On-column trace enrichment by sequential frontal and elution electrochromatography II. Enhancement of sensitivity by segmented capillaries with z-cell configuration--application to the detection of dilute samples of moderately polar and nonpolar pesticides

An on-column trace enrichment method for capillary electrochromatography of dilute samples is described. It involves the sequential use of frontal and elution electrochromatography on a segmented capillary column comprising of two contiguous segments each packed with a different sorbent. While the entering segment is for preconcentration by frontal electrochromatography the second segment is much longer and is meant for separation of the enriched analytes in the subsequent elution electrochromatography step. The preconcentration segment is usually packed with a sorbent that affords the highest affinity towards the solutes of interest while the separation segment is packed with a stationary phase that exhibits the highest selectivity and separation efficiency for the analytes. The detection is performed in the UV using a z-cell configuration for achieving an increased path length for detection. The effectiveness of this on-column trace enrichment is demonstrated on dilute samples of moderately polar solutes (e.g., carbamate insecticides) and nonpolar solutes (e.g., pyrethroid insecticides). Under optimal frontal and elution electrochromatography conditions. 817- and 1100-fold sensitivity increase are achieved for permethrin (a pyrethroid insecticide) and methiocarb (a carbamate insecticide), respectively, with a UV detector. The method is demonstrated with real water samples (e.g., tap and lake water samples) spiked with carbamate and pyrethroid insecticides. The limits of detection for the pesticides achieved in tap and lake waters reached 10(-8) to 10(-9) M.     

Capillary electrochromatography with monolithic silica columns III. Preparation of hydrophilic silica monoliths having surface-bound cyano groups: chromatographic characterization and application to the separation of carbohydrates, nucleosides, nucleic acid bases and other neutral polar species

Two synthetic routes have been introduced and evaluated for the preparation of hydrophilic silica-based monoliths possessing surface-bound cyano functions. In one synthetic scheme, the silica monolith was reacted in a single step with 3-cyanopropyldimethylchlorosilane to yield a cyano phase referred to as CN-monolith. In a second synthetic route, the silica monolith was first reacted with gamma-glycidoxypropyltrimethoxysilane (gamma-GPTS), followed by a reaction with 3-hydroxypropionitrile (3-HPN) to give a stationary phase denoted CN-OH-monolith. Although the gamma-GPTS was intended to play the role of a spacer arm to link the 3-HPN to the silica surface, this spacer arm became an integral part of the hydrophilic stationary phase. Thus, the CN-OH-monolith can be viewed as a double-layered stationary phase (i.e., stratified phase) with a hydroxy sub-layer and a cyano top layer. Due to its stronger hydrophilic character, the CN-OH-monolith yielded higher retention and better selectivity than the CN-monolith. The CN-OH-monolith was demonstrated in the normal-phase capillary electrochromatography (CEC) of various polar compounds including phenols and chloro-substituted phenols, nucleic acid bases, nucleosides, and nitrophenyl derivatives of mono- and oligosaccharides. The CN-OH-monolith yielded a relatively strong electroosmotic flow over a wide range of mobile phase composition, thus allowing rapid separation of the polar compounds studied.     

Capillary electrochromatography with monolithic stationary phases: 1. Preparation of sulfonated stearyl acrylate monoliths and their electrochromatographic characterization with neutral and charged solutes

A novel monolithic stationary phase having long alkyl chain ligands (C17) was introduced and evaluated in capillary electrochromatography (CEC) of small neutral and charged species. The monolithic stationary phase was prepared by the in situ copolymerization of pentaerythritol diacrylate monostearate (PEDAS) and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in a ternary porogenic solvent consisting of cyclohexanol/ethylene-glycol/water. While AMPS was meant to support the electroosmotic flow (EOF) necessary for transporting the mobile phase through the monolithic capillary, the PEDAS was introduced to provide the nonpolar sites for chromatographic retention. Monolithic columns at various EOF velocities were readily prepared by conveniently adjusting the amount of AMPS in the polymerization solution as well as the composition of the porogenic solvent. The monolithic stationary phases thus obtained exhibited reversed-phase chromatography behavior toward neutral solutes and yielded a relatively strong EOF. For charged solutes (e.g., dansyl amino acids), nonpolar as well as electrostatic interaction/repulsion with the monoliths were observed in addition to electrophoretic migration. Therefore, for charged solutes, selectivity and migration can be readily manipulated by changing various parameters including the nature of the monolith and the composition of the mobile phase (e.g., pH, ionic strength and organic modifier). Ultrafast separation on the time scale of seconds of 17 different charged and neutral pesticides and metabolites were performed using short capillary columns of 8.5 cm x 100 microm ID.     

Tandem use of carboxypeptidase Y reactor and displacement chromatograph for peptide synthesis

A packed-bed enzyme reactor with immobilized carboxypeptidase Y was used in tandem with a displacement chromatograph for the preparation of N-benzoyl-L-arginyl-L-methioninamide, from N-benzoyl-L-arginine and L-methioninamide. The pumps and valves of the coupled enzyme reactor and displacement chromatograph were controlled by a microprocessor. The enzyme was immobilized on microparticulate amino-silica by glutaraldehyde and packed into a 60 X 4.6 mm I.D. column. The packed-bed reactor was used in the recirculating mode and components of the reaction mixture were subsequently separated by displacement chromatography on a 250 X 4.6 mm octadecyl-silica column using butoxyethoxyethanol as the displacer. Unreacted L-methioninamide was returned to the reaction mixture. Both the progress of the reaction and the extent of separation by displacement chromatography were monitored by high-performance liquid chromatographic analysis. The system was designed so that enzymatic peptide synthesis, separation by displacement chromatography, and column regeneration were carried out simultaneously by using two identical columns in parallel. An amount of 460 mg of N-benzoyl-L-arginyl-L-methioninamide having purity greater than 99% could be obtained in 24 h with this system. The tandem operation of the enzyme reactor and liquid chromatograph operated in the displacement mode offers a means for the synthesis and purification of peptides.     

Electrically driven microseparation methods for pesticides and metabolites: III. Capillary electrochromatography with novel silica-based stationary phases having a surface-bound surfactant moiety

A silica-based stationary phase with surface bound silylpropyl trialkylammonium functions was introduced and evaluated in the capillary electrochromatography of alkylbenzenes and pesticides. This stationary phase is referred to as octadecyldimethyl(3-trimethoxysilylpropyl) ammonium-silica (ODAS) and has quaternary amine functions that generate an anodic electroosmotic flow (EOF) and octadecyl functions that are responsible for solute retention by a reversed-phase chromatography mechanism. The ODAS stationary phase was characterized over a wide range of elution conditions in term of EOF and retention behavior of alkylbenzene homologous series. The ODAS stationary phase proved useful in the separation of pesticides as well as in the on-column preconcentration of dilute pesticide samples, thus permitting the detection of solution at 7 x 10(-7) M using a UV detector.     

Precursor Carboxy-silica for Functionalization With Interactive Ligands. II. Carbodiimide Assisted Preparation of Silica Bonded Stationary Phases with D-glucamine for Hydrophilic Interaction Liquid Chromatography

Chromatographia - A precursor carboxy-silica support was introduced for grafting a retentive polar ligand, namely D-glucamine, for use in hydrophilic interaction liquid chromatography. This support...     

Poly(carboxyethyl acrylate-co-ethylene glycol dimethacrylate) precursor monolith with bonded octadecyl ligands for use in reversed-phase capillary electrochromatography

A carboxy precursor monolithic column, namely poly(carboxy ethyl acrylate-co-ethylene glycol dimethacrylate) was first produced in a 100 μm i.d. fused-silica capillary and subsequently surface bonded with n-octadecyl (C₁₈) ligands by a post-polymerization functionalization process with octadecylamine in the presence of N,N´-dicyclohexylcarbodiimide. The bonding of octadecyl ligands was achieved via an amide linkage between the carboxy functions of the precursor monolith and the amino group of the octadecylamine compound. The resulting C₁₈ monolith exhibited a very low electroosmotic flow (EOF), a fact that required the incorporation of small amounts of 2-acrylamido-2-methylpropane sulfonic acid (AMPS) in the polymerization solution to produce a precursor monolith with fixed negative charges of sulfonate groups. This may indicate that the conjugation of the carboxy functions with octadecylamine occurred to a large extent so that the amount of residual carboxy functions was sparsely dispersed and not enough to produce a desirable EOF. The EOF velocity of the C₁₈ column having fixed negative charges provided by the incorporated AMPS increased with increasing ACN content of the mobile phase signaling an increased binding of mobile phase ions to the polar amide linkages near the monolithic surface, and a decreased viscosity of the mobile phase, both of which would result in increased EOF velocity. The C₁₈ monolithic column constituted a novel nonpolar sorbent for reversed-phase capillary electrochromatography for nonpolar solutes, e.g., alkylbenzenes, alkylphenyl ketones, and polyaromatic hydrocarbons, and slightly polar compounds including phenol and chlorophenols. The C₁₈ monolithic column exhibited relatively high selectivity toward chlorophenols differing by one chloro substituent.     

Graphene oxide-octadecylsilane incorporated monolithic nano-columns with 50 μm id and 100 μm id for small molecule and protein separation by nano-liquid chromatography

In this study, graphene oxide-octadecylsilane incorporated monolithic nano-columns were developed for protein analysis by nano liquid chromatography (nano LC). The monolithic column with 100 μm id was first prepared by an in situ polymerization using ethylene dimethacrylate (EDMA), 3-chloro-2-hydroxypropylmethacrylate (HPMA-Cl), and methacryloyl graphene oxide nanoparticles (MGONPs). MGONPs were synthesized by the treatment of 3-(trimethoxysilyl)propylmethacrylate (TMSPM) and GO. Tetrahydrofuran (THF) and dodecanol were used as the porogenic solvent. The resulting column was functionalized by dimethyloctadecylch lorosilane (DODCS) for the enhancement of hydrophobicity. The functionalization greatly improved the baseline separation of hydrophobic compounds such as polyaromatic hydrocarbons (PAHs). The optimized monolith with respect to total polymerization mixture was characterized by using Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) X-ray diffraction (XRD) and chromatographic analyses. The blank monoliths without functionalization exhibited poor separation while a good separation performance of MGONPs functionalized monoliths was achieved. The monolith with 100 μm id was evaluated in protein separation in nano LC using RNase A, Cytochrome C, Lysozyme, Trypsin, and Ca isozyme II as the test proteins. It was shown that protein separation mechanism was based on large π-system of GO and hydrophobicity of the monolithic structure. Theoretical plates number up to 57 600 plates were achieved. The nano-column with 50 μm id was also prepared using the same polymerization mixture under the same chemical conditions. These nano-columns were employed for protein separation by nano LC, and the dependence of both nano-column performance on the internal diameter was also discussed.     

High performance liquid chromatography of glycoconjugates

Stationary phases were prepared for the analytical and micropreparative high performance affinity chromatography of carbohydrates and glycoproteins by covalent attachment of lectins and certain biogenic amines to the surface of microparticulate, macroporous diol-silica. Columns packed with immobilized concanavalin A or wheat germ agglutinin afforded rapid separation of both glycoproteins and low molecular weight carbohydrates. The combined use of the two lectins in a mixed bed column or in two columns in series by sequential elution with the appropriate haptenic sugars facilitated the separation of mixtures having a broad range of complex carbohydrates. Chromatographic experiments for the study of the interaction of certain biogenic amines with immobilized glycoproteins used as the stationary phase led to the development of siliceous bonded phases with serotonin, tryptamine or phenylpropanolamine ligates. The sorbents thus obtained were mildly hydrophobic and cationic in contact with eluents of pH 8 or less. They were used for the separation of various carbohydrates; in addition they also proved effective for the separation of protein mixtures under conditions similar to those customarily employed in electrostatic or hydrophobic interaction chromatography.     

Influence of Cyclodextrins on the Kinetics of Oxidation of Amino Acids and BSA by Hydrazyl Radicals

The kinetics of oxidation of amino acids (Arg, His, Lys, Phe, Thr and Tyr), a dipeptide (Gly-His), and BSA (bovine serum albumin) by two persistent water soluble free radicals of the hydrazyl type has been studied.The rate decreases in the order Arg>Lys>Tyr>Thr>HisBSAPheGly-His with bothfree radicals. Addition to the reaction mixture of - and -cyclodextrin decreases the oxidation rate, probably due to amino acidencapsulation in the cyclodextrin cavity. -Cyclodextrin protects more efficiently against oxidation than -cyclodextrin.