Audrius Maruška

(Normal-Phase) Capillary Chromatography Using Acrylic Polymer-Based Continuous Beds

Microchromatographic separations of polar aromatic compounds (pyridine, 4-pyridylmethanol, 4-methoxyphenol, 2-naphthol, catechol, hydroquinone, resorcinol, 2,7-dihydroxynaphthalene) using continuous beds are described. The columns were prepared by a simple one-step in situ polymerization procedure: a solution of acrylic monomers, including the cross-linking agent piperazine diacrylamide, was polymerized in a fused-silica capillary pretreated with 3-(trimetoxysilyl) propyl methacrylate. The continuous bed formed contained a network of channels and was attached covalently to the wall of the silica capillary (100 mm I.D.) via its methacrylate groups. Therefore, the frit used in conventional, packed columns could be omitted. The separation mechanism is discussed, particularly with regard to whether the so-called aromatic adsorption to the matrix itself is involved, an interaction first described by Gelotte [1] (the ligands, isopropyl and sulfonate groups, are not required for separation). This discussion is relevant to the question of whether the separation technique described should be classified as normal-phase or adsorption chromatography. The mobile phase from the HPLC pump was split via an open capillary to get a flow rate through the continuous bed of about 100 nl /min. The beds were tested up to a pressure of 150 bar (8.8 bar /cm). A continuous bed synthesized at a relatively low molar fraction of the cross-linker in the monomer mixture (16.5%) and high total concentration of the monomers (31.9% (w/v)) afforded the highest efficiency for the separation of the polar 21 organic compounds. Plate numbers up to 150 000 m were obtained and the run-to-run reproducibility was high. The selectivity of the separations was adjusted by changing the composition of the mobile phase (hexane–ethanol–methanol). The sample was applied by a diffusion-based injection technique. (Less)

Continuous beds with vancomycin as chiral stationary phase for capillary electrochromatography.

Enantiomeric separations in capillary electrochromatography (CEC) carried out using a continuous‐bed chiral stationary phase (CSP) based on the macrocyclic antibiotic, vancomycin, is presented. The continuous beds were prepared from methacryloxypropyl modified fused silica capillaries (100 νm ID) byin situ copolymerization of N‐(hydroxymethyl)acrylamide and piperazine diacrylamide with vinyl sulfonic acid comonomer used to introduce ionic functionality and thus a strong electroosmotic flow (EOF). The CSP was subsequently prepared by immobilizing the vancomycin stationary phase by reductive amination. Preliminary results have indicated that an extremely strong EOF is obtained in both the nonaqueous polar organic (15.2×10–5 cm2V–1 s–1) and the aqueous reversed‐phase modes of operation (8.5×10–5 cm2V–1 s–1). Enantioselectivity was obtained for four racemic compounds, the best of which was in the case of thalidomide which was separated in 10 minutes with high resolution (Rs = 2.5) and efficiency (120 000 plates meter –1) values.

Capillary electrochromatography: normal-phase mode using silica gel and cellulose-based packing materials

Abstract Cellulose-based packings and native silica gel have been employed as stationary phases in normal-phase capillary electrochromatography. The electroosmotic flow produced with non-aqueous mobile phases (acetonitrile, methanol, hexane-ethanol-methanol) is sufficiently high (mobility up to μ rmeo = 0.152 cm 2 s −1 kV −1 ) and stable for chromatographic separations. Only electroosmotic flow has been used for the propulsion of the mobile phase through the chromatographic packings. The elution order obtained with a mixture of test solutes corresponds to the elution order expected for normal-phase chromatography.

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. Comparative study of in‐line coupled SPME–CZE using RAM and RP capillary inserts was carried out. Using an SPME (RAM) insert, the calculated caffeine, paracetamol and acetylsalicylic acid LODs in a bovine plasma sample were 0.3, 0.8 and 1.9 ng/mL, respectively.

A new approach to human serum albumin chiral stationary phase synthesis and its use in capillary liquid chromatography and capillary electrochromatography

Continuous beds with immobilised human serum albumin as a chiral selector were synthesised in fused-silica capillaries in a modified fashion using additives during the protein allylation and polymerisation steps. Acetyl salicylic acid and L-tryptophan were used as additives to interact with the active centres that are responsible for chiral recognition, thus preserving them during the protein coupling procedure. The beds synthesised under modified conditions exhibit higher resolutions and enantioselectivities in capillary liquid chromatography, especially when L-tryptophan was used as an additive. Monolithic adsorbent synthesised with L-tryptophan possesses much higher efficiency when capillary electrochromatography was employed.

Polyrotaxane approach for synthesis of continuous beds for capillary electrochromatography

The polyrotaxane formation approach was evaluated for synthesis of continuous beds for capillary electrochromatography. This approach has the advantage of generating diverse electroosmotic and chromatographic properties without chemical reactions. The polyrotaxane derivatized continuous beds were formed adding the macrocyclic compounds to the solution of neutral acrylic monomers and crosslinker prior to the initiation of the polymerisation. Cationic and anionic derivatives of beta-cyclodextrin were used as macrocyclic compounds. Investigation of the electroosmotic properties indicated a template directed and enthalpy controlled self-assembly of the polyrotaxanes during the polymerisation of the continuous beds. This process was monomer-composition dependent and favored by the hydrophobicity of the polymeric skeleton. The morphology of the continuous beds was evaluated using high-resolution optical microscopy with CCD camera and atomic force microscopy. Reversed-phase capillary chromatography driven by electroosmosis, originating from the polyrotaxane structure, was performed using several test mixtures. Not primarily designed for the chiral chromatography the polyrotaxane derivatized continuous beds demonstrated enantioselective separation of D,L-metoprolol. The stability of the polyrotaxane derivatized continuous beds was tested. The beds demonstrated reproducible electroosmotic properties in the range from pH 4 to pH 9 (RSD=0.69%).

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. The temperature, time, and stirring were controlled during the fermentation process. Factors considered in SPMPE-GC-FID method development included fiber exposure time, salt addition, temperature of extraction, and temperature of desorption. Various SPME fibers, for example with PDMS, CAR/PDMS, PA, and PDMS/DVB coatings, were tested to obtain the highest recovery of the investigated compounds extracted from yogurt samples. Based on these preliminary experiments, qualitative and quantitative analyses for the determination of biacetyl were performed by SPME-GC-FID. Moreover, a capillary zone electrophoresis method was developed for the determination of carboxylic acids in the yogurt samples. The buffer composition as well as deproteinization by acetonitrile were found to have a crucial effect on the analysis.

The development of microparticulate cellulose-based packing materials and evaluation of potential use in capillary electrochromatography

SummaryExpanding the range of stationary phases employed in capillary electrochromatography, the development of rigid cellulose-based micro-beads, their physical-chemical modification and their use in capillary electrochromatography are described. Cellulose-based stationary phases have been employed in the normal-phase and the reversed-phase modes.

Comparative analysis of radical scavenging and antioxidant activity of phenolic compounds present in everyday use spice plants by means of spectrophotometric and chromatographic methods

Comparative analysis of radical scavenging and antioxidant activities of phenolic compounds present in everyday use spice plants was carried out by means of spectrophotometric and chromatographic methods. Six spice plant samples, namely onion (Allium cepa), parsley (Petroselinum crispum) roots and leaves, celery (Apium graveolens) roots and leaves and leaves of dill (Anethum graveolens) were analyzed. Total amount of phenolic compounds and radical scavenging activity (RSA) was the highest in celery leaves and dill extracts and was the lowest in celery roots. Comparing commonly used spectrophotometric analysis of 2,2-diphenyl-1-picrylhydrazyl (DPPH) RSA of extracts with the results obtained using reversed-phase chromatographic separation with on-line post-column radical scavenging reaction detection, good correlation was obtained (R(2)=0.848). Studies using HPLC system with electrochemical detector showed that bioactive phytochemicals can be separated and antioxidant activities of individual compounds evaluated without the need of a complex HPLC system with reaction detector. The results obtained using electrochemical detection correlate with the RSA assayed using spectrophotometric method (R(2)=0.893).

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. The porous properties were evaluated by inverse size-exclusion chromatography (ISEC) using a wide range of polystyrene standards of different molecular weights. The results have shown, that large pores (ca. 300 nm) dominate in the monolithic bed structure, however much larger flow-through pores must also be present as ca. 1 microm sized S. aureus bacteria were able to migrate through the bed.