Monolithic Silica Column Research Articles

Comments on the Implementation of a Simple Peak Capacity Optimization Procedure and Comparison of Poly(styrene–divinylbenzene) and RP-18 Silica Monolithic Capillary Columns of 5-cm for the Analysis of Protein Digests

For the analysis of protein digests, the peak capacity in reversed-phase liquid chromatography is of paramount importance. A univariate method to maximize the peak capacity as developed by Wang et al. (Anal Chem 78:3406–3416, 20) has been applied and tested for a monolithic RP-18 silica capillary column. In their method, using model peptides representing a bovine serum albumin digest, the gradient time and temperature are kept constant while the flow rate and eluent strength are varied. Despite our criticism on the fixed starting conditions, a long gradient time leading to an unnecessary long analysis time and a high temperature leading to possible degradation products in the chromatogram, and the peak capacity as the only optimization parameter this fast and simple optimization strategy turns out to be applicable to capillary monolithic columns. Furthermore, the influence of the peak capacity on a second optimization parameter, the MS protein identification score, is examined. The procedure is also used to enhance the performance of two popular types of monolithic capillary LC columns (silica-C18 and poly(styrene–divinylbenzene)) of the same length for the analysis of protein digests. Comparison of both columns show that the calculated chromatographic parameters, like productivity and peak capacity, and identification score for both columns are about the same. For a more complicated nine-protein digest the performance of the silica monolith is slightly better.

Direct chromatographic separation and quantification of calcium and magnesium in seawater and sediment porewaters

Direct analysis of Ca2+ and Mg2+ is required for accurate determination of metastable carbonate mineral phase saturation states (ΩCaCO3; ΩMgCO3) in seawater, sediment porewaters, and other high ionic strength brines. To this end, we have implemented a method using High Performance Chelation Ion Chromatography (HPCIC) in which metal ion complexation at the stationary phase renders separation efficiency insensitive to high ionic strength matrix effects common to other ion chromatography (IC) methods. This method, using direct automated on‐column injection, vastly increases sample throughput capacity in comparison to current titration methods. Calcium and magnesium ions in IAPSO standard seawater were selectively separated using a monolithic silica column (100 × 4.6 mm ID) activated with a covalently bonded iminodiacetic acid (IDA) chelator. The colored ion complexes resulting from post‐column reaction (PCR) of the ions with a metallochromic indicator, in this case 4‐(2‐pyridylazo)‐resorcinol (PAR), were detected spectrophotometricaly at 510 nm. Optimization of flow rate, eluent concentration, pH, and sample injection volume allowed baseline separation of Mg2+(0.05474 mol kg−1) and Ca2+ (0.01065 mol kg−1) in less than 8 min using 2 µL seawater sample injections. At a flow rate of 1 mL min−1, peak elutions occurred respectively at 4 and 5 min, using an eluent containing 0.1 M potassium chloride and 1 mM nitric acid adjusted to pH 2.5. Retention time variability below 0.5% for both metals following more than 200 injections indicates long‐term stability of the derivatized monolithic silica column. Method application to marine sediment porewaters is discussed.

Comparison of various silica-based monoliths for the analysis of large biomolecules†

In the present study, three types of silica-based monoliths, i.e. the first and second generations of commercial silica monolithic columns and a wide-pore prototype monolith were compared for the analysis of large biomolecules. These molecules possess molecular weights between 1 and 66 kDa. The gradient kinetic performance of the first-generation monolith was lower than that of the second generation, for large biomolecules (>14 kDa) but very close with smaller ones (1.3-5.8 kDa). In contrast, the wide-pore prototype column was particularly attractive with proteins larger than 19 kDa (higher peak capacity). Among these three columns, the selectivity and retention remained quite similar but a possible larger number of accessible and charged residual silanols was noticed on the wide-pore prototype material, which led to unpredicted small changes in selectivity and slightly broader peaks than expected. The peak shapes attained with the addition of 0.1% formic acid in the mobile phase remained acceptable for MS coupling, particularly for biomolecules of less than 6 kDa. It was found that one of the major issues with all of these silica-based monoliths is the possible poor recovery of large biomolecules (principally with monoclonal antibody fragments of more than 25 kDa).

Polydopamine-functionalized monolithic silica column for mixed-mode chromatography†

Dopamine is easy to self-polymerize under alkaline conditions and the resultant polydopamine is easy to adhere to the surface of many organic and inorganic materials. Based on the characteristics of dopamine, in this paper, a new polydopamine functionalized monolithic silica column was successfully prepared for performing mixed-mode chromatography. The performance of the column was evaluated by the separation of different types of samples including alkylbenzenes, polycyclic aromatic hydrocarbons, aromatic acids, phenols, and bases. The mechanism for the separation of these compounds was studied and appeared to involve the mixed interactions containing π-π, hydrophobic, electrostatic, and hydrophilic interactions.

Limit of the speed-resolution properties in adiabatic supercritical fluid chromatography

Kinetic Poppe plots for small retained compounds were calculated in HPLC (using pure acetonitrile) and SFC (using pure carbon dioxide) for columns having twenty one different lengths (between 3cm and 30m), operated under strict adiabatic conditions (no heat exchange was allowed between the column and the external environment), with a constant pressure drop of 200bar. The outlet pressures were set at 1 and 160bar in HPLC and SFC, respectively. The eluent inlet temperature was set at 312K. The hold-up time t0 and the apparent column efficiency 〈N〉 were calculated by taking into account the longitudinal variations of the eluent pressure, its temperature, density, viscosity, heat capacity, thermal expansion coefficient, equilibrium constant, and diffusion coefficient along the column length. Three different classes of stationary phase were considered: fully porous particles and core–shell particles of different diameter and structure, and silica monolithic columns of the second generation. The reduced plate heights of these stationary phases were taken from experimental data obtained with liquid eluents (acetonitrile/water mixtures). The columns were assumed to be radially homogeneous. The Henry's constant of the compound was fixed at Ka=2.0 at the column inlet. The results demonstrate the potential advantage of using sub-3μm core–shell particles for fast analysis in both LC and SFC, regardless of the intra-particle diffusivity through the stationary phase. In RPLC conditions, the contribution of surface diffusion to intra-particle diffusivity is important and the 4.6 μm core–shell particles can perform as well as sub-2 μm fully porous particles and silica monolithic columns of the second generation. In the absence of surface diffusion or for localized adsorption onto the stationary phase, sub-2 μm particles and silica monolithic column of the second generation outperform the 4.6 μm core–shell particles because the solid–liquid mass transfer resistance controls the column efficiency at high speeds. Eventually, for the same stationary phase and speed of analysis, SFC methods using pure CO2 may provide at least a twice column efficiency than LC methods using pure acetonitrile. For a constant pressure drop and resolution power, SFC methods may generate four times faster analyses than LC methods. Ultimately, a standard commercial 4.6mm × 50mm long column packed with 2.6μm core–shell particles, operated with an inlet flow rate of 25mL/min in fast SFC (200bar back pressure, 40°C) may provide a hold-up time of about 1s requiring data acquisition at a frequency of 400Hz, with a variance of 0.35μL2. This performance will require the use of new, ultra-low dispersion SFC system.

Single run HPLC separation coupled to evaporative light scattering detection unravels wheat flour endogenous lipid redistribution during bread dough making

Wheat flour lipids present a complex mixture of components with varying polarities and have been classified as either starch or non-starch lipids, the latter being further subdivided in free and bound lipids depending on the extraction method and solvents used. A novel separation and detection method for wheat lipids in flour and dough that separates both non-polar and polar lipid classes in a single run HPLC analysis using a monolithic silica column and a quaternary gradient and detects them with evaporative light scattering is presented. The method was applied to study in-depth the changes in lipid distribution during bread dough development, thereby monitoring the fate of the different lipid classes. Full dough development was not necessary to decrease the extractability of both non-polar and polar free lipids and increase their levels in the bound lipid extract. The data for the first time also showed which specific lipid classes were redistributed and transferred from starch to gluten, as confirmed by analyzing starch fractions. In addition, not all redistributed lipids interacted in a similar way with the gluten proteins. Major changes occurred during the first 30 s of mixing after which slight changes were observed for triacylglycerols, free fatty acids, lysophosphatidylcholine, and N-acylphosphatidylethanolamine.

Correction to Aptamer Modified Organic–Inorganic Hybrid Silica Monolithic Capillary Columns for Highly Selective Recognition of Thrombin

Correction to Aptamer Modified Organic–Inorganic Hybrid Silica Monolithic Capillary Columns for Highly Selective Recognition of Thrombin

Synthesis of Silver Nanoparticles Confined in Hierarchically Porous Monolithic Silica: A New Function in Aromatic Hydrocarbon Separations

Silver nanoparticles (Ag NPs) have been homogeneously introduced into hierarchically porous monolithic silica columns with well-defined macropores and SBA-15-type hexagonally ordered mesopores by using ethanol as the mild reductant. Within the cylindrical silica mesopores treated with aminopropyl groups as the host, monocrystalline Ag NPs and nanorods are obtained after being treated in silver nitrate/ethanol solution at room temperature for different durations of reducing time. The loading of Ag NPs in the monolith can be increased to 33 wt % by the repetitive treatment, which also led to the formation of polycrystalline Ag nanorods in the mesopores. Although the bare silica column cannot separate aromatic hydrocarbons, good separation of those molecules by noncharged Ag NPs confined in the porous structure of the monolith has been for the first time demonstrated with the Ag NP-embedded silica column. The NP-embedded monolithic silica would be a powerful separation tool for hydrocarbons with different number, position, and configuration of unsaturated bonds.

Preparation of Silica Monoliths with Macropores and Mesopores and of High Specific Surface Area with Low Shrinkage using a Template Induced Method

In this study we report a new method for the synthesis of a silica monolithic column bed with bimodal pores (throughpores and mesopores). The template induced synthesis method was used to direct bimodal pores simultaneously instead of the usual post base-treating method. Block polymer Pluronic F127 was chosen as a dual-function template to form hierarchically porous silica monolith with both macropores and mesopores. This is a simplification of the method of monolithic column preparation. Poly(ethylene glycol) was used as a partial substitute for F127 can effectively prevent shrinkage during the monolith aging process without losing much surface area (944 <TEX>$m^2/g$</TEX> to 807 <TEX>$m^2/g$</TEX>). More importantly, the resultant material showed a much narrower mesopore size (centered at 6 nm) distribution than that made using only F127 as the template reagent, which helps the mass transfer process. The solvent washing method was used to remove the remaining organic template, and it was proved to be effective enough. The new synthesis method makes the fabrication of the silica monolithic column (especially capillary column) much easier. All the structure parameters indicate that monolith PFA05 prepared by the above method is a good material for separation, with the merits of much higher surface area than usual commercial HPLC silica particles, suitable mesopore volume, narrow mesopore size distribution, low shrinkage and it is easily prepared.

“Mixed” anionic and non-ionic micellar liquid chromatography for high-speed radiometabolite analysis of positron emission tomography radioligands

A mixed micellar liquid chromatographic (LC) method, the mobile phase consisting of anionic and non-ionic surfactants, has been developed for the high-speed direct radiometabolite analysis of positron emission tomography (PET) radioligands in plasma. The addition of Triton X-100 on an anionic surfactant sodium dodecyl sulphate (SDS) mobile phase improved elution strength and peak efficiency for many PET radioligands. Several radioligands could be easily separated from their radioactive metabolites with short run time of only 4min using a “pure” (without organic solvent) mixed micellar mobile phase and semi-preparative monolithic C18-bonded silica column by simple isocratic elution without any treatment of plasma. Moreover, the use of “hybrid” mixed micellar mobile phase containing anionic, non-ionic surfactants and organic solvent was effective to further enhance peak efficiency and elute highly retained hydrophobic PET radioligands. These characteristics enabled significant shorting the radiometabolite analysis procedure of PET radioligands and simplifying the experimental setup.

Speed-resolution properties of columns packed with new 4.6 μm Kinetex-C18 core–shell particles

The achievable separation speed and resolution of columns packed with the new 4.6μm Kinetex particles were characterized by their permeability and their plate heights, measured meticulously. Their specific permeabilities range between 1.81 and 1.95×10−10cm2 and their external porosities between 0.394 and 0.405. The efficiencies of the eluted peaks measured by numerical integration of the whole band concentration provided minimum reduced plate heights for uracil (non-retained) and naphthalene (retained) between 1.3 and 1.5 (H=6.0–6.9μm) for two 4.6mm×150mm replicate columns of Kinetex and between 1.6 and 2.0 (H=7.4–9.2μm) for two narrow-bore 2.1mm×150mm replicate columns. The most efficient 4.6mm×150mm Kinetex column shows a deviation of one reduced plate height h unit with respect to the infinite diameter column (no wall, no inlet, no outlet endfitting) at high speed. Eventually, the separation speed and the resolution of columns packed with 4.6μm core–shell Kinetex particles are better or equivalent to those of columns packed with 2.5μm fully porous particles for hold-up times larger than only 10s. These core–shell materials are virtually equivalent to the second generation of silica monolithic columns with the advantage of operating well at pressure drops larger than 200bar.

Rapid high performance liquid chromatographic method for determination of clarithromycin in human plasma using amperometric detection: application in pharmacokinetic and bioequivalence studies.

A rapid, sensitive and reproducible HPLC method using amperometric detector was developed and validated for the analysis of clarithromycin in human plasma. The separation was achieved on a monolithic silica column (MZ- C8 125×4.0 mm) using acetonitrile-methanol-potassium dihydrogen phosphate buffer (40:6:54,v/v), with pH of 7.5, as the mobile phase at a flow rate of 1.5 mL/min. The assay enables the measurement of clarithromycin for therapeutic drug monitoring with a minimum quantification limit of 20 ng/mL. The method involves simple, protein precipitation procedure and analytical recovery was complete. The calibration curve was linear over the concentration range of 0.1-6 μg/mL. The coefficients of variation for inter-day and intra-day assay were found to be less than 6%. This method was used in bioequivalency and pharmacokinetic studies of the test (generic) product 2 × 500 mg clarithromycin tablets, with respect to the reference product.

Determination of Anilines Pollutant in Water by Monolithic Column-online Solid Phase Microextraction-Liquid Chromatography

For extraction column and valve system compatibility issues in on-line SPE-liquid chromatography analysis,we have built the large inner diameter extraction monolithic column compatible with liquid chromatography and related valve,pumping system.A large inner diameter(530 μm i.d.),uniform and stable silica monolithic capillary column has been prepared by using two hybrid precursor-tetramethoxysilane and methyltrimethoxysilane.After the modification of C 18 group,the results of frontier analysis show that the monolithic column has a larger adsorption capacity,wherein Ν-methylaniline is approximately 214 g/g,and N,N-Dimethylaniline is about 388 g/g.The column permeability is good,so the on-line solid phase extraction can be completed requires only the injection pump.The monolithic column was connected to the sample preparation equipment made by our laboratory and with the combination of liquid chromatography for online enrichment and separation,and it was applied to the determination of trace aniline categories compounds in river water.The mobile phase consisted of 15% methanol and 85% water solution in volume ratio;the flow rate for chromatographic separation was 0.5 mL/min;the detection wavelength was 254 nm.The recoveries were in the range of 81.9%-91.3%,and the relative standard deviation(RSDs) of intraday and interday were not larger than 7.6% at 0.1,1 and 10 g/L three spiked levels for all analytes.

Preparation of a dextran sulfate functionalized monolithic silica column for hydrophilic interaction/cation exchange chromatography

A new mixed-mode hydrophilic interaction/cation exchange monolithic silica stationary phase was prepared by chemical modification of a monolithic silica skeleton by dextran sulfate (DS) using 3-aminopropyl-triethoxysilane as a cross-linking agent. The characteristics of the DS column were evaluated by the separation of various biomolecules including amino acids, nucleosides and bases, nucleotides, and peptides. Good separations for these polar biomolecules were obtained within 11 min on the column using isocratic mode. The investigation for the separation mechanism indicated that strong hydrophilic and electrostatic interactions combined to play a role in the separation of these biomolecules on the DS column.

Chelation ion chromatography of alkaline earth and transition metals a using monolithic silica column with bonded N-hydroxyethyliminodiacetic acid functional groups

A commercially available porous silica monolithic column (Onyx Monolithic Si, 100mm×4.6mm I.D.) was ‘in-column’ covalently functionalised with 2-hydroxyethyliminodiacetic acid (HEIDA) groups, and applied to the simultaneous and rapid separation of alkaline earth and transition metal ions, using high-performance chelation ion chromatography (HPCIC). With a 0.3mM dipicolinic acid (DPA) containing eluent, the baseline separation of various common transition and heavy metal ions and the four alkaline earth metal ions could be achieved in under 14min with a flow rate of just 0.8mL/min. Detection was achieved using spectrophotometric detection at 540nm after post-column reaction (PCR) with 4-(2-pyridylazo)-resorcinol (PAR). Significant effects from variation of eluent nature, concentration and temperature upon selectivity and retention were demonstrated with the new monolithic silica chelating phase. Under optimised conditions (0.165M LiNO3 eluent, pH 2.5), peak efficiencies of 54,000, 60,000 and 64,000N/m, for Zn2+, Mn2+ and Cd2+, respectively, were recorded, far exceeding that previously reported for IDA based chelation ion exchange columns.

Rapid and Deep Profiling of Human Induced Pluripotent Stem Cell Proteome by One-shot NanoLC–MS/MS Analysis with Meter-scale Monolithic Silica Columns

Proteome analyses of human induced pluripotent stem cells (iPSC) were carried out on a liquid chromatography-tandem mass spectrometry system using meter-scale monolithic silica-C18 capillary columns without prefractionation. Tryptic peptides from five different iPSC lysates and three different fibroblast lysates (4 μg each) were directly injected onto a 200 cm long, 100 μm i.d. monolithic silica-C18 column and an 8-h gradient was applied at 500 nL/min at less than 20 MPa. We identified 98,977 nonredundant tryptic peptides from 9510 proteins (corresponding to 8712 genes), including low-abundance protein groups (such as 329 protein kinases) from triplicate measurements within 10 days. The obtained proteome profiles of the eight cell lysates were categorized into two groups, iPSC and fibroblast, by hierarchical cluster analysis. Further quantitative analysis based on an exponentially modified protein abundance index approach combined with UniProt keyword enrichment analysis revealed that the iPSC group contains more "transcription regulation"-related proteins, while the fibroblast group contained more "transport"-related proteins. Our results indicate that this simplified one-shot proteomics approach with long monolithic columns is advantageous for rapid, deep, sensitive, and reproducible proteome analysis.

High-resolution monolithic columns—a new tool for effective and quick separation

This work describes a comparison of three types of commercial high-performance liquid chromatography silica monolithic columns with different inner diameters and generations of monolithic sorbent: a "classic" monolithic column, the first generation (Onyx™ monolithic C(18), 100 mm × 4.6 mm, Phenomenex); a "narrow" monolithic column for fast separation at lower flow rates (Chromolith® Performance RP-18e, 100 mm × 3 mm, Merck); and a recently introduced "high-resolution" monolithic column, the next generation (Chromolith® HighResolution RP-18e, 100 mm × 4.6 mm, Merck). Separation efficiency (number of theoretical plates, height equivalent to a theoretical plate and van Deemter curves), working pressure, the symmetry factor and resolution were critical aspects of the comparison in the case of the separation of ascorbic acid, paracetamol and caffeine. The separations were performed under isocratic conditions with a mobile phase consisting of 10:90 (v/v) acetonitrile-phosphoric acid (pH 2.80). Detailed comparison of the newest-generation monolithic column (Chromolith® HighResolution) with the previously introduced monolithic sorbents was performed and proved the advantages of the Chromolith® HighResolution column.

Development and validation of a LC method for the separation and determination of the anticancer-active FeIII(4-methoxy-salophene) using the new second-generation monolith

LC method with the newly introduced second-generation monolithic silica RP-18e column has been developed for the separation of Fe(III) (salophene) and four methoxy-substituted Fe(III) (salophene) complexes. The method has been validated for the quantitation of Fe(III) (4-OMe-salophene), a highly active anticancer substance in vitro, bound to serum albumin. Our routinely used high-resolution continuum-source atomic absorption spectroscopy method based on the determination of the central iron atom was unsuitable in this case because serum originally contains significant amounts of iron as revealed by a blank sample of serum albumin. The developed LC method depends on detecting the whole complex rather than the bound iron. Two morphologically different first- and second-generation HPLC monolithic columns have been compared for this purpose. The newly introduced second-generation monolithic silica column Chromolith(®) HighResolution RP-18e column (100 × 4.6 mm, Merck) separated the mixture successful within 13 min. A mobile phase consisting of 25 mM phosphate buffer pH 3/methanol (60:40, v/v) was used at a flow rate of 1 mL/min. The dynamic linear working range of the calibration curve for Fe(III) (4-OMe-salophene) was found to be between 1 and 200 μg/mL. Detection and quantitation limits were 0.3 and 1 μg/mL, respectively.

Aptamer Modified Organic–Inorganic Hybrid Silica Monolithic Capillary Columns for Highly Selective Recognition of Thrombin

A novel kind of aptamer modified organic-inorganic hybrid silica monolithic capillary column has been developed, via the covalent bonding of 5'-NH(2)-modified aptamer for human α-thrombin on hybrid silica monolith, prepared by sol-gel method, with tetraethoxysilane and 3-aminopropyltriethoxysilane as precursors. Due to the large specific surface area of the hybrid matrix, the average coverage density of aptamer reached 568 pmol/μL, and the thrombin binding capacity was 1.15 μg/μL, 14 times higher than that of aptamer modified open tubular capillaries. By such an affinity capillary column, the limit of detection of thrombin was decreased to 3.4 nM with a UV detector. Furthermore, even when thrombin was mixed with 1000 times more concentrated human serum, it could be selectively enriched and detected with the signal-to-noise ratio as ca.10. These results indicate that the developed preparation strategy for aptamer based hybrid silica monolithic capillary column might provide an effective method to achieve highly selective recognition of trace targets.

Fast direct determination of strontium in seawater using high-performance chelation ion chromatography

A new method for the fast direct chromatographic determination of strontium in seawater has been developed. Two coupled silica monolithic columns (total dimensions 200×4.6mm I.D.) chemically modified with iminodiacetic acid (IDA) functional groups were used for baseline separation of all alkaline-earth metals in seawater in less than 4min at flow rate of 4.0mLmin−1, using an eluent containing 0.25M sodium chloride and 2mM glycolic acid, pH 5.11. Several colour-forming ligands, including o-cresolphthalein complexone (o-CPC), methylthymol blue (MTB), xylenol orange (XO), 4-(2-pyridylazo)resorcinol (PAR) and ZnEDTA–PAR were evaluated as reagents for post-column reaction (PCR) based spectrophotometric detection. Sensitivity of the developed method for strontium was at the low mgL−1 level in undiluted seawater samples, in the presence of excessive amounts of calcium and magnesium, using post-column reaction detection with o-CPC at 570nm. The method allowed quantitative determination of strontium in seawater samples (LOD 3.23±0.15mgL−1, sr=4.6%), with direct injection of just 4μL of seawater onto the chromatographic column.