Postpolymerization modification of a hydroxy monolith precursor. Part I. Epoxy alkane and octadecyl isocyanate modified poly (hydroxyethyl methacrylate‐co‐pentaerythritol triacrylate) monolithic capillary columns for reversed‐phase capillary electrochromatography

A novel precursor monolithic capillary column referred to as “hydroxy monolith” or OHM was prepared by the in situ copolymerization of hydroxyethylmethacrylate (HEMA) with pentaerythritol triacrylate (PETA) yielding the neutral poly(HEMA‐co‐PETA) monolith. The neutral precursor OHM capillary thus obtained was subjected to postpolymerization modifications of the hydroxyl functional groups present on its surface with 1,2‐epoxyalkanes catalyzed by boron trifluoride (BF₃) ultimately providing Epoxy OHM C‐m capillary column at varying alkyl chain lengths where m = 8, 12, 14, and 16 for RP‐CEC. Also, the same precursor OHM was grafted with octadecyl isocyanate yielding Isocyanato OHM C‐18 column to provide an insight into the effect of the nature of the linkage to the surface hydroxyl groups of the OHM precursor. While the epoxide reaction leaves on the surface of the OHM precursor hydroxy‐ether linkages, the isocyanato reaction leaves carbamate linkages on the same surface of the OHM precursor. This study revealed that changing the alkyl chain length resulted in changing the column phase ratio (?) and also the solute distribution constant (K). While increasing the surface alkyl chain length increased steeply the solute hydrophobic selectivity, i.e. methylene group selectivity, the nature of the ligand linkage produced different retention for the same solutes and affected the selectivity of slightly polar solutes. The various monoliths proved very useful for RP‐CEC of different small solutes at varying polarity over a wide range of mobile phase composition.     

Capillary electrochromatography with novel stationary phases. IV. Retention behavior of glycosphingolipids on porous and non-porous octadecyl sulfonated silica

In this investigation, capillary electrochromatography (CEC) with a novel stationary phase proved useful for the separation of neutral and acidic glycosphingolipids (GSLs). Four different gangliosides, namely G(M1a), G(D1a), G(D1b) and G(T1b), served as the acidic GSLs model solutes. The following four GSLs: galactosylceramide (GalCer), lactosylceramide (LacCer), globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) served as the typical neutral GSLs. The stationary phase, octadecyl sulfonated silica (ODSS), consisted of octadecyl functions bonded to a negatively charged layer containing sulfonic acid groups. Porous and non-porous ODSS stationary phases were examined. The retention behavior of the acidic and neutral GSLs was examined over a wide range of elution conditions, including the nature of the electrolyte and organic modifier and the pH of the mobile phase. The porous ODSS stationary phase yielded the separation of the four different gangliosides using a hydro-organic eluent of moderate eluent strength whereas the non-porous ODSS stationary phase permitted the separation of the four neutral GSLs with a mobile phase of relatively high eluent strength.     

Capillary electrochromatography with monolithic stationary phases. 4. Preparation of neutral stearyl-acrylate monoliths and their evaluation in capillary electrochromatography of neutral and charged small species as well as peptides and proteins

A neutral, nonpolar monolithic capillary column having a relatively strong electroosmotic flow (EOF) yet free of electrostatic interactions with charged solutes was developed for the reversed-phase capillary electrochromatography (RP-CEC) of neutral and charged species including peptides and proteins. The neutral nonpolar monolith is based on the in situ polymerization of pentaerythritol diacrylate monostearate (PEDAS) in a ternary porogenic solvent composed of cyclohexanol, ethylene glycol, and water. PEDAS plays the role of both the cross-linker and the ligand provider, generating a macroporous nonpolar monolith having C17 chains as the chromatographic ligands. Despite the fact that the neutral PEDAS monolith is devoid of fixed charges, the monolithic capillary columns exhibited a relatively strong EOF due to the ability of PEDAS to adsorb sufficient amounts of electrolyte ions from the mobile phase. The adsorbed ions imparted the neutral PEDAS monolith the zeta potential necessary to support the EOF required for mass transport across the monolithic column. The absence of fixed charges on the surface of the neutral PEDAS monolith and in turn the adsorption sites for electrostatic attraction of charged solutes allowed the rapid and efficient separations of proteins and peptides at pH 7.0, with an average plate number of 255,000 and 121,000 plates/m, respectively. To the best of our knowledge, this constitutes the first report on the separation of proteins at neutral pH by RP-CEC using a neutral monolithic column.     

Capillary electrochromatography with monolithic silica column: I. Preparation of silica monoliths having surface-bound octadecyl moieties and their chromatographic characterization and applications to the separation of neutral and charged species

Monolithic silica columns with surface-bound octadecyl (C18) moieties have been prepared by a sol-gel process in 100 microm ID fused-silica capillaries for reversed-phase capillary electrochromatography of neutral and charged species. The reaction conditions for the preparation of the C18-silica monoliths were optimized for maximum surface coverage with octadecyl moieties in order to maximize retention and selectivity toward neutral and charged solutes with a sufficiently strong electroosmotic flow (> 2 mm/s) to yield rapid analysis time. Furthermore, the effect of the pore-tailoring process on the silica monoliths was performed over a wide range of treatment time with 0.010 M ammonium hydroxide solution in order to determine the optimum time and conditions that yield mesopores of narrow pore size distribution that result in high separation efficiency. Under optimum column fabrication conditions and optimum mobile phase composition and flow velocity, the average separation efficiency reached 160 000 plates/m, a value comparable to that obtained on columns packed with 3 microm C18-silica particles with the advantages of high permeability and virtually no bubble formation. The optimized monolithic C18-silica columns were evaluated for their retention properties toward neutral and charged analytes over a wide range of mobile phase compositions. A series of dimensionless retention parameters were evaluated and correlated to solute polarity and electromigration property. A dimensionless mobility modulus was introduced to describe charged solute migration and interaction behavior with the monolithic C18-silica in a counterflow regime during capillary electrochromatography (CEC )separations. The mobility moduli correlated well with the solute hydrophobic character and its charge-to-mass ratio.     

Recent developments in capillary electrophoresis and capillary electrochromatography of carbohydrate species

This review article is concerned with the recent developments in capillary electrophoresis (CE) and capillary electrochromatography (CEC) of carbohydrates. The literature shows that CE possesses impressive potential in the analysis of carbohydrates. On the other hand, CEC has just started to show promise in the analysis of carbohydrates. Advances in separation and detection approaches of derivatized and underivatized carbohydrates are discussed based on the available literature. In addition, important applications are illustrated.     

Recent advances in polymeric monolithic stationary phases for electrochromatography in capillaries and chips

This review article summarizes the advances made over the last two years in polymeric monoliths for capillary electrochromatography (CEC). It covers the scientific literature in the period extending form the second half of 2002 until the end of first half of 2004. Currently, there is an increasing interest in monolithic stationary phases in CEC as an alternative to particulate packed capillary columns due in major part to the simplicity of the in situ preparation of monolithic stationary phases and the availability of a wide chemistry for surface ligands, which allow for tailoring the chromatographic sorbent needed for solving a given separation problem(s). The various approaches, formats, and chemistries used for the preparation of monolithic stationary phases are described.     

Electrically driven microseparation methods for pesticides and metabolites: VI. Surfactant-mediated electrokinetic capillary chromatography of aniline pesticidic metabolites derivatized with 9-fluoroenylmethyl chloroformate and their detection by laser-induced fluorescence

In this report, we describe a surfactant-mediated electrokinetic capillary chromatography (SM-EKC) system for the separation of 9-fluoroenylmethyl chloroformate (FMOC)-derivatized anilines by capillary electrophoresis (CE). The SM-EKC system consisted of dioctyl sulfosuccinate (DOSS)/acetonitrile mixtures and was suited for the CE separation of the relatively hydrophobic FMOC-aniline analytes and other neutral compounds, e.g. alkylphenyl ketones. While the organic modifier acetonitrile (ACN) allowed the solubilization of the hydrophobic solutes and maintained the DOSS surfactant in its monomeric form by inhibiting micellization, the DOSS surfactant associated with the FMOC anilines to a varying degree thus leading to their differential migration and separation. Under these conditions, the FMOC-anilines were readily detected at the 10(-6) M level by UV at 214 nm and at the 10(-8) M level by laser-induced fluorescence (LIF) using a solid-state UV laser operating at 266 nm line as the excitation wavelength. The FMOC precolumn derivatization was also readily performed in lake water spiked with anilines at near the limit of detection (LOD) level. The lake water matrix showed no significant effects on the extent of derivatization at the LOD level as well as on the detection of the analytes due to the selectivity of the FMOC derivatization. The derivatization and detection of spiked lake water necessitated only the removal of microparticles by microfiltration prior to derivatization and detection.     

Chromatographic properties of zirconia-based stationary phases for ion exchange chromatography having surface bound cationic functions

A series of non-porous, microspherical zirconia-based stationary phases with surface bound cationic functions have been introduced and evaluated in ion exchange chromatography of proteins and small acidic solutes. Different surface modification procedures were evaluated in the covalent attachment of weak, strong or hybrid anion exchange moieties on the surface of non-porous zirconia micropar-ticles. N,N-Diethylaminoethanol (DEAE) was used as the weak anion exchange ligand while glycidyltrimethylammonium chloride, which was covalently attached to poly(vinyl alcohol) layer (PVAN) on the zirconia surface, constituted the strong anion exchange moiety. Partially quaternarized poly(ethyleneimine) hydroxyethylated (PEI) was used as the hybrid type of anion exchange coating. DEAE-zir-conia microparticles acted as purely cation exchange stationary phases toward basic proteins indicating the predominance of electron donor-electron acceptor interaction (EDA) with surface exposed zirconium sites as well as cation exchange mechanism via electrostatic interaction with unreacted and unshielded hydroxyl groups. PVAN-zirconia stationary phase exhibited anion exchange chromatographic properties toward acidic proteins, but EDA interaction has stayed as an important contributor to solute retention despite the presence of a relatively thick layer of poly(vinyl alcohol) on the surface of the zirconia particles. The modification of zirconia surface with partially quaternarized PEI proved to be the most effective approach to minimize Lewis acidic metallic properties of the support. In fact, PEI-zirconia stationary phase operated as an anion exchanger toward acidic proteins and other small acidic solutes.