Experimental characterization and modelling of analytical monolithic column

Hydrodynamics, equilibrium and kinetics of adsorption in a silica-based monolithic column Chromolith Performance RP-18e (Merck KgaA, Germany) have been studied. The column permeability was calculated according to the Darcy law for laminar flow. The efficiency of the monolithic column was characterized through the height equivalent to a theoretical plate (HETP) for myoglobin, phenol and progesterone. The 2-D single channel mathematical model has been applied to describe the adsorption dynamics. Parabolic velocity profile, axial and radial diffusion in the monolith channel, linear driving force model for the mass transfer in the monolith channel skeleton wall and linear adsorption equilibrium were assumed. The mathematical model gives good prediction of the experimental elution peaks.     

Preparation and characterization of long alkyl chain methacrylate-based monolithic column for capillary chromatography

This paper describes the fabrication of long alkyl chain methacrylate monolithic materials for using as stationary phases in capillary liquid chromatography. Following deactivation of the capillary surface with 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS), monoliths were formed by co-polymerisation of stearyl methacrylate (SMA) with ethylene glycol dimethacrylate (EDMA) in the presence of the initiator AIBN and a mixture of porogens including iso-amyl alcohol and 1,4-butanediol. The monoliths were prepared in 100 μm i.d. capillaries and the composition of the polymerisation mixtures were optimised in terms of the ratio of SMA/EDMA, the porogen composition and ratio of porogen to monomers. As the porogen weight fraction decreased, the microglobules became smaller and as expected, the total porosity decreased. In order to determine the usability of such materials, the column permeability K was measured by pumping water through the columns at different linear flow rates. Good results were obtained when these capillaries were used to separate mixtures of weak acids, neutral and basic compounds.     

Preparation and characterization of long alkyl chain methacrylate-based monolithic column for capillary chromatography

This paper describes the fabrication of long alkyl chain methacrylate monolithic materials for using as stationary phases in capillary liquid chromatography. Following deactivation of the capillary surface with 3-(trimethoxysilyl)propyl methacrylate (gamma-MAPS), monoliths were formed by co-polymerisation of stearyl methacrylate (SMA) with ethylene glycol dimethacrylate (EDMA) in the presence of the initiator AIBN and a mixture of porogens including iso-amyl alcohol and 1,4-butanediol. The monoliths were prepared in 100 microm i.d. capillaries and the composition of the polymerisation mixtures were optimised in terms of the ratio of SMA/EDMA, the porogen composition and ratio of porogen to monomers. As the porogen weight fraction decreased, the microglobules became smaller and as expected, the total porosity decreased. In order to determine the usability of such materials, the column permeability K was measured by pumping water through the columns at different linear flow rates. Good results were obtained when these capillaries were used to separate mixtures of weak acids, neutral and basic compounds.     

Experimental validation of the ANTS2 code for modelling optical photon transport in monolithic LYSO crystals

In this work the scintillation energy spectra originating from the background radioactivity from polished monolithic lutetium yttrium oxyorthosilicate coupled to position-sensitive silicon photomultipliers (SiPM) was studied using the open source Monte Carlo simulation package ANTS2. Two crystal sizes, fully and partially covering the photosensor area, three surface crystal wrappings (black, specular or diffuse) and the full signal formation process in the photosensor were considered. The simulation results were validated with experimental data acquired under the same geometric and detector operating conditions. In all cases ANTS2 simulated spectra have very good agreement with experimental results, reproducing the expected shape, with correct onset and end at 88 and 1190 keV, respectively, as well as sharp edges at the reference energies of 88, 88 + 202, 88 + 307 and 88 + 202 + 307 keV. The normalized root-mean square error between simulated and measured spectra varied between 4.3% and 10.4%.     

Separation of polyprenol and dolichol by monolithic silica capillary column chromatography

We attempted an analysis of naturally occurring polyprenol and dolichol using a monolithic silica capillary column in HPLC. First, the separation of the polyprenol mixture alone was performed using a 250 x 0.2 mm inner diameter (ID) octadecylsilyl (ODS)-monolithic silica capillary column. The resolution of the separation between octadecaprenol (prenol 18) and nonadecaprenol (prenol 19) exceeded by >or=2-fold the level recorded when using a conventional ODS-silica particle-packed column (250 x 4.6 mm ID) under the same elution conditions. Next, the mixture of the prenol type (polyprenol) and dolichol type (dihydropolyprenol) was subjected to this capillary HPLC system, and the separation of each homolog was successfully achieved. During the analysis of polyprenol fraction derived from Eucommia ulmoides leaves, dolichols were found as a single peak, including all-trans-polyprenol and cis-polyprenol previously identified. This sensitive high-resolution system is very useful for the analysis of compounds that are structurally close to polyprenols and dolichols and that have a low content.     

Two-dimensional protein separation by the HPLC system with a monolithic column

A two-dimensional high-performance liquid chromatography (2D-HPLC) system for protein separation was developed using an ion-exchange column in the first dimension and a reversed-phase monolithic column in the second dimension. The system demonstrated efficient separation of proteins in comparison with conventional systems. For proteomic analysis, proteins extracted from the cell surface of the yeast were separated by 2D-HPLC and evaluated.     

A simple monolithic column electroelution for protein recovery from gel electrophoresis

Protein recovery from gel electrophoresis plays an important role in functional genomics and proteomics but faces a series of issues (e.g., complex procedure, low recovery, long experimental time). In this study, a monolithic column electroelution (MCE) was developed for protein recovery from gel electrophoresis. With the model proteins of bovine serum albumin (BSA), hemoglobin (Hb), and myoglobin (Mb), the developed device and method were compared with common electroelution procedures in agarose gel electrophoresis (AGE). The comparative experiments revealed that (i) the protein recovery achieved with the developed device was greater than 83%, much higher than the 41% to 50% achieved with the common devices; (ii) the running time to obtain 70% recovery was approximately 15min, evidently shorter than the 240min with the common devices; and (iii) the device and procedure were simple and less time-consuming as compared with those of the common devices. It was observed that the serum protein bands cut from polyacrylamide gel electrophoresis could be transferred into solution in 15 to 30min with 82% yield. The device, along with its relevant procedure, has potential use in protein extraction and proteomics as well as in DNA studies.     

HPLC separation of some purine and pyrimidine derivatives on Chromolith Performance Si monolithic column

The chromatographic behavior of some purines and pyrimidines on a monolithic Chromolith Performance Si column under normal-phase high-performance liquid chromatography mode has been studied. Column pressure, column efficiency and selectivity of Chromolith Performance Si column were compared to those of conventional spherical 5 μm silica packed columns Econosphere Silica and Zorbax Rx-SIL. The investigation has shown that application of Chromolith Performance Si column for analysis of polar solutes can reduce the separation time without sacrificing column efficiency and selectivity. Improvement of the monolithic silica column efficiency for polar solutes is observed when ternary mobile phases (mixtures of hexane–isopropanol with ethylene glycol, water or acetonitrile) are applied.     

HPLC separation of some purine and pyrimidine derivatives on Chromolith Performance Si monolithic column

The chromatographic behavior of some purines and pyrimidines on a monolithic Chromolith Performance Si column under normal-phase high-performance liquid chromatography mode has been studied. Column pressure, column efficiency and selectivity of Chromolith Performance Si column were compared to those of conventional spherical 5 microm silica packed columns Econosphere Silica and Zorbax Rx-SIL. The investigation has shown that application of Chromolith Performance Si column for analysis of polar solutes can reduce the separation time without sacrificing column efficiency and selectivity. Improvement of the monolithic silica column efficiency for polar solutes is observed when ternary mobile phases (mixtures of hexane-isopropanol with ethylene glycol, water or acetonitrile) are applied.     

Online multidimensional separation with biphasic monolithic capillary column for shotgun proteome analysis

A biphasic monolithic capillary column with 10 cm segment of strong-cation exchange monolith and 65 cm segment of reversed-phase monolith was prepared within a single 100 microm i.d. capillary. Separation performance of this column was evaluated by a five-cycle online multidimensional separation of 10 microg tryptic digest of yeast proteins using nanoflow liquid chromatography coupled with tandem mass spectrometry, and it took 12 h for whole separation under the operating pressure only approximately 900 psi. Totally, 780 distinct proteins were positively identified through assignment of 2953 unique peptides at false-positive rate less than 1%. The good separation performance of this biphasic column was largely attributed to the good orthogonality of the strong-cation exchange monolith and reversed-phase monolith for multidimensional separation.     

Rapid determination of amino acids in biological samples using a monolithic silica column

A high-performance liquid chromatography method in which fluorescence detection is used for the simultaneous determination of 21 amino acids is proposed. Amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and then separated on a monolithic silica column (MonoClad C18-HS, 150 mm × 3 mm i.d.). A mixture of 25 mM citrate buffer containing 25 mM sodium perchlorate (pH 5.5) and acetonitrile was used as the mobile phase. We found that the most significant factor in the separation was temperature, and a linear temperature gradient from 30 to 49°C was used to control the column temperature. The limits of detection and quantification for all amino acids ranged from 3.2 to 57.2 fmol and 10.8 to 191 fmol, respectively. The calibration curves for the NBD-amino acid had good linearity within the range of 40 fmol to 40 pmol when 6-aminocaproic acid was used as an internal standard. Using only conventional instruments, the 21 amino acids could be analyzed within 10 min. This method was found to be suitable for the quantification of the contents of amino acids in mouse plasma and adrenal gland samples.     

Electrochromatographic enantioseparation of amino acids using polybutylmethacrylate-based chiral monolithic column by capillary electrochromatography

This article describes the development of a polybutylmethacrylate‐based monolithic capillary column as a chiral stationary phase. The chiral monolithic column was prepared by polymerization of butyl methacrylate (BMA), ethylene dimethacrylate (EDMA), and N‐methacryloyl‐l ‐glutamic acid (MAGA) in the presence of porogens. The porogen mixture included N,N‐dimethyl formamide and phosphate buffer. MAGA was used as a chiral selector. The effect of MAGA content was investigated on electrochromatographic enantioseparation of d,l ‐histidine, d,l ‐tyrosine, d,l ‐phenyl alanine, and d,l ‐glutamic acid. The effect of acetonitrile (ACN) content in mobile phase on electro‐osmotic flow was also investigated. It was demonstrated that the poly(BMA‐EDMA‐MAGA) monolithic chiral column can be used for the electrochromatographic enantioseparation of amino acids by capillary electrochromatography (CEC). The mobile phase was ACN/10 mM phosphate buffer (45:55%) adjusted to pH 2.7. It was observed that l ‐enantiomers of the amino acids migrated before d ‐enantiomers. The separation mechanism of electrochromatographic enantioseparation of amino acids in CEC is discussed. Chirality 24:606–609, 2012. © 2012 Wiley Periodicals, Inc.     

Separation of manganese peroxidase isoenzymes on strong anion-exchange monolithic column using pH-salt gradient

Different chromatographic methods including chromatofocusing are used for separation of manganese peroxidase (MnP) isoforms and their isolation from the fungal growth medium. We tested strong anion exchange methacrylate based monolithic columns as a stationary phase for fast separation of MnP's. Sodium acetate buffers of two different pH values (6 and 4) were used for formation of reproducible pH gradient. The entire cycle, involving analysis and column regeneration, was completed in 3 min. Use of pH gradient showed better MnP isoform separation comparing to the salt gradient, while application of combined pH-salt gradient, resulted in further improvement.     

Preparation and evaluation of bovine serum albumin immobilized chiral monolithic column for affinity capillary electrochromatography

A system of capillary silica monolith with bovine serum albumin (BSA) functionalized through two approaches for affinity monolithic capillary electrochromatography (AMCEC) was developed. Covalent immobilization conditions for two different Schiff base methods, which employed 3-glycidopropyl trimethoxysilane (GPTS) and 3-aminopropyl trimethoxysilane (APTS) as starting materials, respectively, were investigated to obtain good and stable chiral separation. The BSA immobilized silica monoliths were evaluated in terms of morphology, electroosmotic flow, retention time, column efficiency and resolution of model compound (±)-tryptophan. The columns exhibited satisfactory run-to-run, column-to-column repeatability and maintained their enantioselectivity for more than 3 months. Both developed methods can baseline separate tryptophan enantiomers, whereas shorter retention time, better column efficiency, and enantiomeric recognition between two pairs of drug enantiomers (pantoprazole and atenolol) were obtained by the GPTS method.     

Experimental modelling of exine self-assembly

Living and fossil megaspores produced by Selaginella (Lycopsida) and its extinct ancestors form distinctive (and occasionally iridescent) exines. Ultrastructural studies of these spores have provided data that demonstrate a colloidal mode of development which in turn implies a degree of self-assembly in the construction of these exines. We present here experimental evidence in support of the theory of selaginellalean megaspore exine construction by depletion flocculation. Iridescent colloidal flocculations of polystyrene latex particles demonstrate an ultrastructural organization virtually indistinguishable from that of the biological system, and clearly demonstrate that self-assembly of complex Selaginella exines by a relatively simple construction process is plausible.     

Preparation of a monolithic capillary column with immobilized alpha-mannose for affinity chromatography of lectins

A simple method for the preparation of an affinity monolithic (also called continuous bed) capillary column for alpha-mannose-specific lectins is described. 2-Hydroxyethyl methacrylate in combination with (+)-N,N -diallyltartardiamide (DATD) and piperazine diacrylamide (PDA, 1,4-bisacryloyl-piperazine) as crosslinkers, were used as monomers for the monolith. After oxidation of DATD with periodate, alpha-mannose with spacer was bound to the aldehyde groups of the polymeric skeleton via reductive amination to form an affinity column for the separation, enrichment or binding studies of mannose-specific lectins. The permeability of the column was excellent. The porosity of the monolith was investigated by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC). The affinity of the monolith was evaluated by frontal analysis (FA) and fluorescence microscopy (FM) using fluorescently labeled concanavalin (Con A). Frontal affinity chromatography showed a specific interaction of two different lectins with the alpha-mannose-modified monolith. According to FM the affinity sites were evenly distributed over the monolithic bed.     

A bifunctional monolithic column for combined protein preconcentration and digestion for high throughput proteomics research

Enzymatic digestion of proteins is a key step in protein identification by mass spectrometry (MS). Traditional solution-based protein digestion methods require long incubation times and are limitations for high throughput proteomics research. Recently, solid phase digestion (e.g. trypsin immobilization on solid supports) has become a useful strategy to accelerate the speed of protein digestion and eliminate autodigestion by immobilizing and isolating the enzyme moieties on solid supports. Monolithic media is an attractive support for immobilization of enzymes due to its unique properties that include fast mass transfer, stability in most solvents, and versatility of functional groups on the surfaces of monoliths. We prepared immobilized trypsin monolithic capillaries for on-column protein digestion, analyzed the digested peptides through LC/FTICR tandem MS, and compared peptide mass fingerprinting by MALDI-TOF-MS. To further improve the digestion efficiency for low abundance proteins, we introduced C4 functional groups onto the monolith surfaces to combine on-column protein enrichment and digestion. Compared with immobilized trypsin monolithic capillaries without C4, the immobilized trypsin-C4 monolith showed improved digestion efficiency. A mechanism for increased efficiency from the combination of sample enrichment and on-column digestion is also proposed in this paper. Moreover, we investigated the effects of organic solvent on digestion and detection by comparing the observed digested peptide sequences. Our data demonstrated that all columns showed good tolerance to organic solvents and maintained reproducible enzymatic activity for at least 30 days.     

In situ characterization of antibody grafting on porous monolithic supports

The efficient immobilization of antibodies on monolithic support is one of the most critical steps when preparing immunoaffinity supports. In this work, the ADECA (amino density estimation by colorimetric assay) method was adapted to tridimensional supports (in a dynamic mode) and proved to be efficient to characterize the antibodies grafting efficiency on 15.3±0.9mg porous glycidyl methacrylate (GMA)-co-ethylene dimethacrylate (EDMA) monolithic columns. The amount of grafted antibodies measured in situ on the monolith by ADECA (8.2±0.2μg of antibodies per milligram of monolith) was consistent with values obtained by bicinchoninic acid assay (BCA) after crushing the monolith. ADECA was shown to be less time-consuming and more versatile than BCA. The ADECA method was further implemented to thoroughly study and optimize the antibody grafting conditions (influence of pH and kinetics of the grafting step) on GMA-based monoliths and to check the covalent nature of the antibody/surface linking and its stability. Using the total amount of grafted antibodies and the amount of recognized antigen, we found that 65±6% of antibodies were able to capture their antigen. Finally, the grafting of Fab and F(ab')(2) fragments demonstrated that no significant improvement of the global binding capacity of the monolith was obtained.