Phenylaminopropyl silica monolithic column for pressure assisted capillary electrochromatography

A novel stationary phase phenylaminopropyl silica (PhA-silica) monolith was successfully prepared for pressure assisted capillary electrochromatography (pCEC). The monolithic silica matrix from a sol-gel process was chemically modified by using [3-(phenylamino)propyl]trimethoxysilane as surface modification reagent to produce the phenylaminoporpyl function. The secondary amino groups on the surface of the monolithic stationary phase contributed to the generation of anodic electroosmotic flow (EOF) under acidic conditions. The phenyl group together with the spacer (-(CH(2))(3)-) in PhA-silica provides sufficient hydrophobic properties. To evaluate the column performance, effects of buffer pH and mobile phase composition on the mobile phase linear velocity and the retention factors of alkylbenzenes, phenols and anilines were investigated in pCEC mode. The monolithic stationary phases exhibit typical reversed-phase (RP) electrochromatographic behavior toward neutral solutes. Hydrophobic as well as electrophoretic migration process within the monoliths was observed for the separation of basic solutes such as anilines without peak tailing.     

Electrochromatographic performance of conventional and polar-embedded C16 silica monolithic stationary phases

A novel monolithic silica column that has a polar‐embedded amide‐secondary amine group linking with C16 functionality for RP‐CEC is described. The amide‐secondary aminealkyloxysilane was synthesized by the reaction of 3‐(2‐aminoethylamino) propyltrimethoxysilane with hexadecanoyl chloride. Then, the silylant agent was bonded to the silica monolith matrix to produce hexadecanamide‐secondary amine bonded silica (HDAIS) monolithic column. The electrochromatographic performance of HDAIS monolithic column for the separation of neutral, basic and polar solutes was studied, which was compared to that using the hexadecyl bonded silica monolithic column. The HDAIS monolithic column displayed reduced hydrophobic retention characteristics in the separation of five hydrophobic n‐alkylbenzenes and four polar phenols when compared to the hexadecyl bonded silica monolithic column. A very much reduced silanol activity of HDAIS monolithic column was observed in the separation of test basic mixture including four aromatic amines, atenolol and metoprolol with 10 mM borate buffer (pH 7.5) containing 30% v/v ACN as the mobile phase. The comparison results indicate good performance for both polar and basic mixtures on HDAIS monolithic column in RP‐CEC, and also show promising results for further applications.     

Monolithic silica columns with mixed mode of hydrophilic interaction and weak anion-exchange stationary phase for pressurized capillary electrochromatography

A silica-based monolithic column as polar stationary phase is proposed for pressurized CEC (pCEC). The monolithic silica matrix from a sol-gel process was chemically modified by 3-aminopropyltrimethoxysilane to produce a column for hydrophilic interaction applications. The amino groups on the surface of the polar stationary phase generated anodic EOF under acidic conditions and served at the same time as a weak anion-exchanger. The anion solutes such as nucleotides were separated by the mixed mode mechanism, which comprised hydrophilic interaction, weak anion-exchange, and electrophoresis. The influences of buffer concentration and organic modifier content on the separation of nucleotides by pCEC have been investigated. In addition, the monolithic silica columns were also able to separate various polar compounds such as phenols, nucleic acid bases, and nucleosides in the hydrophilic interaction CEC mode.     

一种内嵌三嗪环酰胺极性基团新型固定相的制备及其在碱性化合物分离中的应用

以γ-氨丙基三乙氧基硅烷为偶联剂,三聚氯氰为反应物,采用固液表面连续反应法,依次与乙二胺、十二酰氯进行亲核取代反应,制备了一种嵌入三嗪环酰胺极性基团的新型反相色谱固定相,并采用元素分析法进行了表征。用制备的固定相装填色谱柱,以商品化C18色谱柱作为参考,对比考察了碱性化合物的分离情况。结果表明,极性三嗪环酰胺基团被成功地键合到硅胶表面,连续制备3次所得固定相的C、N、H含量的最大相对偏差均小于5%,说明制备工艺重现性良好;用制备的固定相装填的色谱柱分离5种苯胺类、4种吡啶类碱性化合物的选择性好,峰形对称。该结果为进一步推进该新型固定相的商品化提供了参考数据。     

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.     

Preparation and evaluation of a neutral methacrylate-based monolithic column for hydrophilic interaction stationary phase by pressurized capillary electrochromatography

A polar and neutral polymethacrylate-based monolithic column was evaluated as a hydrophilic interaction capillary electrochromatography (HI-CEC) stationary phase with small polar–neutral or charged solutes. The polar sites on the surface of the monolithic solid phase responsible for hydrophilic interactions were provided from the hydroxy and ester groups on the surface of the monolithic stationary phase. These polar functionalities also attract ions from the mobile phase and impart the monolithic solid phase with a given zeta potential to generate electro-osmotic flow (EOF). The monolith was prepared by in situ copolymerization of a neutral monomer 2-hydroxyethyl methacrylate (HEMA) and a polar cross-linker with hydroxy group, pentaerythritol triacrylate (PETA), in the presence of a binary porogenic solvent consisting cyclohexanol and dodecanol. A typical HI-CEC mechanism was observed on the neutral polar stationary phase for both neutral and charged analytes. The composition of the polymerization mixture was systematically altered and optimized by altering the amount of HEMA in the polymerization solution as well as the composition of the porogenic solvent. The monoliths were tested in the pCEC mode. The resulting monoliths had different characteristics of hydrophilicity, column permeability, and efficiency. The effects of pH, salt concentration, and organic solvent content on the EOF velocity and the separation of nucleic acids and nucleosides on the optimized monolithic column were investigated. The optimized monolithic column resulted in good separation and with greater than 140,000 theoretical plates/m for pCEC.     

Silica‐based zwitterionic monolithic stationary phase for separation of neutral and ionized solutes using pressurized CEC

A porous zwitterionic monolith was prepared by in situ covalent attachment of lysine to a γ‐glycidoxypropyltrimethosysilane‐modified silica monolith. The prepared column was used to perform neutral and ionized solutes separations by pressurized (pCEC). Due to the zwitterionic nature of the resulting stationary phase, the monolithic column provided both electrostatic attraction and repulsion sites for electrochromatographic retention for ionized solutes. Separation of several nucleotides was investigated on the monolithic column. It was shown that the nucleotides could be separated based on hydrophilic and electrostatic interactions between the stationary phase and analyte. Besides, the separation property of the zwitterionic silica monolith was compared with the use of diamine‐bonded silica monolith as stationary phase. As expected, the lysine monolith exhibited a lower retention for the five nucleotides, which was due to the dissociation of the external carboxylic acid groups, leading to electrostatic repulsion with negatively charged solutes. Under the same experimental conditions, separation of the five nucleotides on the zwitterionic column was in less than 8 min, while that on the diamine column was in approximately 60 min.     

Preparation and evaluation of a lysine-bonded silica monolith as polar stationary phase for hydrophilic interaction pressurized capillary electrochromatography

A silica-based monolith as polar stationary phase was described for hydrophilic interaction pressurized capillary electrochromatography (HI-pCEC). The polar monolithic column was prepared by on-column reaction of lysine with epoxy groups on a gamma-glycidoxypropyltrimethosysilane-modified silica monolith. The stationary phase yielded strong hydrophilic interaction due to the slightly polar hydroxyl groups, and the strong polar lysine ligand with amino groups and carboxylic groups contained on the surface of the monolith. In order to evaluate the hydrophilic character of lysine ligand, the chromatographic behaviors of epoxy monolith (before lysine bonded) and diol monolith (hydroxyl groups contained) were also investigated. Two groups of comparative experiment were developed in terms of the separation of typical neutral non-polar and polar compounds performed in a mobile phase of aqueous-acetonitrile solution. Results showed that the lysine monolith was much more hydrophilic than the diol monolith, which presented less hydrophobic than the epoxy monolith. For further study on its hydrophilic character, the lysine monolith was demonstrated in the HI-pCEC mode for the separations of various polar compounds such as phenols, nucleic acid bases and nucleosides.     

基于二醛微晶纤维素功能化C18的反相/亲水色谱固定相的制备

通过十八烷基胺的氨基与二醛微晶纤维素的醛基共价键合,制备了基于二醛微晶纤维素(DMCC)官能化C₁₈的新型反相/亲水色谱固定相(C₁₈-DMCC/SiO₂),该色谱固定相被用于反相色谱(RPLC)和亲水相互作用色谱(HILIC)模式。C₁₈-DMCC/SiO₂色谱柱展现了良好的疏水选择性和芳香选择性,在反相色谱模式下可分离烷基苯和多环芳烃(PAHs)。苯胺类、酚类和糖苷类等极性化合物被用于评估该色谱柱在反相色谱模式下的极性选择性,商品C₁₈柱作对照柱,色谱评价结果令人满意。核酸碱基被用于评估C₁₈-DMCC/SiO₂色谱柱的亲水色谱性能。通过考察有机溶剂含量对分析物保留的影响,发现该新型色谱固定相具有反相/亲水色谱的典型特征。     

HPLC Separation of Different Groups of Small Polar Compounds on a Novel Amide-Embedded Stationary Phase

Retention behaviors of an amide-embedded silica base stationary phase, which was recently developed by our group, were studied by using six different groups of small polar compounds including phenolic compounds, substituted anilines, chlorinated herbicides, Sudan dyes and some nucleotides and nucleosides in HPLC. The chromatographic behaviors of the prepared stationary phase for these analytes were compared with those of a commercially available reversed-phase column ACE C18 under same conditions. Among the six groups of analytes studied, the amide-silica stationary phase showed enhanced selectivity towards phenolic compounds, substituted anilines, Sudan dyes and herbicides under reversed-phase conditions and satisfactory selectivity towards nucleosides and nucleotides which could not be separated with ACE C18 column under HILIC conditions. Experimental data provided some evidence that functional groups on the stationary phases might have certain degrees of influence on selectivity possibly through secondary interactions with the model compounds. The retentions of the moderately polar compounds such as phenolic acids, anilines and herbicides on the stationary phase are higher than highly polar compounds such as nucleotides and nucleosides due to both the hydrophobic and hydrophilic interactions between the stationary phase and analytes. The quantitative determination of Sudan dyes (I, II, III, and IV) in red chilli peppers was performed. Many red chilli peppers were screened and three of them contained Sudans dyes.     

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.     

HPLC Separation of Different Groups of Small Polar Compounds on a Novel Amide-Embedded Stationary Phase

Retention behaviors of an amide-embedded silica base stationary phase, which was recently developed by our group, were studied by using six different groups of small polar compounds including phenolic compounds, substituted anilines, chlorinated herbicides, Sudan dyes and some nucleotides and nucleosides in HPLC. The chromatographic behaviors of the prepared stationary phase for these analytes were compared with those of a commercially available reversed-phase column ACE C18 under same conditions. Among the six groups of analytes studied, the amide-silica stationary phase showed enhanced selectivity towards phenolic compounds, substituted anilines, Sudan dyes and herbicides under reversed-phase conditions and satisfactory selectivity towards nucleosides and nucleotides which could not be separated with ACE C18 column under HILIC conditions. Experimental data provided some evidence that functional groups on the stationary phases might have certain degrees of influence on selectivity possibly through secondary interactions with the model compounds. The retentions of the moderately polar compounds such as phenolic acids, anilines and herbicides on the stationary phase are higher than highly polar compounds such as nucleotides and nucleosides due to both the hydrophobic and hydrophilic interactions between the stationary phase and analytes. The quantitative determination of Sudan dyes (I, II, III, and IV) in red chilli peppers was performed. Many red chilli peppers were screened and three of them contained Sudans dyes.

     

Preparation and evaluation of diblock copolymer‐grafted silica by sequential surface initiated‐atom transfer radical polymerization for reverse‐phase/ion‐exchange mixed‐mode chromatography

A novel approach that involved the grafting of diblock copolymer with two types of monomer onto substrate by sequential surface initiated‐atom transfer radical polymerization was proposed to prepare a mixed‐mode chromatographic stationary phase. The distinguishing feature of this method is that it can be applied in the preparation of various mixed‐mode stationary phases. In this study, a new reverse‐phase/ion‐exchange stationary phase was prepared by grafting hydrophobic styrene and cationic sodium 4‐styrenesulfonate by the proposed approach onto silica surface. The chromatographic properties of the prepared stationary phase were evaluated by the separation of benzene derivatives, anilines, and β‐agonists, and by the effect of pH values and acetonitrile content on the retention. Compared with typical RP columns, the prepared stationary phase achieved the better resolution and higher selectivity at a shorter separation time and lower organic content. Moreover, the application of the prepared column was proved by separating widely distributed polar and charged compounds simultaneously.     

Development and application of a new 25,27‐bis(l‐phenylalaninemethylester‐N‐carbonylmethoxy)‐26,28‐dihydroxy‐para‐tert‐butylcalix[4]arene stationary phase

A 25,27‐bis(l ‐phenylalaninemethylester‐N‐carbonylmethoxy)‐26,28‐dihydroxy‐ para‐tert‐butylcalix[4]arene‐bonded silica gel stationary phase was synthesized, structurally characterized and used for LC. Its separation mechanism was studied and compared with octadecyl‐bonded stationary phase, as well as our previously prepared para‐tert‐butylcalix[4]arene‐1,2‐crown‐4 stationary phase. Meanwhile, the chromatographic behaviors were investigated by using polycyclic aromatic hydrocarbons, monosubstituted benzenes, anilines, phenols, Tanaka tests solutes, fluoroquinolones, and flavonoids as probes. Mechanisms involved in the chromatographic separation included hydrophobic, π‐π and π‐electron transfer, hydrogen bonding, and inclusion interactions. Moreover, the column was successfully employed for the analysis of the illegal additive of melamine in milk product.     

Preparation and Characterization of Sol-Gel SE-30-Coated Silica Stationary Phase for Capillary Liquid Chromatography

A sol-gel chemistry-based polymer coating approach was developed for the preparation of a novel polysiloxane-coated silica stationary phase for capillary liquid chromatography. SE-30, a commercial polysiloxane stationary phase used in gas chromatography, was incorporated into the properly designed sol solution. Then the sol-gel mixture was introduced into a silica gel-packed capillary column by pressure. A thin film of sol-gel SE-30-coating is chemically bonded to the surface of silica gel particles by hydrolytic polycondensation under mild conditions without any free radical cross-linking procedures, therefore the sol-gel approach offers a simple and effective pathway to create a hybrid polymer-coated silica stationary phase. Various factors affecting column making were optimized and discussed in this report. The resulting stationary phase showed good permeability, mechanical robustness, high durability to alkaline mobile phase and satisfactory chromatographic performance in separations of polar and non-polar aromatic compounds. Linear solvation energy relationships (LSERs) studies indicate that the stationary phase has a reversed-phased character with SE-30 providing chromatographic functionality. The solute size and the solute hydrogen bond ability are major factors that principally govern the retention of test solutes.     

Phenyl‐bonded stationary phases—The influence of polar functional groups on retention and selectivity in reversed‐phase liquid chromatography

The chromatographic properties of four phenyl‐bonded phases with different structures were studied. The columns used were packed with a stationary phase containing a phenyl ring attached to the silica surface using different types of linkage molecules. As a basic characteristic of the bonded phases, the hydrophobicity and silanol activity (polarity) were investigated. The presence of the polar amino and amide groups in the structure of the bonded ligand strongly influences the polarity of the bonded phase. Columns were compared according to methylene selectivity using a series of benzene homologues and according to their shape and size selectivity using polycyclic aromatic hydrocarbons. The measurements were done using methanol/water and acetonitrile/water mobile phases. The presented results show that the presence of polar functional groups in the ligand structure strongly influences the chromatographic properties of the bonded phase.     

Preparation and Characterization of a New Quercetin‐bonded Stationary Phase for High Performance Liquid Chromatography

A quercetin‐bonded silica gel stationary phase (QUSP) containing natural flavonoid ligand was first prepared via γ‐glycidoxypropyltrimethoxysilane (KH‐560) as a coupling reagent for high‐performance liquid chromatography. Its chemical structure was characterized by Fourier infrared spectroscopy, elemental analysis, thermal thermogravimetry and ¹³C cross polarization/magic angle spinning nuclear magnetic resonance (CP/MAS NMR). The chromatographic property of QUSP was systematically evaluated by using neutral, basic and acidic aromatic compounds as probes. In order to clarify its retention mechanism, a comparative study of QUSP with conventional octadecylsilyl‐bonded stationary phase (ODS) was also carried out under the same conditions. The results showed that the new quercetin‐bonded phase exhibited an excellent reversed‐phase chromatographic property with relatively weak hydrophobicity. However, it has an advantage over ODS in the fast separation of polar aromatic compounds because the quercetin ligand could provide various sites besides hydrophobicity, such as hydrogen bonding, dipole‐dipole, π‐π staking and charge transfer interactions. QUSP was performed in the baseline separations of ionized polar basic or acidic compounds, including pyridines, anilines, pyrimidines, purines and phenols with symmetric peak shape in common mobile phases without buffer salt within relatively short time. The natural ligands from herbs are readily available and contain a variety of active sites, which facilitate the exploration of industrial chromatographic separation materials for green products.     

Study of a monolithic silica capillary column coated with poly(octadecyl methacrylate) for the reversed-phase liquid chromatographic separation of some polar and non-polar compounds

The performance of a monolithic silica capillary column coated with poly(octadecyl methacrylate) (ODM column) for the reversed-phase liquid chromatographic separation of some polar and non-polar compounds was studied, and the results were compared to those obtained by using a monolithic silica capillary column modified with octadecylsilyl-(N,N-diethylamino)silane (ODS column). Benzene and naphthalene derivatives, polycyclic aromatic hydrocarbons (PAHs), steroids, alkyl phthalates, and tocopherol homologues were used as test samples. In general, compounds with aromatic character, rigid and planar structures, and lower length-to-breadth ratios (more compacted structures) seem to have more preference for the polymer coated stationary phase (ODM). Compounds with acidic character have also a higher retention on ODM columns because of the presence of ester groups in the stationary phase. The polymer coated column allowed the separation of some PAHs, alkyl phthalates, steroids, and of beta- and gamma-tocopherol isomers which cannot be separated under the same conditions on ODS columns, while keeping similar column efficiency. These results allowed to suggest ODM columns as a good alternative to conventional ODS columns for reversed-phase liquid chromatography.     

Chromatographic characteristics of poly(1-vinylimidazole)-grafted silica in normal-phase HPLC

A stationary phase based on poly(1-vinylimidazole)-grafted silica has been prepared by the surface radical chain-transfer reaction. The stationary phase was characterized by infrared spectra, X-ray photoelectron spectroscopy and elemental analysis. Chromatographic characteristics of the stationary phase were investigated in normal-phase HPLC. The results showed that both weak polar compounds (polycyclic aromatic hydrocarbons, dialkyl phthalates) and polar compounds (anilines, phenols) could be successfully separated on this stationary phase, implying better separation performance than blank silica and conventional aminopropyl-bonded silica under the same conditions. The excellent performance can be attributed to multiple interactions between surface modifier and the analytes that might include dipole, hydrogen bonding, H-π, electrostatic and inductive interactions.     

Imidazolium‐embedded iodoacetamide‐functionalized silica‐based stationary phase for hydrophilic interaction/reversed‐phase mixed‐mode chromatography

A novel imidazolium‐embedded iodoacetamide‐functionalized silica‐based stationary phase has been prepared by surface radical chain‐transfer polymerization. The stationary phase was characterized by Fourier transform infrared spectrometry, thermogravimetric analysis, and element analysis. Fast and efficient separations of polar analytes, such as nucleosides and nucleic acid bases, water‐soluble vitamins and saponins, were well achieved in hydrophilic interaction chromatography mode. Additionally, a mixed mode of hydrophilic interaction and reversed‐phase could be also obtained in the analysis of polar and nonpolar compounds, including weak acidic phenols, basic anilines and positional isomers, with high resolution and molecular‐planarity selectivity, outperforming the commercially available amino column. Moreover, simultaneous separation of polar and nonpolar compounds was also achieved. In conclusion, the multimodal retention capabilities of the imidazolium‐embedded iodoacetamide‐functionalized silica‐based column could offer a wide range of retention behavior and flexible selectivity toward hydrophilic and hydrophobic compounds.