Monolithic Silica Capillary Columns Research Articles

Studies on π Interactions in Liquid-Phase Separations

π interactions have recently received considerable attentions due to principal factor governing molecular recognitions and self-assemble abilities, as accumulated in the database on proximate arrangements in structures of biological systems and organic functional materials. Therefore, further deep understanding and control of π interactions will greatly facilitate the development of new functional materials. Despite the importance of π interactions, they are still challenging to study because π interactions are much weaker than most other molecular interactions, such as hydrophobic interaction, hydrogen bonding, and electrostatic bonding. On the other hand, liquid chromatography (LC) is a powerful separation technique, which is able to distinguish the partition coefficients of solutes between the mobile and stationary phases, and can sensitively reflect the strength of molecular interactions. We investigated the properties of π interactions by developing new silica-monolithic capillary columns modified with carbon materials providing strong π interactions. In this focusing review, we introduce a few specific π interactions by columns modified with fullerenes and polycyclic aromatic hydrocarbons (PAHs), which showed strong π-π interactions due to spherical recognition and multiple CH-π interactions. Furthermore, π interactions can contribute to the separation of various samples, which are difficult to achieve by the available retention mechanisms. Briefly, we applied various π interactions to specific separation analyses, and we succeeded in separating halogenated compounds, H/D isotopologue pairs, and saccharides by effective π interactions. These results indicate that π interactions contribute to practical separation science, such as removal of environmental pollutants and quantitative determination of medicinal compounds.

Performance of functionalized monolithic silica capillary columns with different mesopore sizes using radical polymerization of octadecyl methacrylate

We report on the possibility to enhance the phase ratio and retention factor in silica monoliths. According to pioneering work done by Núñez et al. [1], this enhancement is pursued by applying a stationary phase layer via radical polymerization with octadecyl methacrylate (ODM) as an alternative to the customary octadecylsilylation (C18-derivatization). The difference in band broadening, retention factor and separation selectivity between both approaches was compared. Different hydrothermal treatment temperatures for the column preparation were applied to produce monolithic silica structures with three different mesopore sizes (resp. 10, 13, and 16 nm, as determined by argon physisorption) while maintaining similar domain size (sum of through-pore and skeleton size). It has been found that the columns with the poly(octadecyl methacrylate)-phase (ODM columns) provided a 60 to 80% higher retention factor in methanol-water mixture compared to the octadecylsilylated (ODS) columns produced by starting from similar silica backbone structures. In acetonitrile-water mixture, the enhancement is smaller (15 to 30% times higher), yet significant. By adjusting the fabrication conditions (for both the preparation of the monolithic backbones and the surface functionalization), the achieved retention factors (up k = 4.89 for pentylbenzene in 80:20% (v/v) methanol/water) are obviously higher than obtained in the pioneering study on ODM monoliths of Núñez et al. [1], and column clogging could be completely avoided. In addition, also separation efficiencies were significantly higher than shown in Ref. [1], with plate heights as low as 5.8 μm. These plate heights are however inferior to those observed on the ODS-modified sister columns. The difference can be explained by the slower intra-skeleton diffusion displayed by the ODM-modified columns, in turn caused by the larger obstruction to diffusion originating from the thicker stationary phase layer.

Preparation of Monolithic Silica and Polymer Capillary Columns with Ultrahigh Column Efficiencies and Comparisons between van Deemter Plots of Alkylbenzenes on These Two Kinds of Columns.

Monolithic silica and polymer capillary columns with ultrahigh column efficiencies were prepared. Permeability and electrochromatography performances of these two kinds of columns were compared. Monolithic silica columns bear higher permeability than polymer counterparts by two orders of magnitude. Furthermore, the van Deemter plots of alkylbenzenes on these two kinds of columns demonstrate that monolithic silica columns produce much lower plate heights of alkylbenzes than polymer columns do. Within the range of electroosmotic flow (EOF) velocity investigated, no uptrend of plate height with the increase of EOF was observed suggesting the great capacity of fast separation and high efficiency. The plate height of thiourea on monolithic silica columns is as low as 2.67μm, representing its corresponding column efficiency is over 430,000plates/m. As far as we know, it is the highest ever column efficiency reported in the literature. Moreover, the separation of butylbenzene isomers was obtained on the monolithic silica column.

Phenyl-bonded monolithic silica capillary column liquid chromatographic separation and detection of fluorogenic derivatized intact proteins.

Prior to the identification of proteins for proteomics analysis in human cells, separation of fluorogenic derivatized proteins with a fluorogenic reagent, 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide, has typically been performed by using a conventional reversed-phase HPLC column. However, the number of proteins in human cells (HepaRG) that are separated by this conventional approach is limited to approximately 500. In this study, a nanoflow liquid chromatography system with an evaluated phenyl-bonded monolithic silica capillary column (0.1 mm i.d., 700 mm length) was used to increase the number of separated fluorogenic derivatized proteins. This system was used to separate derivatized human cell proteins (K562) and yeast (Saccharomyces cerevisiae) proteins as model cell proteomes. More than 1,300 protein peaks were separated/detected from both cell proteomes. We present a straightforward comparison of multiple separation profiles using a novel chromatogram display approach, termed the "spiderweb" chromatogram. In addition, to validate that the detected peaks are derived from proteins, a mass spectrometer was connected to the capillary column and deconvolution of the obtained mass spectra was performed. Furthermore, different molecular weight distribution profiles of the expressed proteins were observed between the two cell proteomes.

Development of Monolithic Silica Capillary Columns for LC/MS Analysis of Intact Proteins

Development of Monolithic Silica Capillary Columns for LC/MS Analysis of Intact Proteins

Performance of small-domain monolithic silica columns in nano-liquid chromatography and comparison with commercial packed bed columns with 2 µm particles

We report on a direct comparison of the separation performance in capillary nano-LC between commercial packed bed columns and the small-domain silica monoliths in applications. Octadecylsilylated monolithic silica capillary columns with a 50 and 100 µm inner diameter (i.d.) were prepared with a procedure providing domain sizes in the sub-2 µm range. The fabricated monolith columns could provide plate heights (H) of 4.0‒4.2 µm for hexylbenzene (retention factor (k) = 3.6) at an optimal linear velocity range under an isocratic condition, while showing a column permeability (Kv0 = 1.6‒1.8 × 10−14 m2) comparable to that of a column packed with 3‒3.5 µm particles. When the peak capacity (np) for a cytochrome C digest was compared for variable gradient times (tG = 15, 30, 60, and 120 min) and constant gradient steepness (b’), the present monolith columns could show a 30‒40% higher np-value than the packed capillary column with 2 µm particles (e.g. np = 180 versus np = 259 at tG = 30 min). The produced monolith columns showed a high chromatographic repeatability for both isocratic and gradient elution (e.g. relative standard deviation (n = 3, RSD (%)) = 0.5% for H, 2,6% for k, and 5.6% for Kv0 in the isocratic mode using the 100 µm i.d.-columns). The present results show that the domain sizes which can now be achieved for capillary silica monoliths are sufficiently small to result in separation efficiencies that can successfully compete with the commercial packed bed columns available for use in nano-LC applications.

Development of a new in-line coupling of a miniaturized boronate affinity monolithic column with reversed-phase silica monolithic capillary column for analysis of cis-diol-containing nucleoside compounds

In-line coupling of capillary columns is an effective means for achieving miniaturized and automated separation methods. The use of multimodal column designed to allow the direct integration of a sample preparation step to the separation column is one example. Herein we propose a novel in-line coupling at the capillary scale between a boronate affinity capillary column (μBAMC unit) and a reversed-phase separation column. This has been made possible due to the elaboration of a new and efficient μBAMC unit. A thiol-activated silica monolithic capillary column was functionalized through thiol-ene photoclick reaction. This simple and fast reaction allows to prepare stable μBAMC units having grafting densities of 1.93 ± 0.17 nmol cm−1. This grafting strategy increases the surface density by a factor 4 compared to our previous strategies and opens the frame to in-line coupling with reversed-phase capillary column. Proof of concept of the in-line coupling was done by coupling a 1-cm length μBAMC unit to a 7-cm length reversed phase capillary column. The conditions of loading, elution and separation were optimized for cis-diol nucleosides analysis (uridine, cytidine, adenosine, guanosine). A loading volume (at pH 8.5) of up to 21 hold volume (i.e 1 μl) of the μBAMC unit can be loaded without sample breakthrough. For the least retained nucleoside (uridine) a limit of detection of 50 ng mL−1 was estimated. Elution and full separation of the four nucleosides was triggered by flushing the multimodal column with an acetic acid (50 mM) / methanol (98/2, v/v) mobile phase.

Hyphenation of short monolithic silica capillary column with vacuum ultraviolet spectroscopy detector for light hydrocarbons separation

Compared to conventionnal bench top instruments, on-line GC analyzers require specific characteristics. On one hand, for some applications operating with a reactor pressure as high as several tens of bars, sample pressure has to be reduced before GC separation, or specific valves and columns have to be designed to perform separation with high carrier gas inlet pressure. On the other hand, informative detectors such as mass spectrometer are valuable but low maintenance detectors are prefered. To fit these two requirements (sampling at high pressure without decompression stage, and informative detector with low maintenance), short monolithic silica capillary column operated with inlet pressure as high as 60 bar has been hyphenated to VUV detector.Injection and column performance have been first investigated. The system has been optimized by adjusting split ratio at high pressure and by tuning two main VUV detector parameters (“average number” linked to data point averaging and make-up gas pressure) to decrease the limit of quantification. The optimization stage led to a set of experimental parameters which is a good compromise between signal-to-noise ratio and chromatographic efficiency. Finally, the hyphenated monolithic column has be used to partially separate a mixture of methane, ethane, carbon monoxide and carbon dioxide within 15 s, and the VUV deconvolution capabilities have been exploited to overcome coelution and finally separate individual signals.

Differentiating π Interactions by Constructing Concave/Convex Surfaces Using a Bucky Bowl Molecule, Corannulene in Liquid Chromatography.

Convex-concave π conjugated surfaces in hemispherical bucky bowl such as corannulene (Crn) have shown increasing utility in constructing self-assembled new functional materials owing to its unique π electrons and strong dipole. Here, we investigate these specific molecular recognitions on Crn by developing new silica-monolithic capillary columns modified with Crn and evaluating their performance in the separation of different aromatic compounds by liquid chromatography (LC). We synthesized two Crn derivatives and conjugated them onto the surface of a silica monolith. The first Crn derivative was edge functionalized, which can undergo free inversion of a convex-concave surface. The second Crn derivative was synthesized by modifying the spoke of Crn, which suppresses the convex-concave inversion. Results of LC suggest that each surface showed different shape recognition based on π interaction. Furthermore, the concave surface of Crn showed strong CH-π interaction with a planar molecule, coronene, demonstrated by the shifts of the 1H NMR signals of both Crn and coronene resulting from the multiple interactions between Crn and π electrons in coronene. These results clearly demonstrated the presence of CH-π interactions at multiple points, and the role of shape recognition.

Monolithic Silica Capillary Columns with Improved Retention and Selectivity for Amino Acids

A strategy for the preparation of silica-based monolithic capillary columns (150 × 0.1 mm) with high selectivity to amino acids is presented. The zwitterionic columns were prepared by coating the silica monolith with [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide via 3-(trimethoxysilyl)propyl methacrylate. The columns were evaluated under isocratic conditions in hydrophilic interaction liquid chromatography. The best separation of amino acids was obtained on the monolithic column prepared by a stepwise modification procedure where the modification step was repeated four times. The mixture of fifteen amino acids was separated within 13 min using the mobile phase consisting of 75% acetonitrile and 25% 5 mmol/L ammonium acetate at pH 4.5.

Molecular and Physiological Study of Candida albicans by Quantitative Proteome Analysis.

Candida albicans is one of the major pathogens that cause the serious infectious condition known as candidiasis. C. albicans was investigated by proteome analysis to systematically examine its virulence factors and to promote the development of novel pharmaceuticals against candidiasis. Here, we review quantitative time-course proteomics data related to C. albicans adaptation to fetal bovine serum, which were obtained using a nano-liquid chromatography/tandem mass spectrometry system equipped with a long monolithic silica capillary column. It was revealed that C. albicans induced proteins involved in iron acquisition, detoxification of oxidative species, energy production, and pleiotropic stress tolerance. Native interactions of C. albicans with macrophages were also investigated with the same proteome-analysis system. Simultaneous analysis of C. albicans and macrophages without isolating individual living cells revealed an attractive strategy for studying the survival of C. albicans. Although those data were obtained by performing proteome analyses, the molecular physiology of C. albicans is discussed and trials related to pharmaceutical applications are also examined.

Exploring the pressure resistance limits of monolithic silica capillary columns

We report on an experimental approach to measure the pressure stability and mechanical strength of monolithic silica capillary columns with different diameters (50 and 100μm i.d.) and considering two different domain sizes, typical for the second generation monoliths or smaller. The approach consists of exposing the capillaries to ultra-high pressures (gradually stepwise increased from 20 to 80MPa), with intermediate measurements of the column efficiency, permeability and retention factors to check the mechanical stability of the bed. It was observed that all tested columns withstood the imposed pressure stress, i.e., all the tested parameters remained unaffected up till the maximal test pressure of 80MPa. The applied pressure gradient corresponded to 320MPa/m. The two 100μm i.d.-capillary columns were also exposed to pressures between 80 and 90MPa for a prolonged time (8h), and this did not cause any damage either.

Effect of Acidic Additives on Peak Capacity and Detectivity in Peptide Analysis Using Nano-Flow LC/MS with Low-Density ODS Modified Monolithic Silica Capillary Columns

Effect of Acidic Additives on Peak Capacity and Detectivity in Peptide Analysis Using Nano-Flow LC/MS with Low-Density ODS Modified Monolithic Silica Capillary Columns

Putative Alginate Assimilation Process of the Marine Bacterium Saccharophagus degradans 2-40 Based on Quantitative Proteomic Analysis.

Quantitative proteomic analysis was conducted to assess the assimilation processes of Saccharophagus degradans cultured with glucose, pectin, and alginate as carbon sources. A liquid chromatography-tandem mass spectrometry approach was used, employing our unique, long monolithic silica capillary column. In an attempt to select candidate proteins that correlated to alginate assimilation, the production of 23 alginate-specific proteins was identified by statistical analyses of the quantitative proteomic data. Based on the analysis, we propose that S. degradans has an alginate-specific gene cluster for efficient alginate utilization. The alginate-specific proteins of S. degradans were comprised of alginate lyases, enzymes related to carbohydrate metabolism, membrane transporters, and transcription factors. Among them, the short-chain dehydrogenase/reductase Sde_3281 annotated in the alginate-specific cluster showed 4-deoxy-L-erythro-5-hexoseulose uronic acid reductase (DehR) activity. Furthermore, we found two different genes (Sde_3280 and Sde_0939) encoding 2-keto-3-deoxy-D-gluconic acid (KDG) kinases (KdgK) that metabolize the KDG derived from alginate and pectin in S. degradans. S. degradans used Sde_3280 to phosphorylate the KDG derived from alginate and Sde_0939 to phosphorylate the KDG derived from pectin. The distinct selection of KdgKs provides an important clue toward the elucidation of how S. degradans recognizes and processes polysaccharides.

メートル長モノリス型シリカカラムを用いた LC-MS/MSシステムによるヒトプロテオーム解析

メートル長モノリス型シリカカラムを用いた LC-MS/MSシステムによるヒトプロテオーム解析

Description of the interaction between Candida albicans and macrophages by mixed and quantitative proteome analysis without isolation.

Candida albicans is an opportunistic pathogen that causes fatal diseases in immunocompromised hosts. Host resistance against C. albicans relies on ingestion of the pathogen by macrophages. Analysis of the escaping behavior of C. albicans from macrophages is required to understand the onset of systemic candidiasis. In this study, native interactions of C. albicans with macrophages were investigated by proteome analysis using high efficiency of long monolithic silica capillary column. Using this system, we developed a method of “mixed and quantitative proteome analysis” in which C. albicans and macrophages were simultaneously analyzed by nanoLC–MS/MS without the need to isolate the two individual living cells. Two hundred twenty-seven proteins from C. albicans and five proteins from macrophages were identified as candidate interaction-specific molecules. C. albicans seemed to produce glucose through a β-oxidation pathway, a glyoxylate cycle, and gluconeogenesis for escape from macrophages. Up-regulation of stress-related and candidate pathogenic proteins in C. albicans indicated how C. albicans endured the harsh environment inside the macrophages. Down-regulation of apoptosis-associated protein NOA1- and chaperone HSPA1A-syntheses in macrophage indicated that C. albicans was able to escape from macrophages in part by suppressing the production of these macrophage proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-015-0127-2) contains supplementary material, which is available to authorized users.

Elucidation of the recognition mechanisms for hemicellulose and pectin in Clostridium cellulovorans using intracellular quantitative proteome analysis.

Clostridium cellulovorans is an anaerobic, cellulolytic bacterium, capable of effectively degrading and metabolizing various types of substrates, including cellulose, hemicellulose (xylan and galactomannan), and pectin. Among Clostridia, this ability to degrade and metabolize a wide range of hemicellulose and pectin substrates is a unique feature; however, the mechanisms are currently unknown. To clarify the mechanisms of hemicelluloses and pectin recognition and metabolism, we carried out a quantitative proteome analysis of C. cellulovorans cultured with these substrates. C. cellulovorans was cultured in the medium of glucose (control), xylan, galactomannan (Locus bean gum, LBG), or pectin for 36 h. Xylan and galactomannan were used to search for the common recognition mechanisms of hemicellulose, and pectin was used to search for unique recognition systems in C. cellulovorans. Using an isobaric tag method and liquid chromatograph/mass spectrometer equipped with a long monolithic silica capillary column, we identified 734 intracellular proteins from all substrates. We performed KEGG analyses and cluster analyses of the resulting proteins. In the KEGG analyses, we found common degradation mechanisms for hemicellulose and pectin. In the cluster analysis corresponding to the genome analysis, we detected substrate-specific clusters that include genes involved in substrate recognition, substrate degradation, and metabolism. Combining the results of the KEGG analyses and cluster analyses, we propose the mechanisms involved in the recognition and metabolism of hemicellulose and pectin in C. cellulovorans.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-015-0115-6) contains supplementary material, which is available to authorized users.

Application of Frontal Affinity Chromatography to Study the Biomolecular Interactions with Trypsin.

Trypsin is a serine protease that has been proposed as a potential therapeutic target for metabolic disorders and malignancy diseases, thus the identification of biomolecular interactions of compounds to trypsin could be of great therapeutic importance. In this study, trypsin was immobilized on a monolithic silica capillary column via sol-gel. The binding properties of four small molecules (daidzin, genistin, matrine and oxymatrine) to trypsin were examined using the trypsin affinity columns by frontal analysis. The results indicate that the matrine (dissociation constant, Kd = 7.904 μM) has stronger interaction with trypsin than the oxymatrine (Kd = 8.204 μM), whereas daidzin and genistin were nearly have no affinity with trypsin. The results demonstrated that the frontal affinity chromatography can be used for the direct determination of protein-protease inhibitor binding interactions and have several significant advantages, including easy fabricating, reproducible, minimal technological requirements and potential to become a reliable alternative for quantitative studies of biomolecular interactions.

Versatile ene-thiol photoclick reaction for preparation of multimodal monolithic silica capillary columns

This paper presents a photografting process of monolithic silica capillary columns based on the ene-thiol click chemistry. This study is performed on a “generic” vinyl-functionalized silica monolith (Hmin 6±1μm). The photoclick reaction is investigated using different thiol monomers (octadecanethiol, cysteine and sodium mercaptoethanesulfonate) to prepare capillary columns dedicated to various chromatographic modes (reversed-phase, HILIC and strong cation exchange). Whatever the monomer used, the photografting reaction is achieved in less than 5min with a relatively high thiol monomer content. This allows preparing highly retentive and efficient monolithic columns while avoiding polymerization and/or column clogging. In addition to the aforementioned properties (duration, versatility, efficiency), this photo-triggered chemical reaction allows addressing several appropriate surface functionalizations inside a single monolithic column in order to prepare nanovolume multimodal capillary columns. A multimodal biphasic monolithic column with a 1cm length cation-exchange segment followed by a 9cm length reversed-phase segment (SCX-RP) is prepared through two successive photografting reactions using a UV-mask to localize the reactions. This multimodal biphasic column is investigated using a model sample for the selective fractionation and separation of cationic and neutral compounds and is applied to the on-line preconcentration and separation of β-blockers.

Immobilized β-cyclodextrin-based silica vs polymer monoliths for chiral nano liquid chromatographic separation of racemates

The enantioselectivity of immobilized β-cyclodextrin phenyl carbamate-based silica monolithic capillary columns was compared to our previously described polymer counterpart. 2,3,6-Tris(phenylcarbamoyl)-β-cyclodextrin-6-methacrylate was used as a functional monomer for the preparation of β-cyclodextrin (β-CD)-based silica and polymer monoliths. The silica monoliths were prepared via the sol–gel technique in fused silica capillary followed by modification of the bare silica monoliths with an anchor group prior to polymerization with β-CD methacrylate using either 2,2′-azobis(isobutyronitrile) or benzoylperoxide as radical initiators. On the other hand, the polymer monoliths were prepared via the copolymerization of β-CD methacrylate and ethylene glycol dimethacrylate in different ratios in situ in fused silica capillary. The prepared silica/polymer monoliths were investigated for the chiral separation of different classes of pharmaceuticals namely; α- and β-blockers, anti-inflammatory drugs, antifungal drugs, dopamine antagonists, norepinephrine-dopamine reuptake inhibitors, catecholamines, sedative hypnotics, diuretics, antihistaminics, anticancer drugs and antiarrhythmic drugs. Baseline separation was achieved for alprenolol, bufuralol, carbuterol, cizolertine, desmethylcizolertine, eticlopride, ifosfamide, 1-indanol, propranolol, tebuconazole, tertatolol and o-methoxymandelic acid under reversed phase conditions using mobile phase composed of methanol and water. The silica-based monoliths showed a comparative enantioselectivity to the polymer monoliths.