Poly (N-acryloxysuccinimide-co-ethylene glycol dimethacrylate) precursor monolith and its post polymerization modification with alkyl ligands, trypsin and lectins for reversed-phase chromatography, miniaturized enzyme reactors and lectin affinity chromatography, respectively.

This investigation was aimed at introducing a monolithic precursor that can be conveniently grafted with the desired chromatographic ligand via the process of post polymerization modification (PPM). The precursor was obtained by the in-situ polymerization of N-acryloxysuccinimide (NAS) and ethylene glycol dimethacrylate (EDMA) in a narrow bore stainless steel column of 1 mm i.d. yielding a poly(NAS-co-EDMA) monolithic column designated as the poly(NAS-co-EDMA) monolith (NASM) column. In a first PPM, the NASM column was bonded with octadecyl (OD) ligands yielding a nonpolar NASM-OD column that proved useful for reversed phase chromatography (RPC) of proteins in gradient elution at increasing %ACN in the mobile phase. NASM-OD resulted from the reaction between the N-hydroxysuccinimide of NASM with octadecyl amine. In a second PPM, NASM was surface immobilized with trypsin generating a proteolytic narrow bore enzyme reactor called NASM-trypsin immobilized enzyme reactor (IMER) that permitted the online digestion of proteins in a 20-min single pass through the IMER incorporated in a setup equipped with a short RPC column to achieve simultaneously a peptide tryptic map. This constituted a rapid turnover whereby ∼95% of the protein was hydrolyzed by the immobilized trypsin. In a third PPM, the NASM column was surface immobilized with three different lectins (LCA, Con A and RCA) having complementary affinities toward serum glycoproteins thus permitting the capture of a wide range of glycoproteins/glycoforms. The three NASM-lectin columns when operated in a tandem format led to assessing the level of the various glycoforms in human serum via LC-MS/MS analysis of the captured protein fractions by each NASM-lectin column.

Selective precolumn derivatization of fatty acids with the fluorescent tag 6-aminoquinoline and their determination in some food samples by reversed-phase chromatography.

Fatty acids (FAs) have been selectively derivatized with a fluorescent tag, 6-aminoquinoline (6AQ), which yielded fluorescent FA-6AQ derivatives that have excitation (λexc = 270 nm) and emission (λemi = 495 nm) wavelengths that are farther apart. This precolumn derivatization is characterized by its simplicity occurring at room temperature between the carboxylic acid group of the FA and the amino group of 6AQ in the presence of a nonaqueous soluble carbodiimide coupling agent such as the N,N´-dicyclohexylcarbodiimide. The FAs extracts are readily derivatized in chloroform and can be analyzed without any further sample cleanup that minimizes sample loss. The FA-6AQ derivatives derived from standard FAs as well as from extracted FAs from food samples were separated by reversed phase chromatography on a homemade naphthyl methacrylate monolithic (NMM) column and C4 silica-based column. While the NMM column provided excellent separation for saturated FA-6AQ derivatives, the C4 silica column was able to separate simultaneously saturated and unsaturated FA-6AQ derivatives. The MNN column permitted the analysis and quantitation of the saturated FA-6AQ derivatives extracted from coconut oil. The C4 column provided the selectivity needed to analyze and quantify saturated and unsaturated derivatized with 6AQ and extracted from meat. The limits of detection and quantitation were 5 and 20 nM, respectively, with a linear dynamic range extending from 20 nM to 40 µM. The 40 µM upper limit was due to the limited solubility of the FA-6AQ derivatives in the diluting mobile phase, which is the initial mobile phase used in gradient runs.

Polar and nonpolar organic polymer-based monolithic columns for capillary electrochromatography and high-performance liquid chromatography.

This review article is a continuation of the previous reviews on the area of monolithic columns covering the progress made in the field over the last couple of years from the beginning of the second half of 2014 until the end of the first half of 2016. It summarizes and evaluates the evolvement of both polar and nonpolar organic monolithic columns and their use in hydrophilic interaction LC and CEC and reversed-phase chromatography and RP-CEC. The review article discusses the results reported in a total of 62 references.

Robust naphthyl methacrylate monolithic column for high performance liquid chromatography of a wide range of solutes.

An organic monolithic column based on the co-polymerization of 2-naphthyl methacrylate (NAPM) as the functional monomer and trimethylolpropane trimethacrylate (TRIM) as the crosslinker was introduced for high performance reversed-phase liquid chromatography (RPC). The co-polymerization was performed in situ in a stainless steel column of 4.6mm i.d. in the presence of a ternary porogen consisting of 1-dodecanol and cyclohexanol. This monolithic column (referred to as naphthyl methacrylate monolithic column or NMM column) showed high mechanical stability at relatively high mobile phase flow velocity indicating that the column has excellent hydrodynamic characteristics. To characterize the NMM column, different probe molecules including alkyl benzenes, and aniline, benzene, toluene and phenol derivatives were chromatographed on the column and the results in terms of k, selectivity and plate counts were compared to those obtained on an octadecyl silica (ODS) column in order to assess the presence of π-π and hydrophobic interactions on the NMM column under otherwise the same elution conditions. The NMM column offered additional π-π interactions with aromatic molecules in addition to hydrophobic interactions under RPC elution conditions. Run-to-run and column-to-column reproducibility of solute k values were evaluated, and percent relative standard deviation of <1% and ∼2-3.5%, respectively, were obtained. Six standard proteins were readily separated on the NMM column using shallow (30min at 1.0mL/min), steep (10min at 1.0mL/min) and ultra steep (1min at 3.0mL/min) linear gradient elution at increasing ACN concentration in the mobile phase using a 10cm×4.6mm i.d. column in case of shallow and steep linear gradients and a 3cm×4.6mm i.d. column for ultra steep linear gradient.

Recent advances in nonpolar and polar organic monoliths for HPLC and CEC.

This article is aimed at providing a review of the progress made in the field over the period 2011 to present in order to expand in parts on two previous reviews (S. Karenga and Z. El Rassi, Electrophoresis, 2011, 32, 90-104; D. Gunasena and Z. El Rassi, Electrophoresis, 2012, 33, 251-261). In brief, this review article describes progress made in nonpolar and polar monoliths used in RP HPLC and CEC and in hydrophilic interaction LC/CEC, respectively. This article is by no means an exhaustive review of the literature; it is rather a survey of the recent progress made in the field with 69 references published on nonpolar and polar polymeric monoliths.