Silica Packing Research Articles

Heart-cutting two-dimensional liquid chromatography methods for quantification of 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole in Class III caramel colours

Heart-cutting two-dimensional liquid chromatography/UV detection methods were developed to determine 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole (noted as THI) in Class III caramel colours. In the first dimension, the caramel sample diluted in water was fractionated either by reversed-phase chromatography on C18 silica packing or by ion-exchange chromatography using strong cationic exchanger (SCX) stationary phase. In each case, THI elution domain was recovered in a loop and transferred to a second column packed with porous graphitic carbon (PGC) for the selective separation of THI. From the accuracy profiles with acceptance limit fixed at 20% and β-expectation tolerance interval fixed at 90%, the two methods, C18–PGC and SCX–PGC, were validated for THI/caramel ratios included between 5–50 and 10–50 ppm, respectively. Finally, the two methods were compared for the analysis of numerous caramel samples.

When Size Matters—Near Infrared Reflection Spectroscopy of Nanostructured Materials

This article evaluates the applicability of near infrared (NIR) reflection spectroscopy for the physico-chemical and morphological characterisation of matter on a scale smaller than 1 micrometre (normally between 1 and 100 nanometres). The investigated materials comprise porous and non-porous silica particles, carbon based materials such as C60 fullerenes, nano-crystalline diamond (NCD) and dendrimers, all of them having diameters and/or pore sizes in the nanometre range, respectively. In case of the silica packings and differently derivatised C60 fullerenes, absorbance signals could be clearly assigned to corresponding surface modifications. Identification or classification of the material can be achieved successfully by principal component analysis. Nano crystalline diamond surfaces, whether H- or O-terminated, could be differentiated by a computed partial least squares (PLS) regression model with around 80% precision. Generations 0–7 of poly(amidoamine) (PAMAM) dendrimers with functionalised surface amine groups are characterised in respect of particle diameter and molecular weight. The established PLS models showed a standard error of prediction of only 0.43 nm and 12.30 kDa, respectively. NIR spectroscopy has developed as a flexible analytical method that can be utilized for fast, reliable and highly reproducible screening of matter even in the nanometer range.

Direct catalytic oxidation of methane to formaldehyde: New investigation opportunities provided by an improved flow circulation method

An improved flow circulation reactor with reaction mixture quenching was used to study the direct oxidation of methane to formaldehyde at 600–770°C, a CH4/O2 volume ratio of 1: 1 in the starting mixture, and gas mixture flow rates of 0.4–2.0 l/h. It was found that, in the presence of a surface containing SiO2 (quartz reactor walls, the surface of silica packing materials, or the surface of SiO2 as a catalyst constituent), the process occurred by a heterogeneous-homogeneous mechanism with chain continuation in the volume of a gas phase. The process was controlled by the size and shape of the free reaction volume, the contact surface area, and the residence time of a mixture in the reaction volume. The introduction of typical oxidation catalysts containing, for example, Pt or V2O5 as an active component along with SiO2 resulted in a decrease in the yield of and selectivity for formaldehyde.

Fused-Core Silica Column High-Performance Liquid Chromatography/Tandem Mass Spectrometric Determination of Rimonabant in Mouse Plasma

A high-performance liquid chromatography (HPLC) method using a fused-core silica particle packing was evaluated to allow fast and efficient separation for the analysis of pharmaceutical compounds. Fused-core particles are produced by "fusing" a porous silica layer onto a solid silica particle. The efficiencies of columns packed with 2.7 microm "fused-core" particles (a 0.5 microm porous shell fused to a solid 1.7 microm silica core particle) and 1.7 microm porous particles were compared in reversed-phase HPLC using rimonabant as an analyte. The fused-core silica materials providing the shorter diffusional mass transfer path for solutes are less affected in resolving power by increases in mobile-phase velocity than the sub-2 microm porous silica packings resulting in faster separations and higher sample throughput. This fast HPLC technology is comparable with ultrahigh-pressure liquid chromatography (UHPLC) in terms of chromatographic performance but demands neither expensive ultra-high-pressure instrumentation nor new laboratory protocols. The column effluent was directly connected to the atmospheric pressure chemical ionization (APCI) source prior to tandem mass spectrometric detection. In this work, the described fast HPLC-MS/MS and UHPLC-MS/MS approaches requiring approximately 1.5 min per sample were applied and compared for the determination of the rimonabant in mouse plasma samples at the low nanograms per milliliter region in support of a pharmacodynamic study.

Fully Packed Capillary Electrochromatographic Microchip with Self-Assembly Colloidal Silica Beads

A fully packed capillary electrochromatographic (CEC) microchip showing improved solution and chip handling was developed. Microchannels for the CEC microchip were patterned on a cyclic olefin copolymer substrate by injection molding and packed fully with 0.8-microm monodisperse colloidal silica beads utilizing a self-assembly packing technique. The silica packed chip substrate was covered and thermally press-bonded. After fabrication, the chip was filled with buffer solution by self-priming capillary action. The self-assembly packing at each channel served as a built-in nanofilter allowing quick loading of samples and running buffer solution without filtration. Because of a large surface area-to-volume ratio of the silica packing, reproducible control of electroosmotic flow was possible without leveling of the solutions in the reservoirs resulting 1.3% rsd in migration rate. The capillary electrophoretic separation characteristics of the chip were studied using fluorescein isothiocyanate (FITC)-derivatized amino acids as probe molecules. A mixture of FITC and four FITC-derivatized amino acids was successfully separated with 2-mm separation channel length.

Liquid chromatographic method development for steroids determination (corticoids and anabolics): Application to animal feed samples

A LC isocratic separation study of a complex mixture containing 18 steroids (corticoids and anabolics), used potentially as growth promoters, was carried out. For this purpose, using a Hypersil ODS column at controlled temperature, mobile phases (from binary to quaternary) prepared from water and MeOH, ACN or THF as organic modifiers and UV detection at 245 nm, were employed (dehydroepiandrosterone was detected at 200 nm). The optimum separation was achieved using water/acetonitrile (65:35, v/v) as mobile phase at 30 °C, allowing the separation of 16 out of 18 steroids in about 30 min. The retention scale using optimized binary mobile phases was related with steroids hydrophobicity and structure, allowing a classification into three groups for these compounds. To improve the separation several alkyl–silica packings were tested: Type A (Lichrospher C8) and Type B (Luna C18, Kromasil C18, Purospher C18 and Synergy C12). Taking into account resolution, number of separated compounds and run time analysis the Hypersil column was selected as the best choice for further applications. Calibration graphs were obtained using fluorocortisone, fluoxymesterone or methylprednisolone as internal standard. The optimized separation was applied to the analysis of piglet feed samples spiked with steroids. The sample preparation process included solvent extraction using diethyleter and solid phase extraction using silica cartridges. The recoveries were in the range 70–92%. Decision limits and detection capability were in the range 34–198 and 41–249 μg/kg, respectively. Repeatability was also evaluated.

Preparation of a Concanavalin A Immobilized Affinity Column and Its Application in the Structural Analysis of Ribonuclease B

The research on glycoproteomes represents an interesting field in the functional proteomics research. Affinity chromatography and mass spectrometry are powerful techniques that are used for gaining valuable information on glycoproteomes because glycoproteins and their unusual forms resulting from protein glycosylation can be important indicators of several diseases. In this study, the concanavalin A (Con A) immobilized silica packing was prepared and used for the separation of glycoprotein and glycopeptides. A very low, non-specific adsorption on the Con A affinity column was demonstrated by mass recovery of bovine serum albumin at more than 98.5%. The effect of concentration of methyl-alpha-D-mannopyranoside (alpha-Me-D-Man) in the mobile phase and the effect of flow rate on the retention behavior of ribonuclease B (RNase B) were also investigated. The standard glycoprotein RNase B was separated under optimized conditions using 0.2 mol/L alpha-Me-D-Man in the mobile phase at a flow rate of 0.5 mL/min. Meanwhile, the oligosaccharides and glycopeptides were enriched using a Con A column after digestion of the purified RNase B with peptide-N-glycosidase F (PNGase F) and trypsin. The structure of N-linked glycan and the rate and the site of glycosylation of RNase B were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Glycoproteins and glycopeptides in human serum and digest solution could be separated by this method. The results showed that this method is rapid and sensitive for the purification and characterization of glycoproteins and glycopeptides.

Silica particles encapsulated poly(styrene-divinylbenzene) monolithic stationary phases for μ-high performance liquid chromatography

In the paper we demonstrate a new approach for the preparation and application of continuous silica bed columns that involve encapsulation (entrapment) of functionalized silica microparticles, which can be used as packing material in micro high performance liquid chromatography (μ-HPLC) and capillary electrochromatography (CEC). Like traditional packed columns, these capillaries possess characterized silica particles that offer high phase ratio and narrow pore size distribution leading to high retention and separation efficiency, respectively. More importantly, immobilization of the microparticles stabilizes the separation bed and eliminates the need for retaining frits. The developed capillary columns were fabricated in exactly the same way as a packed capillary column (slurry packing) but with an additional entrapment step. This immobilization of the packed bed was achieved by in situ polymerization of styrene and divinylbenzene in presence of decanol as a porogen and azobisisobutyronitrile as thermal initiator. Silica particles with different particle sizes and pore sizes ranging from 60 to 4000 Å were studied. In addition different modified silica was used, including C-18 reversed phase, anion exchange and chiral stationary phases. Efficient separation of polyphenolic compounds, peptides, proteins and even DNA mutation were achieved using the developed technique depending on the properties of the silica particles used (particles pore size). For example, using 3 μm ProntoSIL C-18 particles with 300 Å pore size, separation efficiencies in the range of 120,000–200,000 plates/m were obtained for protein separation, in a 6 cm × 200 μm i.d. capillary column. Using encapsulated silica C-18 with 1000 Å pore size, separation of DNA homo and hetero duplexes were achieved under denaturing HPLC conditions for mutation detection. In addition, nucleotides were separated using anion exchange material encapsulated with poly(styrene-divinylbenzene) (PS/DVB), which indicated that the chromatographic properties of the silica packing material were still active after polymerization. The prepared capillary columns were found to be stable and could easily be operated continuously up to a pressure of 350 bar without column damage and capillary can be cut to any desired length.

Scientific achievements of Jack Kirkland to the development of HPLC and in particular to HPLC silica packings—a personal perspective

Joseph (Jack) Kirkland is one of the outstanding protagonists of modern column liquid chromatography (HPLC). He started in 1953 as an analytical chemist at the Experimental Station of Du Pont de Nemours & Co, Wilmington, Delaware, analyzing pesticides by gas chromatography (GC). He early recognized the potential of HPLC as a powerful separation technique at the end of 1960. His major contributions to HPLC were in the field of silica based packings and stationary phases. At the beginning of the 1970’s he manufactured Porous Layer Beads and later microparticulate porous silicas based at the Zorbax technology. He made outstanding contributions in the field of instrument design for HPLC and in the field of Sedimentation Field Flow Fractionation (SFFF), in HPLC method development and optimization strategies. In 1992 Jack resigned from Du Pont de Nemours & Co, Wilmington, Delaware, and joined Rockland Technologies, Newport, Delaware, as a Director of Research and Development. During this period he successfully developed a series of novel, designed reversed phase silicas. He resigned from Rockland technologies, now Agilent Technologies, Newport, Delaware, in 2001, but always remained dedicated to HPLC.

Effect of solvent strength and temperature on retention for a polar-endcapped, octadecylsiloxane-bonded silica stationary phase with methanol–water mobile phases

Synergi™ Hydro-RP is a new type of polar-endcapped, octadecylsiloxane-bonded silica packing for reversed-phase liquid chromatography. Its retention properties as a function of solvent strength and temperature are evaluated from the change in retention factors over the composition range (0–70% v/v methanol) and temperature range (25–65 °C) using the solvation parameter model and response surface methodologies. The main factors that affect retention are solute size and hydrogen-bond basicity, with minor contributions from solute hydrogen-bond acidity, dipole-type and electron lone pair interactions. Within the easily accessible range for both temperature and solvent strength, the ability to change selectivity is much greater for solvent strength than temperature. Also, a significant portion of the effect of increasing temperature is to reduce retention without changing selectivity. Response surfaces for the system constants are smooth and non-linear, except for cavity formation and dispersion interactions ( v system constant), which is linear. Modeling of the response surfaces suggests that solvent strength and temperature are not independent factors for the b, s and e system constants and for the model intercept ( c term).

Studies on a poly(styrene-divinylbenzene)-encapsulated sub-micrometer sized organic–inorganic hybrid silica packing, its synthesis, some of its characteristics and applications

The hydrolyzed product of a mixture of tetraethoxysilane (TEOS) and vinyltriethoxysilane (VTEOS) was used to produce organic–inorganic hybrid silica by sol–gel processing. The product was a kind of composite with vinyl groups incorporated into the structure of the final particles through CSi bonds. These hybrid silica particles were spherical and the average diameter was about 350 nm as measured by scanning electron microscopy (SEM). The particles were non-porous as proved by BET method. The hybrid silica particles were subsequently encapsulated with a layer of PS–DVB, a polymeric mixture of styrene and divinylbenzene, with the vinyl groups on the particle surface as anchors of the polymer chains. The encapsulated packing was used as stationary phases in CEC, and the chromatographic behaviors were studied. Experimental results showed that, as expected, the smaller diameter stationary phase resulted in higher column efficiencies. In comparison with the columns packed with 5 μm diameter encapsulated-silica stationary phases of the same kind and other sub-micron stationary phases reported, it can be concluded that the EOF velocity is independent of the particle diameter when using 350 nm phase, but determined by the surface nature of the stationary phase.

Reversed-phase chromatography on a polar endcapped octadecylsiloxane-bonded stationary phase with water as the mobile phase

SynergiTM Hydro-RP is a polar endcapped octadecylsiloxane-bonded silica packing for reversed-phase chromatography compatible with water as a mobile phase. The effect of temperature on retention and selectivity for a varied group of compounds is evaluated from plots of the retention factors against temperature and by application of the solvation parameter model. Retention is shown to increase with the size and capability of the solute for electron lone pair interactions and is reduced by hydrogen-bonding and dipole-type interactions. Increasing temperature over the range 25 to 65°C reduces retention with only a small affect on selectivity. Increasing temperature effectively increases the phase ratio for the column and diminishes the relative contribution of electron lone pair interactions with the stationary phase. The SynergiTM Hydro-RP column in suitable for the separation of small polar molecules with good peak shape and reasonable efficiency in an acceptable separation time.

Effect of Silica Packing on Ozone Synthesis from Oxygen-Nitrogen Mixtures

The effect of silica packing in an ozone generator fed with oxygen-nitrogen mixtures has been studied. The measurements were carried out in an ozone generator with a double-sided cooled discharge gap, in which silica packing has been placed. In the presence of the packing considerably higher ozone concentrations were achieved than in the ozone generator without packing. An especially clear effect of silica packing was observed in air-fed conditions. It has been found that the increase in the concentration of the obtained ozone coincides with the increase of N2O in the post-reaction gas.

Retention studies of sulphated glycosaminoglycan disaccharides on porous graphitic carbon capillary columns

The retention behaviour of highly polar and charged disaccharide isomers has been studied on porous graphitic carbon columns and experimental parameters were varied over wide a range, including concentration and type of mobile phase constituents as well as temperature. The hydrophilic and anionic sugar analytes were highly retained on this stationary phase compared to the octadecyl-derivatized silica packings more commonly used. E.g., an increase in retention with polarity of a solute and with temperature was observed. By isotherm measurements and nonlinear fitting of Langmuirian expressions to the experimental data the graphite surface appeared homogeneous with only one kind of active adsorption site for these kinds of compound which was furthermore supported by the linear Van't Hoff plots obtained by varying the temperature. The gain in free energy was found to be entropically driven after determination of the ΔH° and ΔS° values. However, enthalpy-entropy compensation behavior was not met.

Performance of metal complex substituted polysiloxanes in capillary electrophoresis and capillary electrochromatography

Two novel polysiloxanes containing the metal complex, Co(TACN) 2 3+ (TACN=1,4,7-triazacyclononane) were used as coatings for capillary electrophoresis (CE) and capillary electrochromatography (CEC). Through crosslinking and covalent bonding, the positively charged polymers were bonded to silica supports. In both CE and CEC, these coatings exhibited strong, pH-independent, and anodic electroosmotic flow (EOF), and had excellent long-term stability. Successful separations of aromatic acids were achieved in CE. In CEC, separation of alkylbenzenes (7 min) and basic compounds (20 min) was achieved with higher resolving power than conventional octadecyl silica packings. These polymers represent a new class of coatings for CE and CEC that generate pH-independent EOF.

Gas chromatography silica packings with chemically bonded complexes of Cu(II) and Cr(III)

In the presented work, copper(II) and chromium(III) complexes chemically bonded to silica surface were prepared and used in gas chromatography. Chlorides of these metals were bonded to the silica surface by the use of the ketoimine group originally from 2-(3-triethoxysililpropylimino)-3-( n-butyl)-pentanone-4. Packings obtained in such way were investigated to evaluate their usefulness in the analysis of aliphatic and aromatic halogenated hydrocarbons as well as ethers, thioethers and ketones. Moreover, packings were subjected to elemental analysis and differential scanning calorimetry (DSC), thus some retention parameters were determined to characterize specific interactions between bonded metal and adsorbate molecules. Attempts were also made to separate some mixtures of the nucleophilic compounds.

Reversed-Phase High-Performance Liquid-Chromatographic Determination Systems Specific to Ultratrace Hard Metal Ions with Tridentate Schiff Bases and Pyridylhydrazones

Abstract Two Schiff-base ligands derived from salicylaldehyde and two 2-pyridylhydrazones were examined for the ultratrace determination of metal ions by reversed-phase high-performance liquid chromatography (HPLC). Although these ligands have been thought to be rather unattractive in inorganic analysis, their renewal in the HPLC methodology was demonstrated in this work. In these ligand systems, certain hard metal ions such as Al(III), Ga(III), V(V), Fe(III), and Co(III) ions, were effectively separated on octadecyl- or cyanopropyl-bonded silica packing materials. A highly sensitive fluorimetric detection of Al(III) ion was achievable with N-salicylidene-2-hydroxyaniline having an O,N,O donor set; although the contamination problem actually limited the detection capability, the calculated detection limit for Al(III) ion was down to 0.1 nM (3 ppt) on a 3 σ blank basis. The chromatography of chelates with 4-N,N-diethylaminosalicylaldehyde-2-pyridylhydrazone seems to be attractive for actual analysis; Al(III), Ga(III), V(V), Fe(III), and Co(III) ions at the sub-ppb to ppb levels have been photometrically detected with a relative standard deviation of less than 5%. The fluorimetric detection using 4-hydroxysalicylaldehyde-2-pyridylhydrazone was very effective to access trace levels of Al and Ga ions, giving detection limits of 3.0 nM and 1.3 nM, respectively (M = mol dm−3).

Impact of the post-treatment conditions of parent silica on the silanization of n-octadecyl bonded silica packings in reversed-phase high-performance liquid chromatography

Native mesoporous silica beads were subjected to a sequence of post-treatment procedure including hydrochloric acid treatment, calcination and subsequent rehydroxylation. The post-treated silica beads were converted into RP-18 silica by silanization with monochloro- and dimethoxy- n-octadecylsilanes, respectively. The influence of post-treatments and silanization conditions on the physico–chemical characteristics and on the chromatographic behaviour of the RP-silicas was studied. Also the changes of the pore structural parameters and the silanol group densities during the post-treatment and silanization were assessed.

Protein mapping by two-dimensional high performance liquid chromatography

Current developments in drug discovery in the pharmaceutical industry require highly efficient analytical systems for protein mapping providing high resolution, robustness, sensitivity, reproducibility and a high throughput of samples. The potential of two-dimensional (2D) HPLC as a complementary method to 2D-gel electrophoresis is investigated, especially in view of speed and repeatability. The method will be applied for proteins of a molecular mass <20 000 which are not well resolved in 2D-gel electrophoresis. The 2D-HPLC system described in this work consisted of anion- or cation-exchange chromatography in the first dimension and reversed-phase chromatography in the second dimension. We used a comprehensive two-dimensional approach based on different separation speeds. In the first dimension 2.5 μm polymeric beads bonded with diethylaminoethyl and sulfonic acid groups, respectively, were applied as ion exchangers and operated at a flow-rate of 1 ml/min. To achieve very high-speed and high-resolution separations in the second dimension, short columns of 14×4.6 mm I.D. with 1.5 μm n-octadecyl bonded, non-porous silica packings were chosen and operated at a flow-rate of 2.5 ml/min. Two reversed-phase columns were used in parallel in the second dimension. The analyte fractions from the ion-exchange column were transferred alternatively to one of the two reversed-phase columns using a 10-port switching valve. The analytes were deposited in an on-column focusing mode on top of one column while the analytes on the second column were eluted. Proteins, which were not completely resolved in the first dimension can, in most cases, be baseline-separated in the second dimension. The total value of peak capacity was calculated to 600. Fully unattended overnight runs for repeatability studies proved the applicability of the system. The values for the relative standard deviation (RSD) of the retention times of proteins were less than 1% ( n=15), while the RSDs of the peak areas were less than 15% ( n=15) on average. The limit of detection was 300 ng of protein on average and decreased to 50 ng for ovalbumin. The 2D-HPLC system offered high-resolution protein separations with a total analysis time of less than 20 min, equivalent to the run time of the first dimension.

Towards the ultimate minimum particle diameter of silica packings in capillary electrochromatography

Porous silica beads with an average particle diameter between 0.2 and 3 μm have been applied as packing material in capillary electrochromatography (CEC). The experiments were directed to investigate whether it is really feasible and as promising as expected to use such small particles. In CEC, plate heights of H≈1–2 d p can be achieved which is smaller than the plate heights usually attained in high-performance liquid chromatography. Using a capillary packed with 0.5 μm silica beads we achieved a plate height of H=3 d p indicating the presence of dispersive effects like Joule heating. Calculations demonstrate that at a field strength of about 800 V cm −1 one third of the plate height can be lost by Joule heating effects if the heat is not removed by a cooling system. Additionally, the H( u) curve is still descending at the maximum electroosmotic flow (EOF) velocity we generated with the modified capillary electrophoresis instrument. To fully exploit the potential of submicron size silicas higher field strengths, i.e., higher EOF velocities, must be attained. To study the influence of the kind of packing on the EOF porous as well as nonporous silicas have been applied. The experiments clearly indicate that the EOF of porous and nonporous silicas is the same. Since the EOF is more or less exclusively generated by the packing material the zeta potential of n-octyl bonded 0.5 μm silica has been determined. The dependence of the zeta potential on the pH is identical to the dependence of the EOF on the pH in a packed capillary. The point of zero charge of the silica is at pH 2–3.