谱性能。结果表明，对于不同的分析物，3种固定相表现出完全不同的分离选择性和色谱行为。可以根据分析物的特征选取不同电荷比例的固定相，表明此种固定相在极性化合物的分离上具有良好的应用前景。     

Preparation and evaluation of rigid porous polyacrylamide-based strong cation-exchange monolithic columns for capillary electrochromatography

A CEC monolithic column with strong cation-exchange (SCX) stationary phase based on hydrophilic monomers was prepared by in situ polymerization of acrylamide, methylenebisacrylamide, and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in a complete organic binary porogenic solvent consisting of DMSO and dodecanol. The sulfonic groups provided by the monomer AMPS on the surface of the stationary phase generate an EOF from anode to cathode, and serve as an SCX stationary phase at the same time. The monolithic stationary phase exhibited normal-phase chromatographic behavior for neutral analytes. For charged analytes, electrostatic interaction/repulsion with the monolith was observed. The strong SCX monolithic column has been successfully employed in the electrochromatographic separation of basic drugs, peptides, and alkaloids extracted from natural products.     

Selectivity differences of water‐soluble vitamins separated on hydrophilic interaction stationary phases

In this study, the retention behavior and selectivity differences of water‐soluble vitamins were evaluated with three types of polar stationary phases (i.e. an underivatized silica phase, an amide phase, and an amino phase) operated in the hydrophilic interaction chromatographic mode with ESI mass spectrometric detection. The effects of mobile phase composition, including buffer pH and concentration, on the retention and selectivity of the vitamins were investigated. In all stationary phases, the neutral or weakly charged vitamins exhibited very weak retention under each of the pH conditions, while the acidic and more basic vitamins showed diverse retention behaviors. With the underivatized silica phase, increasing the salt concentration of the mobile phase resulted in enhanced retention of the acidic vitamins, but decreased retention of the basic vitamins. These observations thus signify the involvement of secondary mechanisms, such as electrostatic interaction in the retention of these analytes. Under optimized conditions, a baseline separation of all vitamins was achieved with excellent peak efficiency. In addition, the effects of water content in the sample on retention and peak efficiency were examined, with sample stacking effects observed when the injected sample contained a high amount of water.     

Impact of column temperature and mobile phase components on selectivity of hydrophilic interaction chromatography (HILIC)

The retention mechanism and chromatographic behavior for different polar analytes under hydrophilic interaction chromatography (HILIC) conditions have been studied by application of different mobile phases and stationary phases to various analytes at different temperatures. In addition to the commonly accepted mechanism of analyte liquid-liquid partitioning between mobile phase and water-enriched solvent layer which is partially immobilized onto the surface of the stationary phase, hydrogen-bonding, hydrophobic interaction, and ion-exchange interactions may also be involved. The predominant retention mechanism in HILIC separation is not always easily predictable. It can depend not only on the characteristics of the analytes but also on the selection of mobile and stationary phase compositions. The objective of this review is to evaluate the potential application of column temperature and mobile phase composition toward improving HILIC selectivity. The functional groups from analyte structures, stationary phase materials and organic mobile phase solvents will be highlighted.     

Preparation and chromatographic performance of calix[4]crown-5 macrocycle-bonded silica stationary phase

A new calix[4]crown-5 macrocycle-bonded silica stationary phase (CL-CIMS) was prepared and applied at the same time to develop a chromatographic procedure to separate aromatic amines, phenols and drugs in this study. The chromatographic behaviors of the prepared stationary phase for these analytes were studied and compared with those of ODS (octadecylsilane). The effect of organic modifier content and pH of the mobile phase on retention and selectivity of these compounds were investigated. Some aromatic amines, phenols or drugs on CL-CIMS were successfully separated. The results show that CL-CIMS exhibits high selectivities for the above analytes in high aqueous mobile phases and a bright prospect in routine, fast separation of aromatic amines, phenols and drug compounds. From chromatographic data, it can be concluded that hydrophobic interaction is mainly responsible for the retention behavior as well as hydrogen-bonding interaction, π-π and dipole-dipole interaction.     

The use of derivatized cyclodextrins as solubilizing agents in the preparation of macroporous polymers employed as amphiphilic continuous beds in capillary electrochromatography

Employing solubilization by complexation with CDs, new mixed-mode monolithic stationary phases for CEC and micro-LC were synthesized. Free radical copolymerization was performed in aqueous solution with a CD-solubilized hydrophobic monomer, a water-soluble crosslinker (piperazinediacrylamide), and a charged monomer (vinylsulfonic acid). Different hydrophobic methacrylate monomers (isobornyl, adamantyl, cyclohexyl, and phenyl methacrylate) were investigated. Chromatographic properties of the synthesized monoliths were studied with aqueous and nonaqueous mobile phases with hydrophobic and polar analytes. Due to the amphiphilic nature of the polymers synthesized, the elution orders obtained correspond to the RP mode and to the normal-phase mode dependent on the polarity of the mobile phase. However, observations made with polar solutes and polar mobile phase can only be explained by a mixed-mode retention mechanism. The influence of the total monomer concentration (%T) on the chromatographic properties and on the specific permeability was elucidated. Run-to-run, day-to-day, and capillary-to-capillary reproducibility of electroosmotic mobility and retention factors were determined. Comparison of retention data with those of a commercial octadecyl silica gel HPLC column reveals that the methylene selectivity of the monolithic capillaries prepared in this study is very similar to that of routinely used octadecyl silica gels.     

Capillary electrochromatography with novel stationary phases: II. Studies of the retention behavior of nucleosides and bases on capillaries packed with octadecyl-sulfonated-silica microparticles

An octadecyl-sulfonated silica (ODSS) stationary phase specially designed for performing capillary electrochromatography (CEC) at relatively strong electroosmotic flow (EOF) proved useful for the separations of some nucleosides and bases. The ODSS stationary phase is composed of a hydrophilic, negatively charged sublayer to which a nonpolar top layer containing octadecyl ligands is covalently attached. The charged sublayer contains sulfonic acid groups which ensure a relatively strong EOF. Due to the presence of permanently charged sulfonic acid groups in the sublayer, the hydrophilic nature of the sublayer and the hydrophobic character of the top octadecyl layer, retention and selectivity of charged and relatively polar nucleosides and bases on the ODSS stationary phase are based on electrostatic interaction, hydrophilic interaction, and reversed-phase mechanisms. This yielded for the ODSS stationary phase a unique selectivity towards the nucleosides and bases, thus allowing their rapid separation. To gain insight into the chromatographic behavior of nucleosides and bases on the ODSS stationary phase, the results were compared to those obtained on an octadecyl-silica (ODS) capillary under otherwise the same elution conditions. Due to the difference in the nature of the organic layers on the surface of the ODSS and ODS stationary phases, the elution order on both stationary phases differed significantly, and the ODSS capillary proved more suitable for the separation of the nucleosides and bases than the ODS capillary.     

Methacrylate-based monolithic column with mixed-mode hydrophilic interaction/strong cation-exchange stationary phase for capillary liquid chromatography and pressure-assisted CEC

A novel porous polymethacrylate-based monolithic column by in situ copolymerization of 3-sulfopropyl methacrylate (SPMA) and pentaerythritol triacrylate in a binary porogenic solvent consisting of cyclohexanol/ethylene glycol was prepared. The monolith possessed in their structures bonded sulfonate groups and hydroxyl groups and was evaluated as a hydrophilic interaction and strong cation-exchange stationary phases in capillary liquid chromatography (cLC) and pressure-assisted CEC using small polar neutral and charged solutes. While the SPMA was introduced as multifunctional monomer, the pentaerythritol triacrylate was used to replace ethylene glycol dimethacrylate as cross-linker with much more hydrophilicity due to a hydroxyl sub-layer. The different characterization of monolithic stationary phases were specially designed and easily prepared by altering the amount of SPMA in the polymerization solution as well as the composition of the porogenic solvent for cLC and pressure-assisted CEC. The resulting monolith showed the different trends about the effect of the permeabilities on efficiency in the pressure-assisted CEC and cLC modes. A typical hydrophilic interaction chromatography mechanism was observed at higher organic solvent content (ACN%>70%) for polar neutral analytes. For polar charged analytes, both hydrophilic interaction and electrostatic interaction contributed to their retention. Therefore, for charged analytes, selectivity can be readily manipulated by changing the composition of the mobile phase (e.g., pH, ionic strength and organic modifier). With the optimized monolithic column, high plate counts reaching greater than 170 000 plates/m for pressure-assisted CEC and 105 000 plates/m for cLC were easily obtained, respectively.     

Single-step preparation and characterization of polymeric monolith for pressurized capillary electrochromatography of typical homologs

A monolithic stationary phase was prepared in a single step by in situ copolymerization of iso-butyl methacrylate (IBMA), ethylene dimethacrylate (EDMA), and N,N-dimethylallylamine (DMAA) in a binary porogenic solvent consisting of N,N-dimethylformamide (DMF) and 1,4-butanediol. As the frame structures of monoliths, the amino groups are linked to support the EOF necessary for driving the mobile phase through the monolithic capillary, while the hydrophobic groups are introduced to provide the nonpolar sites for the chromatographic retention. To evaluate the column performance, separations of typical kinds of neutral or charged homologs, such as alkylbenzenes, phenols (including isomeric compounds of hydroquinone, resorcin, and catechol), and anilines (including isomeric compounds of o-phenylenediamine and 1,4-phenylenediamine), were performed, respectively on the prepared column under the mode of pressurized pCEC. Effects of the buffer pH and the mobile phase composition on the linear velocity of mobile phase and the retention factors of these compounds were investigated. It was found that the retention mechanism of charged solutes could be attributed to a mixed mode of hydrophobic interaction and electrophoresis, while an RP chromatographic behavior on the monolithic stationary phases was exhibited for neutral solutes. Especially, basic compounds such as anilines were well separated on the monolithic columns in the "counterdirectional mode," which effectively eliminated the electrostatic adsorption of basic analytes on the charged surface of the stationary phases.     

Cucurbit(6)uril immobilized on silica: A novel high‐performance liquid chromatographic stationary phase

In the present study, one of the new generation of host molecules, cucurbit(6)uril (CB(6)), was immobilized onto silica (CB(6)/SiO₂) by a sol–gel approach. CB(6)/SiO₂ was characterized by NMR spectroscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, and elemental analysis. It was used as a high‐performance liquid chromatographic stationary phase and its chromatographic performance was systematically investigated with different types of analytes as probes. The results revealed that the CB(6)/SiO₂ stationary phase exhibited weak hydrophobic and strong hydrophilic properties. Hence, the variables for hydrophilic interaction liquid chromatography, including components and pH of the mobile phase, were further investigated to explore the retention mechanism of this CB(6)/SiO₂ stationary phase. For less polar analytes, both hydrophobic and hydrophilic interactions could contribute to the retention, while for polar analytes, hydrophilic interaction may be predominant. Compared to the tetraethoxylsilane‐coated SiO₂ stationary phases, the CB(6)/SiO₂ stationary phase exhibited a different retention behavior toward basic analytes with excellent stability. It is a novel promising hydrophilic interaction liquid chromatography stationary phase.     

Preparation of Cyclodextrin Type Stationary Phase Based on Graphene Oxide and Its Application in Enantioseparation and Hydrophilic Interaction Chromatography

Graphene oxide (GO) was covalently coupled to the surface of amino silica gel by amide bond. β-cyclodextrin (β-CD) was further chemically bonded with GO to prepare a novel chiral stationary phase. The resulting material was characterized by Fourier transform-infrared (FT-IR) spectra, scanning electron microscopy (SEM), transmission electron microscopy (TEM), elemental analysis and thermogravimetric analysis (TGA). The separation of seven enantiomers was improved in varying degrees. Meanwhile, the stationary phase showed typical characteristics of hydrophilic interaction chromatography (HILIC), and four small nucleoside molecules were separated with the mobile phase of methanol-acetonitrile-water (45:45:10, V/V) in the HILIC mode. In addition, the separation mechanism of the stationary phase was further explored by studying the effects of mobile phase composition, temperature and pH value on the analyte retention. The low temperature was conducive to the separation of analytes at 20–60 °C. The addition of protonated solvent methanol significantly decreased the retention time of the four analytes. The change of pH affected the degree of protonation of the analyte, the interaction between analytes and the stationary phase, and retention time of analytes. The results showed that GO and β-CD played a synergistic effect in the chiral resolution of the chromatographic stationary phase. The retention of analytes in HILIC was attributed to their mixed-mode retention mechanisms including hydrophilic interaction, electrostatic interaction, hydrogen bonding, π-π stacking and so on.     

Preparation of a Novel Amino-Phosphate Zwitterionic Stationary Phase for Hydrophilic Interaction Chromatography

A new stationary phase which contains both negatively charged phosphate groups and positively charged amino groups was successfully synthesized by modification of amino-functionalized silica particles with trichlorophosphine oxide (POCl₃) for hydrophilic interaction chromatography (HILIC). The composition of the surface grafts was determined by Fourier transform infrared spectroscopy and elemental analysis. Various parameters, such as column temperature, water content, pH values and ionic strength of the mobile phase were investigated to study the retention mechanism. The results demonstrated that the stationary phase involved a complex retention process including surface adsorption, partitioning and electrostatic interactions. Under optimized conditions, the separation of nucleobases and nucleosides, water-soluble vitamins, organic acids on the novel stationary phase could be achieved in the HILIC mode.     

Study of retention behaviour and mass spectrometry compatibility in zwitterionic hydrophilic interaction chromatography for the separation of modified nucleosides and nucleobases

A study has been made of the chromatographic behaviour of modified nucleosides and nucleobases using different stationary phases with functional groups of polar nature, all of them compatible with aquoorganic mobile phases. The stationary phases assayed were a pentafluorophenylpropyl (PFP) column for reverse phase separation, and another two for hydrophilic interaction chromatography (HILIC) separation. Six modified nucleosides and nucleobases (hydroxylated and methylated derivatives) were chosen as the target analytes. In the study, chromatographic resolution as well as the sensitivity in detection by mass spectrometry were taken into account. The results obtained showed that the zwitterionic (ZIC-HILIC) column was the most suitable one for the separation of these analytes. From the study of the different parameters affecting separation it may be concluded that in the ZIC-HILIC column separation is based on a mechanism of partition and interaction through weak electrostatic forces.     

Preparation of a Novel Amino-Phosphate Zwitterionic Stationary Phase for Hydrophilic Interaction Chromatography

A new stationary phase which contains both negatively charged phosphate groups and positively charged amino groups was successfully synthesized by modification of amino-functionalized silica particles with trichlorophosphine oxide (POCl₃) for hydrophilic interaction chromatography (HILIC). The composition of the surface grafts was determined by Fourier transform infrared spectroscopy and elemental analysis. Various parameters, such as column temperature, water content, pH values and ionic strength of the mobile phase were investigated to study the retention mechanism. The results demonstrated that the stationary phase involved a complex retention process including surface adsorption, partitioning and electrostatic interactions. Under optimized conditions, the separation of nucleobases and nucleosides, water-soluble vitamins, organic acids on the novel stationary phase could be achieved in the HILIC mode.

     

Preparation of a novel carboxyl stationary phase by “thiol‐ene” click chemistry for hydrophilic interaction chromatography

A novel carboxyl‐bonded silica stationary phase was prepared by “thiol‐ene” click chemistry. The resultant Thiol‐Click‐COOH phase was evaluated under hydrophilic interaction liquid chromatography (HILIC) mobile phase conditions. A comparison of the chromatographic performance of Thiol‐Click‐COOH and pure silica columns was performed according to the retention behaviors of analytes and the charged state of the stationary phases. The results indicated that the newly developed Thiol‐Click‐COOH column has a higher surface charge and stronger hydrophilicity than the pure silica column. Furthermore, the chromatographic behaviors of five nucleosides on the Thiol‐Click‐COOH phase were investigated in detail. Finally, a good separation of 13 nucleosides and bases, and four water‐soluble vitamins was achieved.