Diatom biosilica for liquid chromatography.

This work presents, for the first time, the preparation method and subsequent use of biosilica in column liquid chromatography in reverse-phase mode. Diatom biosilica consists of the siliceous exoskeletons (frustules) of unicellular algae. Controlled cultivation of Pseudostaurosira trainorii diatoms resulted in frustules with an average diameter of approximately 4 µm, sidewall thickness of 1 µm, and a bottom thickness of 110-150 nm.

Immobilized enzyme microreactors for analysis of tryptic peptides in β-casein and β-lactoglobulin.

In this study, our primary objective was to develop an effective analytical method for studying trypsin-digested peptides of two proteins commonly found in cow's milk: β-casein (βCN) and β-lactoglobulin (βLG). To achieve this, we employed two distinct approaches: traditional in-gel protein digestion and protein digestion using immobilized enzyme microreactors (μ-IMER). Both methods utilized ZipTip pipette tips filled with C18 reverse phase media for sample concentration.

Cholesterol Stationary Phase in the Separation and Identification of siRNA Impurities by Two-Dimensional Liquid Chromatography-Mass Spectrometry.

The aim of this research was to develop a simple and efficient ion-pair reagent-free chromatographic method for the separation and qualitative determination of oligonucleotide impurities, exemplified by synthesis of raw products of the two single strands of patisiran siRNA. The stationary phases with mixed hydrophobic/hydrophilic properties (cholesterol and alkylamide) were firstly used for this purpose with reversed-phased high-performance liquid chromatography. Several different chromatographic parameters were tested for their impact on impurities separation: type, concentration, pH of salt, as well as organic solvent type in the mobile phase.

Optimization of the packing process of microcolumns with the embedded phosphodiester stationary phases.

The development of new home-made stationary phases involves their packaging procedure and is crucial to obtain satisfactory working parameters. The parameter that illustrates the quality of the packed bed is its efficiency measured as the height equivalent to the theoretical plate. According to the Van Deemetr's equation, it depends on three factors, but only one of them, eddy diffusion, does not depend on the linear flow velocity.

CE-DAD-MS/MS in the simultaneous determination and identification of selected antibiotic drugs and their metabolites in human urine samples.

In this study, a new analytical method was developed and validated for the simultaneous analysis of antibiotic drugs (amoxicillin, cefotaxime, ciprofloxacin, clindamycin, linezolid, metronidazole) and their metabolites (amoxycilloic acid, amoxicillin diketopiperazine, 3-desacetyl cefotaxime lactone, clindamycin sulfoxide, ciprofloxacin piperazinyl-N4-sulfate, linezolid N-oxide, metronidazole-OH) in human urine. Capillary electrophoresis (CE) along with the tandem mass spectrometry (MS/MS) was used to determine and identify all analytes. Appropriate conditions for MS/MS measurements along with the use of the central composite design were optimized.

Synthesis and application of stationary phase for DNA-affinity chromatographic analysis of unmodified and antisense oligonucleotide.

The goal of the research was the synthesis and application of an oligonucleotide immobilized stationary phase for the analysis of unmodified and antisense oligonucleotides. The method for attaching these molecules to aminopropyl silica modified with pentanedioic acid was developed. Each step of the synthesis was carefully controlled with the application of spectroscopic, elemental, and chromatographic analyses.

Analysis of Natural Dyes from Historical Objects by High Performance Liquid Chromatography and Electromigration Techniques.

Based on material published between 1989 and 2018 in this paper high performance liquid chromatography and electromigration techniques used in studies of natural dyes that can be found in historical objects are rewieved. Different aspects of analysis have been discussed: the stationary and mobile phase, the choice of sample solvent, methods of extraction and detection, including sensitivity parameters, such as LOD and/or LOQ. The discussed dyes have been divided into three categories (a) red antraquinone dyes along with dyes extracted from bark and tree juices, (b) yellow flavonoid dyes and saffron and (c) blue indigoid dyes.

Preparation of an improved hydrophilic monolith to make trypsin-immobilized microreactors.

In the present work the preparation of capillary-based microreactors with immobilized trypsin was investigated. The monolithic support was synthesized from 2-hydroxyethyl methacrylate (HEMA) as a functional monomer and N,N'-methylenebis(acrylamide) (MBA) as a hydrophilic crosslinker. Two monomers contents in the polymerization mixture (27% and 35%) at the ratio of HEMA:MBA=3:2 were tested.

Application of a cholesterol stationary phase in the analysis of phosphorothioate oligonucleotides by means of ion pair chromatography coupled with tandem mass spectrometry.

The main aim of this study was the investigation of the influence of several ion pair reagents towards both the retention and the mass spectrometry sensitivity of phosphorothioate oligonucleotides. A cholesterol stationary phase was applied for the first time in the analysis of this group of compounds. The mobile phase composition was modified by changing the concentration and the type of amines and acetates or 1,1,1,3,3,3-hexafluoroisopropanol.

Preparation and evaluation of dual-enzyme microreactor with co-immobilized trypsin and chymotrypsin.

The preparation of capillary microfluidic reactor with co-immobilized trypsin and chymotrypsin with the use of a low-cost commercially available enzymatic reagent (containing these proteases) as well as the evaluation of its usefulness in proteomic research were presented. The monolithic copolymer synthesized from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) was used as a support. Firstly, the polymerization conditions were optimized and the monolithic bed was synthesized in the fused silica capillary modified with 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS).

Cholesterol-based polymeric monolithic columns for capillary liquid chromatography. Part II.

This paper presents a second part of our research devoted to cholesterol-based polymeric monolithic stationary phase. The obtained capillary columns were successfully used for separations of alkylbenzenes, steroid hormones and polycyclic aromatic hydrocarbons during isocratic or gradient elutions. The columns showed excellent thermal stability.

Cholesterol-based polymeric monolithic columns for capillary liquid chromatography.

A novel, cholesterol-based polymeric monolithic stationary phase for capillary liquid chromatography, was prepared by thermally initiated in-situ polymerization. Cholesteryl methacrylate (CholMA) was used as a functional monomer and trimethylolpropane trimethacrylate (TRIM) was a cross-linker, while azobisisobutyronitrile (AIBN) was an initiator. Isooctane and toluene were chosen as "poor" and "good" solvent, respectively, as constituents of the porogen solvent.

Monolithic molecularly imprinted polymeric capillary columns for isolation of aflatoxins.

Monolithic molecularly imprinted polymers extraction columns have been prepared in fused-silica capillaries by UV or thermal polymerization in a two-step process. First, a poly-(trimethylolpropane trimethacrylate) (polyTRIM) core monolith was synthesized either by UV or thermal polymerization. Then it was grafted with the mixture of methacrylic acid (MAA) as a functional monomer, ethylene dimethacrylate (EDMA) as a cross-linking agent, 5,7-dimethoxycoumarin (DMC) as an aflatoxin-mimicking template, toluene as a porogen solvent and 2,2-azobis-(2-methylpropionitrile) (AIBN) as an initiator of the polymerization reaction.

Preparation of Monolithic Capillary Chromatographic Columns Using Supercritical Fluid as a Porogen Solvent.

Monolithic polymeric beds were synthesized in fused silica capillaries using either trimethylolpropane trimethacrylate (TRIM) or a mixture of butyl methacrylate (BMA) with ethylene glycol dimethacrylate (EDMA) as monomers. Carbon dioxide at temperature and pressure conditions above its critical values was used as a porogen solvent. The purpose of using the supercritical carbon dioxide was to have the possibility of changing the solvation power (and thus the porosity of the resulting monolith) of the porogen by pressure and temperature changes instead of changing the porogen composition.

Supramolecular recognition of estrogens via molecularly imprinted polymers.

The isolation and preconcentration of estrogens from new types of biological samples (acellular and protein-free simulated body fluid) by molecularly imprinted solid-phase extraction has been described. In this technique, supramolecular receptors, namely molecularly imprinted polymers (MIPs) are used as a sorbent material. The recognition sites of MIPs were prepared by non-covalent multiple interactions and formed with the target 17beta-estradiol as a template molecule.

Effect of zeta potential value on bacterial behavior during electrophoretic separation.

The aggregation and/or adhesion of bacterial cells is a serious disadvantage of electrophoretic separations. In this study, physicochemical surface characteristics of bacteria were measured to establish their role in bacterial adhesion and aggregation on the basis of electrophoretic behavior of different clinical strains of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria. The number and the shape of peaks obtained on the electropherograms were connected with the zeta potential measurements and in-line microscope observation using specially designed CE fluorescence stereomicroscope setup.

Differentiation of Staphylococcus aureus strains by CE, zeta potential and coagulase gene polymorphism.

Staphylococcus aureus is a common cause of infection in both hospitals and the community, and it is becoming increasingly virulent and resistant to antibiotics. Possibilities of fast, sensitive and cheap determination of these pathogenic bacteria are extremely important in antimicrobial therapy. In the present study, CE with chemically modified capillary and zeta potential measurements were used for differentiation of three different clinical strains of S.

Migration of bacteria through a monolith.

The separation of bacteria by electromigration techniques was a subject of several of our previous papers. This contribution presents the results of investigation of the porosity of the monolithic bed and migration of Staphylococcus aureus cells through it. The gigaporous monolith was thermally synthesized using glycidyl methacrylate, triethylene glycol dimethacrylate and trimethylolpropane trimethacrylate as the monomers in the presence of porogen solvent containing 1-decanol, polyethylene glycol and 2-methoxyethanol.

Effect of temperature during photopolymerization of capillary monolithic columns.

Polymeric monolithic capillary columns were synthesized using butyl methacrylate (BMA), ethylene glycol dimethacrylate (EDMA), and 2-acrylamido-2-methylpropanesulfonic acid (AMPS) as monomers and 1,4-butanediol, 1-propanol, and water as a porogen mixture. The synthesis was performed over a wide temperature range from -15 degrees C to +70 degrees C using UV radiation to trigger the polymerization process initiated by benzoin methyl ether (BME). The columns exhibited different hydrodynamic properties (permeability) as well as efficiency.

EOF in monolithic poly(styrene-co-divinylbenzene) capillary columns.

In this paper, we present the result of our study on generation of EOF on uncharged poly(styrene-co-divinylbenzene)-based monolithic capillary columns. Three types of neutral monoliths were used: unmodified poly(styrene-co-divinylbenzene) and two modified with octadecyl chains: using grafting process or Friedel-Crafts reaction. The electroosmotic mobilities (mu(EOF)) versus pH profiles are compared, as well as molecular modeling using HyperChem software was employed to describe the observed phenomena.

Determination of volatile and non-volatile products of milk fermentation processes using capillary zone electrophoresis and solid phase microextraction coupled to gas chromatography.

The aim of the investigations was to develop analytical methods for the determination of selected volatile and non-volatile organic compounds numbering among the final products of milk fermentation. The analyzed compounds were as follows: biacetyl and carboxylic acids (formic, acetic, citric, and lactic). The model yogurt was prepared under controlled conditions in our laboratory by addition of the selected bacteria (Lactobacillus bulgaricus and Streptococcus thermophilus) to the milk sample.

Coupling of solid-phase microextraction continuous bed (monolithic) capillaries with capillary zone electrophoresis for direct analysis of drugs in biological fluids.

Hyperlink robust biocompatible solid-phase microextraction (SPME) devices were prepared using continuous bed (monolithic) restricted-access media (RAM) as the SPME capillary insert. The RAM-based SPME approach was able to simultaneously separate proteins from a biological sample, while directly extracting the active components of caffeine, paracetamol and acetylsalicylic acid from the drug NeoCitramonum. The devices were interfaced with a CZE system and fully automated analysis for sample preconcentration, desorption, separation and quantification of analytes was evaluated.

Alkylated poly(styrene-divinylbenzene) monolithic columns for mu-HPLC and CEC separation of phenolic acids.

Macroporous poly(styrene-divinylbenzene) monolithic columns were prepared in fused silica capillaries of 100 microm id by in-situ copolymerization of styrene with divinylbenzene in the presence of propan-1-ol and formamide as the porogen system. The monoliths were subsequently alkylated with linear alkyl C-18 groups via Friedel-Crafts reaction to improve the retention and chromatographic resolution of strongly polar phenolic acids. A new thermally initiated grafting procedure was developed in order to shorten the time of the alkylation process.

Preparation and application of monolithic beds in the separation of selected natural biologically important compounds.

The importance of monolithic (continuous) beds is connected with their easy preparation and the far-reaching possibilities of modification of their surface and porous properties. These properties make them particularly attractive for the analysis of biologically important compounds characterized by a wide spectrum of physicochemical properties. This review summarizes their preparation methods as well as their application as continuous beds for determination of such biologically important compounds as catecholamines, vitamins, flavonoids, amino acids, peptides, and proteins.