Capillary electrochromatography with monolithic silica columns. IV. Electrochromatographic characterization of polar bonded monolithic stationary phases having surface-bound cyano functionalities

Two polar ligands, namely 3-hydroxypropionitrile and 1H-imidazole-4,5-dicarbonitrile (IDCN) were covalently attached to epoxy-activated silica-based monolithic capillary columns via an epoxide ring-opening reaction to yield CN-OH-Monolith and 2CN-OH-Monolith, respectively. The silica monolith was prepared by a sol-gel process, and the resulting "rod-like" stationary phase was subjected to pore tailoring with an alkaline solution to convert small pore domains to mesopore domains, thus yielding a monolith with bimodal pore structure consisting of flow through pores (i.e., flow channels for mobile-phase flow) and mesopores that provide most of the adsorption capacity of the monolith toward the separated solutes. The two polar monoliths, CN-OH-Monolith and 2CN-OH-Monolith, were evaluated in normal-phase CEC with organic-rich mobile phases less polar than the stationary phase. The 2CN-OH-Monolith bearing more polar functions than the CN-OH-Monolith exhibited more retention and improved selectivity toward model polar solutes.     

Single-step approach to beta-cyclodextrin-bonded silica as monolithic stationary phases for CEC

A novel single-step sol-gel approach for the preparation of beta-CD-bonded silica monolithic electrochromatographic columns is established. The porous silica networks were fabricated inside fused-silica capillaries using sol-gel processing of tetramethoxysilane and an organfunctional silicon alkoxide that contains beta-CD. Scanning electron micrographs and nitrogen adsorption-desorption data showed that these functional monolithic columns have double pores structures with micrometer-size co-continuous through-pores and silica skeletons with open mesopores. The beta-CD monolithic columns have successfully been applied to the separation of several neutral and negatively charged isomers by CEC. The column performance was evaluated by using positional isomers of naphthalenedisulfonic acid as model compounds. A plate height of less than 10 mum for the first eluted isomer of naphthalenedisulfonic acid was obtained at an optimal flow rate (0.47 mm/s) of the mobile phase. Moreover, the columns have been proved to be stable for more than 100 runs during 3 months period and show reasonable column reproducibility.     

Electrochromatographic enantioseparation using chiral ligand exchange monolithic sol-gel column

This paper describes the development of a monolithic sol-gel column modified with l-hydroxyproline as a ligand exchange chiral stationary phase. It has been demonstrated that the monolithic chiral stationary phase can be used for the enantioseparation of dansyl amino acids, free amino acids, hydroxy acids, and dipeptides by capillary electrochromatography and micro-liquid chromatography. The recommended mobile phase was acetonitrile/0.50 mM Cu(Ac)₂-50 mM NH₄Ac (7:3) adjusted to pH 6.5. The characteristics of the monolithic column using hydroxyproline as chiral selector in CEC have been discussed.     

Polymerised bicontinuous microemulsions as stationary phases for capillary electrochromatography

Summary Polymerisation of bicontinuous microemulsions yields porous monolithic structures with well defined pore sizes that are potentially suitable for use as stationary phases for capillary electrochromatography (CEC). A variety of pore sizes can be achieved by altering the composition of the microemulsion, which typically consists of butyl methacrylate (BMA) and ethylene glycol dimethacrylate (EGDMA) as the polymerisable oil phase. The aqueous phase consists of water, a surfactant (sodium dodecyl sulphate, SDS) and a co-surfactant (1-propanol). 2-acrylamido-2-methyl-1-propane sulfonic acid (AMPS) is also added to provide charges along the polymer backbone to allow electroosmotic flow (EOF) to occur. SEM analysis shows that in-situ polymerisation yields a monolithic structure with a porous topography. Investigations have shown that these monoliths are easy to prepare, robust and suitable for the separation of phthalates. They generate higher linear velocities than are achieved using the silica based HPLC packings normally used for CEC.     

Capillary electrochromatography with monolithic silica columns. V. Study of the electrochromatographic behaviors of polar compounds on monolithic silica having surface bound cyano functionalities

In this report, a novel polar monolithic capillary column is described for normal phase CEC (NP-CEC) of representative polar compounds including mono- and oligosaccharides, peptides, and basic drugs. The polar monolithic column, which was described in detail in the preceding paper, consisted of silica-based monolith bonded with 1H-imidazole-4,5-dicarbonitrile (IDCN) and is denoted as 2CN-OH-Monolith. Various retention parameters for neutral polar solutes (e.g., mono- and oligosaccharides) and charged polar solutes (e.g., peptides and basic drugs) were evaluated over a wide-range of elution conditions. These retention parameters yielded quantitative assessment for the polar interactions between the model solutes and the stationary phase under investigation as well as the effect of electromigration of charged solutes on their overall migration in NP-CEC. Furthermore, this investigation demonstrated that despite the possibility of achieving isocratic separation in NP-CEC for widely differing polar species, multistep-gradient elution in NP-CEC is preferred to bring about the rapid separation of a large number of polar species in a single run.     

Preparation of polar group derivative β-cyclodextrin bonded hydride silica chiral stationary phases and their chromatography separation performances

Three new polar group-substituted β-cyclodextrin derivatives were synthetized and they covalent bonded to hydride silica to obtain chiral stationary phases. Their separation results of 35 chiral pyrrolidine compounds were also presented and discussed.     

On-line concentration of neutral and charged species in capillary electrochromatography with a methacrylate-based monolithic stationary phase

A method involving self-concentration, on-column enrichment and field-amplified sample stacking for on-line concentration in capillary electrochromatography with a polymer monolithic column is presented. Since monolithic columns eliminate the frit fabrication and the problems associated with frits, the experimental conditions could be more flexibly adjusted to obtain higher concentration factor in comparison with conventional particulate packed columns. With self-concentration effect, the detection sensitivity of benzene and hexylbenzene is improved by a factor of 4 and 8, respectively. With on-column enrichment and ultralong injection, improvement as high as 22,000 times in detection sensitivity of benzoin is achieved. Furthermore, a combination of the three above-mentioned methods yields up to a 24,000-fold improvement in detection sensitivity for caffeine, a charged compound. Parameters affecting the efficiency of on-line concentration are investigated systematically. In addition, equations describing on-line concentration process are deduced.     

A novel monolithic column for capillary electrochromatographic separation of oligopeptides

A monolithic column was prepared using l-phenylalanine as template and a covalent approach through the formation of Schiff base with o-phthalaldehyde (OPA). OPA, allylmercaptan, l-phenylalanine, and triethylamine were stirred at first, then methacrylic acid, 2-vinylpyridine, ethyleneglycol dimethacrylate, α,α-azobisisobutyronitrile, and 1-propanol were added to the reaction mixture. The resulting material was introduced into a capillary column. Following thermal polymerization, the template was then extracted with a mixture of HCl and methanol. The column was employed for the capillary electrochromatographic separation of oligopeptides. A capillary column of 75 (50) cm × 75 μm ID with a mobile phase of phosphate buffer (pH 7.0, 40 mM)/methanol (5%, v/v), an applied voltage of +15 kV, and detection at 214 nm, could baseline separate angiotensin I, angiotensin II, [Sar¹, Thr⁸] angiotensin, oxytocin, vasopressin, tocinoic acid, β-casomorphin bovine, β-casomorphin human, and FMRF amide within 20 min. The separation behavior of the templated polymer was also compared with that of the non-templated polymer. As a result, it can be concluded that the electrochromatographic separation of this set of peptides was mediated by a combination of electrophoretic migration and chromatographic retention involving hydrophobic, hydrogen bonding, electrostatic as well as the Schiff base formation with OPA in the cavity of the templated polymer.     

Reversed-phase and weak anion-exchange mixed-mode silica-based monolithic column for capillary electrochromatography

A silica-based CEC monolithic column with mixed modes of RP and weak anion-exchange (WAX) was successfully prepared by using the sol-gel technique at mild temperature. The synthesizing procedure was optimized by changing the ratios of tetraethoxysilane (TEOS), aminopropyltriethoxysilane (APTES), and octyltriethoxysilane (C(8)-TEOS) in the mixture. While serving as WAX group, the amino group dominated the charge on the surface of the capillary column and generated an EOF from cathode to anode at low pH. At pH above 7.5, a cathodic EOF was observed due to the full ionization of silanol group and the suppression in the ionization of amino group. The morphology of monolithic columns was examined by SEM, and the performance of column was evaluated in detail by separating different kinds of compounds. As expected, the monolithic column exhibited RP chromatographic behavior for neutral solutes. Fast and efficient separation of six aromatic acids was obtained using acidic mobile phase with column efficiency up to 160,000 plates/m. Symmetrical peaks can be obtained for aromatic amines because positively charged amino groups on the surface can effectively minimize the adsorption of positively charged analytes to the stationary phase.     

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.     

Development of acrylate-based monolithic stationary phases for electrochromatographic separations

Organic monolithic stationary phases were synthesized in fused-silica capillaries. They were prepared by in situ polymerization under UV irradiation of various alkyl acrylates, 1,3-butanediol diacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid in a ternary porogenic solvent. The resulting stationary phases were tested in CEC. The influence of UV irradiation energy on the resulting separative performances of the monoliths was studied. It was thus demonstrated that the use of hexyl acrylate rather than butyl acrylate and lauryl methacrylate gives highly efficient monoliths (more than 300 000 plates per meter) with optimized EOF. It was also confirmed that the mobile phase ionic strength may affect significantly the separation efficiency. The influence of the nature of the mobile phase organic modifier (ACN or methanol) on EOF, retention, efficiency, and selectivity was studied and differences were observed. Finally, the performances of monolithic stationary phases developed and optimized for CEC separations were evaluated in nanoLC.     

Recognition of oxytocin by capillary electrochromatography with monolithic tetrapeptide-imprinted polymer used as the stationary phase

Using YPLG (Tyr-Pro-Leu-Gly), a tetrapeptide, as the template, an imprinted monolithic column was prepared and applied to the selective recognition of oxytocin based on the epitope approach and capillary electrochromatography (CEC). By optimizing the polymerization solution in terms of functional monomer, cross-linking reagent, porogen, and imprinted template via CEC evaluations of synthesized columns, an imprinted monolith with good recognition capacity (the imprinting factors for YPLG and oxytocin were 4.499 and 4.013, respectively) and high column efficiency (theoretical plates for YPLG and oxytocin were 22,995 plates/m and 16,952 plates/m, respectively) was achieved. In addition, the effects of various experimental parameters on the recognition of oxytocin, including the organic modifier content, the buffer concentration, and the pH value, were studied systematically. Furthermore, a mixture of oxytocin and other proteins was analyzed using this monolithic CEC column, and oxytocin was eluted much more slowly than other large biomolecules, which demonstrated the high selective recognition ability of such an imprinted monolith for oxytocin with PLG (Pro-Leu-Gly) as the epitope. Figure Separation of a mixture of oxytocin, BSA, bovine hemoglobin, ovalbumin, and lysozyme on the open column, the blank monolithic column, and the monolithic YPLG-imprinted column     

Characterization of polymer monolithic stationary phases for capillary HPLC

Monolithic capillary columns have been prepared in fused‐silica capillaries by radical co‐polymerization of ethylene dimethacrylate and butyl methacrylate in the presence of porogen solvent mixtures containing various concentration ratios of 1‐propanol, 1,4‐butanediol, and water with azobisisobutyronitrile as the initiator of the polymerization reaction. The through pores in organic polymer monolithic columns can be characterized by “equivalent permeability particle size”, and the mesopores with stagnant mobile phase by “equivalent dispersion particle size”. Increasing the concentration of propanol in the polymerization mixture diminishes the pore volume and size in the monolithic media and improves the column efficiency, at a cost of decreasing permeability. Organic polymer monolithic capillary columns show similar retention behaviour to packed alkyl silica columns for compounds with different polarities characterized by interaction indices, I_x, but have different methylene selectivities. Higher concentrations of propanol in the polymerization mixture increase the lipophilic character of the monolithic stationary phases. Best efficiencies and separation selectivities were found for monolithic columns prepared using 62–64% propanol in the porogen solvent mixture. To allow accurate characterization of the properties of capillary monolithic columns, the experimental data should be corrected for extra‐column contributions.     

Use of a polar-embedded stationary phase for the separation of tocopherols by CEC

A polar-embedded stationary phase (ULTIMA C18) has been investigated for the separation of alpha-, beta-, gamma- and delta-tocopherols by CEC in comparison with commercially available C(18) and C(30) n-alkyl RPs. The behavior of this stationary phase was tested for different mobile phases based on methanol, ACN, or mixtures thereof and different separation parameters such as retention factors and resolution were evaluated. The main feature of this stationary phase is the improved selectivity for the separation of beta- and gamma-tocopherols (positional isomers) when compared with the pure n-alkyl C(18) material, which was unable to resolve these compounds. Additionally, it is possible to observe a reversal in the elution order of the beta- and gamma-tocopherol isomers with respect to that obtained on the C(30) column. The resulting data indicate that the enhanced selectivity obtained with the polar-embedded stationary phase, with respect to the conventional C(18) material, is due to the participation of both hydrophobic and polar interactions: these latter are of the hydrogen bridge type with the amide group of the polar-embedded stationary phase, which increases the retention of the tocopherols and facilitates the discrimination between the beta- and gamma-isomers. Adequate separation of the four tocopherols was obtained by CEC using the polar-embedded stationary phase and 95:5 v/v methanol/water (5 mM Tris, final concentration) as the mobile phase.     

Preparation and characterization of monoliths covalently bonded chelating groups for capillary electrochromatographic separation of metal ions

In this work, a novel polymer-based monolithic column was prepared using an o-phthalaldehyde-l-phenylalanine Schiff base complex as the reactive center and a mixture of methanol and n-propanol as the porogen. The monolithic column was employed for the separation of a metal ion mixture including Pb(II), Mn(II), Cu(II), Ni(II), Cr(III), Fe(III) and Cr(VI). Tetrabutylammonium bromide (TBAB) was used as a mobile phase additive to enhance the separation efficiency of metal ions by EDTA precomplexation. Using a phosphate buffer (20 mM, pH 3.0), TBAB (10 mM), MeOH (15%, v/v), an applied voltage of −15 kV, and detection at 220 nm, the metal ion mixture was satisfactorily resolved. The average theoretical plate number was 17,900 plates/m. The separation was also carried out in the absence of TBAB, leading to dissimilar elution order and shorter retention time. The separation behavior of the monolithic column was also compared with that of the blank polymer. The unique properties of the monolithic column might be mediated by a combination of electrophoretic behavior and chromatographic retention involving hydrophobic and hydrophilic interactions, as well as ligand exchange.     

Preparation and evaluation of amphiphilic silica‐based monolithic column having surface‐bound octanoyl‐aminopropyl moieties for capillary electrochromatography

A novel amphiphilic silica‐based monolithic column having surface‐bound octanoyl‐aminopropyl moieties was successfully prepared by a one‐step in situ derivatization process. As expected, the amphiphilic monolithic column exhibited RP chromatographic behavior toward non‐polar solutes (e.g., alkyl benzenes) with high column performance. As the pH of the buffer inside the column increases, the EOF changed from −2.65×10⁻⁸m² V⁻¹s⁻¹ at pH 3.0 to 1.20×10⁻⁸ m² V⁻¹s⁻¹ at pH 8.0 with the reversion of EOF at about pH 6.4. Using acidic mobile phase, five aromatic acids can be efficiently separated in less than 6 min under co‐EOF conditions. For basic compounds, symmetrical peaks were obtained due to the existence of hydrophilic acyl amide group, which can effectively minimize the adsorption of the positively charged basic analyte to the silica‐based surface of the capillary column.     

A novel ionic liquid monolithic column and its separation properties in capillary electrochromatography

A novel ionic liquid (IL) monolithic capillary column was successfully prepared by thermal free radical copolymerization of IL (1-vinyl-3-octylimidazolium chloride, ViOcIm⁺Cl⁻) together with lauryl methacrylate (LMA) as the binary functional monomers and ethylene dimethacrylate (EDMA) as the cross-linker in binary porogen. The proportion of monomers, porogens and cross-linker in the polymerization mixture was optimized in detail. The resulting IL-monolithic column could not only generate a stable reversed electroosmotic flow (EOF) in a wide pH range (2.0–12.0), but also effectively eliminate the wall adsorption of the basic analytes. The obtained IL-monolithic columns were examined by scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR). These results indicated that the IL-monolithic capillary column possessed good pore properties, mechanical stability and permeability. The column performance was also evaluated by separating different kinds of compounds, such as alkylbenzenes, thiourea and its analogues, and amino acids. The lowest plate height of ∼6.8 μm was obtained, which corresponded to column efficiency (theoretical plates, N) of ∼147,000 plates m⁻¹ for thiourea. ILs, as a new type of functional monomer, present a promising option in the fabrication of the organic polymer-based monolithic columns in CEC.     

Preparation of a stationary phase with s‐triazine ring embedded group for reversed phase high‐performance LC

This paper describes the synthesis and chromatographic evaluation of a new polar‐embedded stationary phase, which utilized 2,4,6‐trichloro‐1,3,5‐triazine as the spacer. The resulting materials were characterized by elemental analysis, IR, and solid‐state ¹³C NMR. Empirical test mixtures were utilized to evaluate the column, and showed that it had good performance for basic compounds and high selectivity for polyaromatic hydrocarbons. Moreover, the novel stationary phase has unique property, especially in the separation of “homologous alkaloids” from natural products.     

A new method for in situ crosslinking of stationary phases on fused silica capillary columns

A new mixed crosslinking agent composed of dicumyl peroxide and tetra(methylvinyl)cyclotetrasiloxane was used to prepare fused silica capillary columns with in situ crosslinked stationary phases including PEG-20M, SE-54, and OV-1. These columns proved to have good thermostability and inertness. As examples of potential applications a mixture of isomers of nitrotoluene and dinitrotoluene, and pyrolyzates of polystyrene were separated by using these columns.     

Poly(divinylbenzene-alkyl methacrylate) monolithic stationary phases in capillary electrochromatography

In this study, a series of poly(divinylbenzene-alkyl methacrylate) monolithic stationary phases, which were prepared by single step in situ polymerization of divinylbenzene and various alkyl methacrylates (butyl-, octyl-, or lauryl-methacrylate), were developed as separation columns of benzophenone compounds for capillary electrochromatography (CEC). In addition to the presence of plenty of benzene moieties, the stationary phases contained long and flexible alkyl groups on the surface. With an increase in the molecular length of alkyl methacrylate, the polymeric monolith, which had higher hydrophobicity, effectively reduced the peak tailing of benzophenones, but a weaker retention was observed. The unusual phenomenon was likely due to the π–π interaction between the aromatic compound and the polymeric material. The usage of longer alkyl methacrylate as reaction monomer limited the retention of aromatic compounds on the stationary phase surface, thus the π–π interaction between them was possibly reduced. Consequently, the retention time of aromatic compounds was markedly decreased with an increase in carbon length of alkyl methacrylate that was carried on the polymeric monolith. Compared to previous reports on polystyrene-based columns in which the peak-tailing problem was reduced by decreasing the benzene moieties on the stationary phase, this study demonstrated that the undesirable retention (peak-tailing) could also be improved by the inclusion of long alkyl methacrylate to the polystyrene-based columns.