Silica Packing Materials Research Articles

Bridged Ethylene Polyethylene Oxide Surfaces to Improve Packing Materials for Widepore Size Exclusion Chromatography.

Here, we describe the preparation of bridged ethylene polyethylene oxide (BE-PEO) surface-modified silica packing materials for size exclusion chromatography. BE-PEO surface-modified silica was hydrolyzed and subsequent 1H nuclear magnetic resonance analysis of hydrolysis products confirmed the successful formation of BE-PEO bonded surface. Silica particles exhibiting 3µm diameters and 1000 Å nominal pore diameters were selected as a base material for this work out of the critical need to improve analytical capabilities for the testing of cell and gene therapy drug products. Accelerated high pH aging study revealed significant enhancement in column stability. Multi-angle light scattering noise measurements showed inordinately lower baseline noise. Moreover, we evaluated the chromatographic performance of BE-PEO silica-packed columns through separations of a protein test mixture, DNA ladder, monoclonal antibody-based therapeutics, and adeno-associated viruses. BE-PEO silica columns demonstrated high resolution, high recovery separations that were confirmed to be reproducible and capable of extended column lifetimes and exhibited low ionic and hydrophobic secondary interactions. In summary, BE-PEO silica particles have yielded a new level of performance, improved base stability, and inherently lower baseline noise. These novel widepore particles will facilitate more sensitive size-based detection and characterization of large biologics in the form of advanced gene therapy products.

Synthesis and evaluation of ginsenosides imprinted polymer-based chromatographic stationary phase.

Molecular imprinting technique has aroused great interest in preparing novel stationary phases, and the resulting materials named molecularly imprinted polymers coated silica packing materials exhibit good performance in separating diverse analytes based on their good characteristics (including high selectivity, simple synthesis, and good chemical stability). To date, mono-template is commonly used in synthesizing molecularly imprinted polymers-based stationary phases. The resulting materials always own the disadvantages of low column efficiency and restricted analytes, and the price of ginsenosides with high purity was very high. In this study, to overcome the weaknesses of molecularly imprinted polymers-based stationary phases mentioned above, the multi-templates (total saponins of folium ginseng) strategy was used to prepare ginsenosides imprinted polymer-based stationary phase. The resulting ginsenosides imprinted polymer-coated silica stationary phase has a good spherical shape and suitable pore structures. Additionally, the total saponins of folium ginseng were cheaper than other kinds of ginsenosides. Moreover, the ginsenosides imprinted polymer-coated silica stationary phase packed column performed well in separation of ginsenosides, nucleosides, and sulfonamides. The ginsenosides imprinted polymer-coated silica stationary phase possesses good reproducibility, repeatability, and stability for seven days. Therefore, a multi-templates strategy for synthesizing the ginsenosides imprinted polymer-coated silica stationary phase is considered in the future. This article is protected by copyright. All rights reserved.

Characterization of the Interfacial Liquid Layer Formed on Hydrophobic Packing Material Surfaces by Liquid Chromatographic Analysis of the Distribution of Ions and Molecules.

We determine the bulk liquid phase volumes in octadecyl-bonded silica (C18 silica) columns equilibrated with acetonitrile–water and methanol–water (0–19%(v/v)) binary mixed solvents by a liquid chromatographic method with inorganic ions used as probes. The solvent composition and the thickness of the interfacial liquid layer formed on the C18-bonded silica surface are then determined from the bulk liquid phase volume, the total liquid phase volume, the surface area of the C18 silica packing material, and the retention volumes of the isotopically labeled eluent components for the columns. We used two C18 silica packing materials having identical bonding structures but different pore sizes and surface areas. Our results show that various hydrophilic organic compounds as well as inorganic ions recognize the interfacial liquid layer as being different from the bulk phase. The behavior of the solute compounds exhibiting substantially weak retention in reversed-phase liquid chromatography or the so-called negative adsorption, such as urea, sugars, and inorganic ions, can rationally be interpreted with a partition between the bulk liquid phase and the interfacial solvation liquid layer. The structural properties of the solvent layer on the C18-bonded layer determined by liquid chromatography are consistent with the molecular dynamics simulation results that have been obtained by other researchers.

Polyethyleneimine-functionalized carbon dots and their precursor co-immobilized on silica for hydrophilic interaction chromatography

A new strategy based on synergistic effect of the carbon dots and their precursor was proposed to enhance the selectivity of hydrophilic interaction liquid chromatography (HILIC). In this work, polyethyleneimine (PEI) and PEI-functionalized carbon dots (PEICDs) mix-grafted silica packing material was prepared to act as a novel HILIC stationary phase. Both inner and outer surface of the porous silica are decorated with the mixture of PEI and carbon dots. This stationary phase, namely Sil-PEI/CDs, demonstrate enhanced retention ability and selectivity toward polar analytes, with which 11 nucleosides and nucleobases and 9 ginsenosides can be nicely separated. The Sil-PEI/CDs (RSD 0.12% – 0.54%) exhibited even better stability than the traditional PEI modified stationary phase (RSD 0.39% – 0.87%) within 40 h of continuously elution. And excellent column efficiency was observed from Sil-PEI/CDs (∼65,000 plates/m for cytidine). The strategy of mixed stabilization revealed a new method to prepare good performanced carbon dots based chromatographic column.

Preparation of mixed-mode stationary phase for high performance liquid chromatography with ground hybrid silica monolithic material

With polyethylene glycol as a porogen, vinyltrimethoxysilane (VTMS) and tetramethoxysilane (TMOS) as silica precursors, a hybrid silica monolithic material was obtained under the catalysis of acetic acid and thermally decomposed urea. The silica monolithic material was ground by a ballmill, treated with tris(hydroxymethyl)aminomethane (Tris), then washed and dried to obtain silica particles with particle size~3 μm. The effects of different reaction conditions on the particle size, surface morphology and dispersibility of silica particles were investigated. When the volume ratio of TMOS to VTMS was 3:1, it was observed that silica particles with a pore diameter of 7.5 nm and a specific surface area of 245 m2/g were obtained. The resultant silica particles were modified by binding with chlorodimethyloctadecylsilane (C18) and by the thiol-ene click reaction to obtain a mixed-mode type stationary phase. The test results showed that the silica packing materials prepared in this work has certain applicability.

Quantitative Determination of Alkaloids in Lotus Flower (Flower Buds of Nelumbo nucifera) and Their Melanogenesis Inhibitory Activity.

A quantitative analytical method for five aporphine alkaloids, nuciferine (1), nornuciferine (2), N-methylasimilobine (3), asimilobine (4), and pronuciferine (5), and five benzylisoquinoline alkaloids, armepavine (6), norarmepavine (7), N-methylcoclaurine (8), coclaurine (9), and norjuziphine (10), identified as the constituents responsible for the melanogenesis inhibitory activity of the extracts of lotus flowers (the flower buds of Nelumbo nucifera), has been developed using liquid chromatography-mass spectrometry. The optimum conditions for separation and detection of these 10 alkaloids were achieved on a πNAP column, a reversed-phase column with naphthylethyl group-bonded silica packing material, with CH3CN–0.2% aqueous acetic acid as the mobile phase and using mass spectrometry equipped with a positive-mode electrospray ionization source. According to the protocol established, distributions of these 10 alkaloids in the petal, receptacle, and stamen parts, which were separated from the whole flower, were examined. As expected, excellent correlations were observed between the total alkaloid content and melanogenesis inhibitory activity. Among the active alkaloids, nornuciferine (2) was found to give a carbamate salt (2′′) via formation of an unstable carbamic acid (2′) by absorption of carbon dioxide from the air.

1.9 μm superficially porous packing material with radially oriented pores and tailored pore size for ultra-fast separation of small molecules and biomolecules

In this work, 1.9μm reversed-phase packing materials with superficially porous structure were prepared to achieve the rapid and high efficient separation of peptides and proteins. The silica particles were synthesized via three steps, nonporous silica particle preparation by a modified seeded growth method, mesoporous shell formation by a one pot templated dissolution and redeposition strategy, and pore size expansion via acid-refluxing. By such a method, 1.9μm superficially porous materials with 0.18μm shell thickness and tailored pore diameter (10nm, 15nm) were obtained. After pore enlargement, the formerly dense arrays of mesoporous structure changed, the radially oriented pores dominated the superficially porous structure. The chromatographic performance of such particles was investigated after C18 derivatization. For packing materials with 1.9μm diameter and 10nm pore size, the column efficiency could reach 211,300 plates per m for naphthalene. To achieve the high resolution separation of peptides and proteins, particles with pore diameter of 15nm were tailored, by which the baseline separation of 5 peptides and 5 intact proteins could be respectively achieved within 1min, demonstrating the superiority in the high efficiency and high throughput analysis of biomolecules. Furthermore, BSA digests were well separated with peak capacity of 120 in 30min on a 15cm-long column. Finally, we compared our columns with a 1.7μm Kinetex C18 column under the same conditions, our particles with 10nm pore size demonstrated similar performance for separation of the large intact proteins. Moreover, the particles with 15nm pore size showed more symmetrical peaks for the separation of large proteins (BSA, OVA and IgG) and provided rapid separation of protein extracts from Escherichia coli in 5min. All these results indicated that the synthesized 1.9μm superficially porous silica packing materials would be promising in the ultra-fast and high-resolution separation of biomolecules.

Scalable PEO-PPO-PEO triblock copolymer purification from Pluronics through competitive adsorption

A new scalable method for large scale purification of Pluronic block copolymers has been developed using a slurry of cyano(CN)-modified silica particles and Pluronic block copolymers in a solution. We have taken advantage of the competitive adsorption of polymers onto nanoporous CN-modified silica particles in solution to purify polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) triblock copolymers from as-received (AR) Pluronic samples, such as F108, F127 and F68. Interaction chromatography and size exclusion chromatography revealed that significant levels of low molecular weight (MW) impurities exist in AR Pluronics. We found that more than 20 wt.% impurities exists in AR F108 and AR F127, while the purified PEO-PPO-PEO triblock copolymers contained less than 2 wt.% of the low MW impurities after using the silica slurry method. In the case of AR F68, which contains about 9 wt.% impurities, it is possible to completely remove the low MW impurities. A multiple injection interaction chromatography technique has been employed to reveal the surface displacement of pre-adsorbed low MW impurities by the triblock copolymers in solution. By tuning the solvent quality, this report finally highlights that the impurities can be effectively removed in laboratory for a large scale purification using the silica slurry. For example, using about 10 g of the bulk silica packing material, it was possible to obtain 1 g of purified triblock copolymers.

Analytical Method for Quantification β-Cyclodextrin in Milk, Cream and Butter by LC

A simple, rapid and sensitive liquid chromatographic method using a refractive index detector has been developed and validated for identification and quantification of β-cyclodextrin in milk, cream and butter. The chromatographic system consists of a YMC ODS-AQ column packed with C18 reversed phase silica packing material as stationary phase using a mixture of methanol and water 7:93% (v/v) as the mobile phase. Linearity was established for the β-cyclodextrin concentration in the range 0.01–4 mg mL−1, with a coefficient (r) of 0.9998. Repeatability of the method was assessed; the coefficient of variation for β-cyclodextrin range were 1.24, 3.01, and 5.36% for milk, cream and butter. Recoveries ranged between 99.91 and 94.53%. This method was highly reproducible and reliable for quantification of treated milk with β-cyclodextrin for removal cholesterol from the milk fat.

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.

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.

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).

Fast, generic gradient high performance liquid chromatography coupled to fourier transform ion cyclotron resonance mass spectrometry for the accurate mass analysis of mixtures

Fast gradient high performance liquid chromatography (HPLC) has been combined with a commercially available Fourier transform ion cyclotron resonance (FTICR) mass spectrometer for the routine and high performance analysis of mixtures. With this combination we were able to separate and detect, under high mass accuracy conditions, a six-component drug mixture in less than 5 minutes. The fast gradients described are now possible due to the development of mechanically robust, ultra pure silica packing materials, which allow relatively high flow rates (ca. 1 mL/min for a 2 mm diameter column). For the six compounds present in the model mixture, relative mass errors of less than 1 ppm were obtained (based on an external calibration) providing sufficient mass accuracy to make unequivocal assignments of empirical formulae. Preliminary results of fast gradient HPLC/FTICR-MS/MS are also shown for the same six-component mixture. Copyright 2000 John Wiley & Sons, Ltd.

Effects of Mobile-Phase Composition and Temperature on the Selectivity of Poly(N-isopropylacrylamide)-Bonded Silica Gel in Reversed-Phase Liquid Chromatography

We developed a simple preparation method for poly(N-isopropylacrylamide) (poly(NIPAM))-bonded silica using a water-organic two-phase system to directly copolymerize N-isopropylacrylamide with 3-(methacryloxy)propyl moieties on the silica surfaces. The stationary phase showed a phase transition with the change in temperature or in methanol content, resulting in notable changes in selectivity which cannot be expected with alkyl-type stationary phases. The packing material showed an increase in retention and column efficiency with the increase in temperature between 30 and 40 °C or in methanol content between 10 and 20%. The temperature-responsive properties were reduced with the increase in methanol content of the mobile phase and lost at 40% or higher. Significant changes in retention selectivity by the addition of methanol were observed for benzene derivatives below the transition temperature of poly(NIPAM). The preferential retention of hydrogen bond acceptors was observed at low temperatures in the mobile phase of low methanol content, while dominant contribution of the hydrophobic interaction to the retention of solutes was observed at the higher temperature in aqueous mobile phase or at the higher methanol concentration. The temperature increase brought about similar changes in retention behavior of the poly(NIPAM)-bonded silica packing materials to the addition of methanol to the mobile phase.

Measurement of the degree of internal friction of two native silica packing materials

The degree of internal friction of two different packing materials for liquid chromatography (Kromasil NP10 and DuPont Pro Sil) has been measured using conventional methods of the mechanics of continuous solids applied in the study of the storage, handling and processing of powders. The former material was found to have a lower angle of internal friction and to flow more freely than the latter one. Possible relationships between these properties and the qualities of the column beds obtained are discussed.

Selectivity of electron-donor- and electron-acceptor-bonded silica packing materials for hydrophobic environmental contaminants in polar and non-polar eluents

Electron-acceptor-bonded stationary phases, 2-(nitrophenyl)-ethylsilyl (NPE) 3-( p-nitrophenoxy)propylsilyl (NPO), and electron-donor-bonded phases, 3-(N-carbazolyl)propylsilyl (CZP), 2-(1-pyrenyl)ethylsilyl (PYE), and 5-coronenylpentysilyl (COP), were prepared from silica particles and their selectivities were examined in both polar and non-polar solvents for specific isomers of polychlorodibenzo- p-dioxins (PCDDs), hexachloronaphthalenes (HxCNs) and planar and non-planar polychlorobiphenyl (PCB) congeners. Although no single stationary phase was able to separate all the isomer pairs that are coproduced during the synthesis of the PCDDs and HxCNs, pairs can be separated by selecting a suitable stationary phase and solvent. The separation of mixtures of PCDD isomers were found to be most successful with PYE and NPO phases, which yielded the opposite elution orders for each isomer pair that is produced as a mixture. Similar results were obtained for the HxCN isomers that were separated on PYE and CZP phases. The COP phase provided easier separation of non- ortho-substituted and mono- ortho-substituted PCBs from the other PCBs based on the planarity than PYE phase.

High performance liquid chromatography characterization of macromolecules

AbstractA new method, temperature gradient interaction chromatography(TGIC) is employed for the characterization of macromolecules. Fine and reproducible control of interaction between polymer chains and the alkyl chain bonded silica packing material can be achieved by varying the temperature of the column. This method provides a far superior resolution to the conventional size exclusion chromatography. In addition, this method has a high sample loading capacity to be effective for preparative purpose. Furthermore, this method can be used to characterize binary polymer mixtures, where one component of a polymer mixture is separated by the size exclusion mechanism and the other is by the interaction mechanism simultaneously from single isocratic elution.

Perfusive electroosmotic transport in packed capillary electrochromatography: Mechanism and utility

In this study, silica-based packing materials with pore sizes herein ˚ . referred to primarily as channel diameters, d ranging from 60 to 4000 A were ch utilized to elucidate the role of pore size in CEC. Separations were conducted using a commercially available CE system, the inlet and outlet buffer reservoir compartments having been modified to allow constant application of ;150 psi of helium head pressure. This application of pressure was sufficient to prevent nucleation of gas bubbles, a common practical hindrance in CEC. On-column UV detection was performed following the outlet frit of the packed section of the capillary. Electrokinetic sample introduction was employed. The experimental results of these studies indicate that the use of wide-pore media in CEC may provide for notable increases in efficiency. When wide-channel, corpuscular silica packing materials are employed in CEC, the microbeads which comprise the larger particle begin to dictate the ''true'' or effective diameter of the packing particles, and analytes experience two distinct regions of pore space through which eluent . flows: the intraparticle region inside the particles of packing material and the . interparticle region between adjacent particles of packing material . The percent of total pore volume contributed by through-pores is likely to be relatively small in the packing materials used, suggesting that particles with a greater degree of perfusive character could provide even further increases in both efficiency and loading capacity. These larger diameter packing materials are easier to work with, less expensive, and offer increased probability of success in the manufacture of packed capillaries for CEC. Q 1997 John Wiley & Sons, Inc. JM icro Sep9: 389)397, 1997 ()

Rapid analysis of organic microcontaminants in environmental water samples by trace enrichment and liquid chromatography on a single short column

On-column trace enrichment and liquid chromatography using a single short (20 mm length) high-pressure packed column was optimized for the rapid simultaneous identification and quantification of a wide range of organic microcontaminants in environmental water samples. The quality of different C 18 bonded silica packing materials in terms of their performance (i.e., peak capacity, repeatability) and enrichment/extraction characteristics (i.e., recovery, selectivity) was tested. With 15-ml samples, limits of detection of 0.1–0.4 μg/1 and 0.5–1.1 μg/1 were achieved for tap and surface water, respectively. The applicability of the system was demonstrated by the screening of real environment samples and the provisional identification of unknowns.

Characterization of a Range of Alkyl-Bonded Silica HPLC Stationary Phases: Correlation of Quantitative Surface Analysis Data with the Retention Behavior of Neutral, Acidic, and Basic Solutes

An investigation was made of the correlation between quantitative surface analytical data obtained by XPS and static SIMS and the chromatographic performance of a range of n-alkyl-bonded silica (C1-C18) packing materials. A series of acidic, basic, and neutral solutes was used to study the retention behavior. For comparison, analysis of bulk total percentage carbon (%C) and alkyl surface density of the bonded silica particulates were also included. Significant correlations were observed, in the majority of cases, between the retention factor (k) and the XPS C:Si atomic ratio, which was similar to that obtained between k and the bulk %C or k and the bonded alkyl chain length. Similar significant correlations were also obtained between k and the static SIMS alkyl:Si ion peak area ratios. XPS alkyl:Si atomic ratios were calculated as an estimate of alkyl surface coverage of the silica support, and these correlated well with the surface density calculated from the bulk %C and the surface area of the packing material. The XPS alkyl:Si ratio also demonstrated a significant correlation with the peak asymmetry factor derived for basic solutes. These studies confirm that both XPS and static SIMS can generate surface chemical data from chromatography particulates, which has direct relevance to the prediction of chromatographic behavior. We believe that these techniques will prove to be effective tools to assist in the characterization of chromatographic supports and stationary phases.