Amino-bonded Silica Research Articles

Further Evaluation of the Base Stability of Hydrophilic Interaction Chromatography Columns Packed with Silica or Ethylene-Bridged Hybrid Particles

One of the fundamental attributes of a liquid chromatography column is its stability when exposed to acidic and basic mobile phases. However, there have been relatively few reports to date on the stability of hydrophilic interaction chromatography (HILIC) columns. Here, we report the results of stability evaluations carried out for HILIC columns packed with ethylene-bridged hybrid or silica particles using accelerated conditions, employing a 100% aqueous pH 11.3 ammonium bicarbonate mobile phase at 70 °C. Under these conditions, the primary mode of column failure was a loss of efficiency due to the formation of voids resulting from the hydrolysis of the particles. We investigated the dependence of stability on the surface area of both unbonded and sulfobetaine-bonded ethylene-bridged hybrid stationary phases. The results show a clear trend of stability increasing as the surface area decreases. Several commercially available HILIC columns that are recommended for use with high-pH mobile phases were also evaluated. The results show times to 50% loss of the initial efficiency ranging from 0.3 to 9.9 h. Columns containing unbonded, sulfobetaine-bonded or diol-bonded ethylene-bridged hybrid stationary phases had longer lifetimes than amino-bonded silica or sulfobetaine-bonded, hybrid-coated, superficially porous silica columns.

Size exclusion chromatography of aromatic compounds in high-boiling petroleum samples

Knowledge of aromatic compounds in petroleum samples is of paramount interest from processing and environmental viewpoints. Considering the complexity of such samples, a selective analytical strategy viz. aromatic specific size exclusion chromatographic methodology using amino-bonded silica phase, was adopted to obtain the average molecular weights of distilled cuts, residue and the corresponding crude oil. The method was found to be suitable from middle distillates up to the residue. It was found that the increase in boiling temperature of distilled cuts was reflected in an increase in high molecular weight aromatic species. The increase in the molecular weight of aromatic species is linked to the increase in alkylation as verified by studying the elution pattern of alkylated aromatic compounds. The accuracy of the molecular weights obtained from the size exclusion chromatographic method depends on the elution profile of aromatic species in petroleum samples. It was found that the size and shape of the parent aromatic compounds negatively influence the size exclusion elution profile. However, such influence can be assumed to be negligible as all the aromatic species of each distilled cut, residue, and crude oil eluted in the permeation zone of the chromatogram. This method has the potential to be used in the second dimension of a 2D-LC method where the aromatic compounds are separated based on the size (or alkylation). The first dimension will separate compounds based on the number of aromatic rings, thereby eliminating the interference of parent aromatic compounds of different sizes in the total elution profile.

Size exclusion chromatography of petroleum samples using amino-bonded silica phase

Amino-bonded silica phase, frequently used in adsorption chromatographic mode for petroleum analysis, was evaluated for the separation of petroleum components based on polymer liquid chromatographic modes (viz. size exclusion, critical condition, and interaction). After verification of the stationary phase for polymer chromatography using polystyrene standards at different compositions of cyclohexane and dichloromethane, a similar approach was attempted for a distilled fraction of a crude oil. The approach was successful in size exclusion chromatographic mode whereas other modes were partially achievable because of the complex nature of petroleum samples. Subsequently, size exclusion mode was applied in crude oils with different American Petroleum Institute (API) gravity values. An inverse linear relationship between the API gravity value and the average molecular weights of the aromatic compounds of crude oils was established. It was found that the weight average molecular weight (Mw) was more sensitive to changes in API gravity values of the crude oils in comparison to number average molecular weight (Mn). Additionally, crude oil with lower API gravity value reflected broader distribution of species. Further studies on crude oils with lower API gravity value (<20°) and of different geographical origins will confirm the universality of the method. This method will provide reliable average molecular weights of the aromatic content for aromatic rich refinery streams to optimize process conditions. Furthermore, the developed method can potentially be extended to a two-dimensional LC method using the same stationary phase with different mobile phase compositions for comprehensive characterization of petroleum samples.

Preparation and evaluation of a new type of amino-bonded silica chromatographic stationary phase for the determination of lactose

A method for the separation and determination of lactose and sucrose using high performance liquid chromatography-refractive index (HPLC-RI) detection base on the prepared amino-bonded silica gel chromatographic stationary phase was established. The method was accorded to the content determination method in Pharmacopoeia of the People's Republic of China (2015). Then, a series of chromatographic behaviors of lactose and sucrose such as the retention times, resolutions and peak area stabilities were investigated on three different types of amino-bonded silica columns (300 mm×4.6 mm, 5 μm). The mobile phase was acetonitrile-water (70:30, v/v) with isocratic elution, the flow rate was 1.0 mL/min and the injection volume was 10 μL. The resolution of lactose and sucrose was 3.03 by isopropyl side chain protected amino column, and the peak shape of each target compound was good. The relative standard deviation (RSD) of peak area of lactose was 1.14%, less than 2.0% prescribed in the Pharmacopoeia of the People's Republic of China (2015). Therefore, this method meets the requirements of lactose content determination, and is suitable as the quality control method in the above mentioned Pharmacopoeia.

Post-synthetic modification of MIL-101(Cr) with pyridine for high-performance liquid chromatographic separation of tocopherols

Effective separation of tocopherols is challenging and significant due to their structural similarity and important biological role. Here we report the post-synthetic modification of metal–organic framework (MOF) MIL-101(Cr) with pyridine for high-performance liquid chromatographic (HPLC) separation of tocopherols. Baseline separation of four tocopherols was achieved on a pyridine-grafted MIL-101(Cr) packed column within 10min using hexane/isopropanol (96:4, v/v) as the mobile phase at a flow rate of 0.5mLmin−1. The pyridine-grafted MIL-101(Cr) packed column gave high column efficiency (85,000platesm−1 for δ-tocopherol) and good precision (0.2–0.3% for retention time, 1.8–3.4% for peak area, 2.6–2.7% for peak height), and also offered much better performance than unmodified MIL-101(Cr) and commercial amino-bonded silica packed column for HPLC separation of tocopherols. The results not only show the promising application of pyridine-grafted MIL-101(Cr) as a novel stationary phase for HPLC separation of tocopherols, but also reveal a facile post-modification of MOFs to expand the application of MOFs in separation sciences.

Fabrication of a TCEP-immobilised monolithic silica microchip for reduction of disulphide bonds in proteins

Protein identification by mass spectrometry forms the cornerstone of proteomics. Commonly, the identification of proteins is based on digestion of proteins into peptides using proteolytic enzymes. Cleavage of the disulphide bonds in proteins before enzymatic digestion and mass spectral analysis is important in order to facilitate the accessibility of the enzyme to cleave the proteins into peptides. As a result, the protein sequence coverage will be increased. In this study, a novel approach for immobilisation of the reducing reagent on the surface of a silica-based monolith in order to use it for reduction of disulphide bonds in proteins was successfully developed. This was carried out by silanisation of the surface of the silica-based monolith with (3-aminopropyl)triethoxysilane (APTES), followed by immobilisation of the reducing reagent, tris(2-carboxyethyl)phosphine hydrochloride (TCEP) on the surface of the amino-bonded silica monolith. The fabricated monolith was characterised using SEM analysis, EDX analysis, IR spectroscopy, and BET model. The performance of the fabricated glass microchip containing the TCEP-immobilised silica monolith to reduce the disulphide bonds in proteins was checked by injection of 100 μL of denatured insulin inside the microchip using a syringe pump at a flow rate of 10 μL min−1, followed by sealing both ends of the ETFE tubes with Blu-Tak. The microchip was kept in a humidified chamber for 30 min at 60 °C. After the reduction reaction, the reduced cysteine residues were alkylated with IAA at 60 °C for 30 min, followed by using a MALDI-TOF-MS instrument for qualitative confirmation. The results show that the fabricated microchip-based silica monolith has the ability to reduce disulphide bonds in insulin. In addition, the method is simple, reduces the risk of contamination, and results in lower amounts of the sample and reagents compared with the conventional techniques for proteomics sample preparation. A future study investigating reduction of the disulphide bonds in proteins from a real sample using this new microfluidic device would be very interesting.

Comprehensive two-dimensional chromatography with coupling of reversed phase high performance liquid chromatography and supercritical fluid chromatography

A 2D comprehensive chromatographic separation of blackberry sage fragrant oil was performed by using HPLC in the first dimension and SFC in the second. A C(18)-bonded silica column eluted with an ACN gradient was used in the HPLC dimension and an amino-bonded silica column eluted with ACN as a modifier in the SFC dimension. This 2D separation was completed in the off-line mode, the fractions from the HPLC column being collected and injected in the SFC column. The retention factors on the two columns have a -0.757 correlation coefficient. The method provides a practical peak capacity of 2400 in 280 min. The first eluted peaks in HPLC are the last ones eluted in SFC and vice versa. The results demonstrate that the coupling of an HPLC and an SFC separation have a great potential for 2D chromatographic separations.

Hydrophilic interaction chromatography separation mechanisms of tetracyclines on amino‐bonded silica column

Effects of mobile-phase variations on the chromatographic separation on amino-bonded silica column in hydrophilic interaction chromatography (HILIC) were investigated for four zwitterionic tetracyclines (TCs): oxytetracycline, doxycycline, chlortetracycline, and tetracycline. A mixed-mode retention mechanism composed of partitioning, adsorption, and ion exchange interactions was proposed for the amino HILIC retention process. Buffer type and pH significantly influenced the retention of TCs, but showed similar separation selectivity for the tested analytes. Experiments varying buffer salt concentration and pH demonstrated the presence of ion exchange interactions in TCs retention. The type and concentration of organic modifier also affected the retention and selectivity of the analytes, providing direct evidence supporting the Alpert retention model for HILIC. The retention time of the analytes increased in the following order of organic modifiers: tetrahydrofuran < methanol < isopropanol < acetonitrile. The linear relationships of logk' versus %water (v/v) curve and logk' versus logarithm of %water (v/v) in the mobile phase indicated that TCs separation on the amino phase was controlled by partitioning and adsorption. The developed method was successfully utilized in the detection of TCs in both river water and wastewater samples using solid-phase extraction (SPE) for sample cleanup.

Analysis of volatile and semi-volatile compounds in tobacco using off-line combination of liquid chromatography and capillary gas chromatography-mass spectrometry

A method of off-line combination of liquid chromatography and capillary gas chromatography-mass spectrometry (LC-CGC/MS) was established for the analysis of volatile and semi-volatile compounds in tobacco. The separation mechanism of volatile and semi-volatile compounds was studied. An LC column of 250 mm x 2.0 mm packed with 5 microm amino-bonded silica was used as stationary phase for the separation. n-Hexane/dichloromethane/acetonitrile (90: 6.6: 3.4, v/v/v) was used as mobile phase. Five fractions from one LC run were collected in five nitrogen-blowing flasks, separately. The same fractions from 10 LC runs were combined together and concentrated to 1 mL by nitrogen blowing, and then were analyzed by CGC/MS. The column of DB-5MS (60 m x 0.25 mm x 0.25 microm) was used for CGC/MS analysis. Comparing with direct CGC/MS analysis of the same sample, the LC-CGC/MS system is suitable for a complex sample, and has a higher reliability of qualitative analysis.

Quantification of monosaccharides through multiple‐reaction monitoring liquid chromatography/mass spectrometry using an aminopropyl column

A simple, sensitive, and reproducible quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was designed for the simultaneous quantification of monosaccharides derived from glycoprotein and blood serum using a multiple-reaction monitoring (MRM) approach. Sialic acids and neutral monosaccharides were efficiently separated using an amino-bonded silica phase column. Neutral monosaccharide molecules were detected as their aldol acetate anion adducts [M + CH(3)CO(2)](-) using electrospray ionization in negative ion MRM mode, while sialic acids were detected as deprotonated ions [M-H](-). The new method did not require a reduction step, and exhibited very high sensitivity to carbohydrates with limits of detection of 1 pg for the sugars studied. The linearity of the described approach spanned over three orders of magnitude (pg to ng). The method was validated for monosaccharides originating from N-linked glycans attached to glycoproteins and glycoproteins found in human blood serum. The method effectively quantified monosaccharides originating from as little as 1 microg of glycoprotein and 5 microL of blood serum. The method was robust, reproducible, and highly sensitive. It did not require reduction, derivatization or postcolumn addition of reagents.

Selective Enrichment of Phenols from Coal Liquefaction Oil by Solid Phase Extraction Method

This study focuses on the solid phase extraction method for the enrichment and separation of phenol from coal liquefaction oil. The phenols' separation efficiency was compared on different solid phase extraction (SPE) cartridges, and the effect of solvents with different polarity and solubility parameter on amino-bonded silica was compared for selection of optimal elution solution. The result showed that amino-bonded silica has the highest selectivity and best extraction capability due to two factors, weak anion exchange adsorption and polar attraction adsorption.

Determination of Sucralose in Soft Drinks by High-Performance Thin-Layer Chromatography: Interlaboratory Study

An interlaboratory comparison was carried out to evaluate the effectiveness of a method based on HPTLC in which reagent-free derivatization is followed by UV/fluorescence detection. The method was tested for the determination of sucralose (C12H19C13O8; (2R,3R,4R,5S,6R)-2-[(2R,3S,4S,5S)-2,5-bis(chloromethyl)-3,4-dihydroxyoxolan-2-yl]oxy-5-chloro-6-hydroxymethyl)oxane-3, 4-diol; CAS Registry No. 56038-13-2) in carbonated and still beverages at the proposed European regulatory limits. For still beverages, a portion of the sample was diluted with methanol-water. For carbonated beverages, a portion of the sample was degassed in an ultrasonic bath before dilution. Turbid beverages were filtered after dilution through an HPLC syringe filter. The separation of sucralose was performed by direct application on amino-bonded (NH2) silica gel HPTLC plates (no cleanup needed) with the mobile phase acetonitrile-water. Sucralose was determined after reagent-free derivatization at 190 degrees C; it was quantified by measurements of both UV absorption and fluorescence. The samples, both spiked and containing sucralose, were sent to 14 laboratories in five different countries. Test portions of a sample found to contain no sucralose were spiked at levels of 30.5, 100.7, and 299 mg/L. Recoveries ranged from 104.3 to 124.6% and averaged 112% for determination by UV detection; recoveries ranged from 98.4 to 101.3% and averaged 99.9% for determination by fluorescence detection. On the basis of the results for spiked samples (blind duplicates at three levels), as well as sucralose-containing samples (blind duplicates at three levels and one split level), the values for the RSDr ranged from 10.3 to 31.4% for determinations by UV detection and from 8.9 to 15.9% for determinations by fluorescence detection. The values for the RSDR values ranged from 13.5 to 31.4% for determinations by UV detection and from 8.9 to 20.7% for determinations by fluorescence detection.

Elution of Cationic Species with/without Ion Pair Reagents from Polar Stationary Phases via SFC

Traditionally, the application of supercritical fluid chromatography has been limited to compounds of relatively low polarity. Thus, we report here that one secondary amine and two quaternary amine salts were successfully eluted from a Deltabond Cyano-bonded silica column with the addition of sodium alkylsulfonate to the methanol-modified, carbon dioxide-based mobile phase. A possible ion-pairing interaction between the positively charged analytes and the anionic part of the sulfonate additive has been proposed. In another set of experiments, the three amine salts readily eluted from both Ethyl-pyridine-bonded silica and Amino-bonded silica phases without the need of additive although the peak shapes were less than desirable. The addition of sulfonate salt to the mobile phase again sharpened the peaks. In the presence of a mixture of methanol and CO2, we suggest that these stationary phases are positively charged. We describe here also for the first time the employment of a strong silica-based anion exchange (SAX) column for supercritical fluid separation of cationic species. Two elution mechanisms were proposed for the three amine salts depending upon whether the ionic additive was incorporated into the mobile phase. Separation as the ion-pair was proposed with an ionic additive in the mobile phase; whereas separation of the intact amine salt from the positively charged basic stationary phases was suggested to be operational without the ionic additive.

Retention mechanism of poly(ethylene oxide) in reversed-phase and normal-phase liquid chromatography

The retention behavior of low- and high-molecular-mass poly(ethylene oxide) (PEO) in reversed-phase (RP) and normal-phase (NP) liquid chromatography was investigated. In RPLC using a C 18 bonded silica stationary phase and an acetonitrile–water mixture mobile phase, the sorption process of PEO to the stationary phase showed Δ H°>0 and Δ S°>0. Therefore, PEO retention in RPLC separation is an energetically unfavorable, entropy-driven process, which results in an increase of PEO retention as the temperature increases. In addition, at the enthalpy–entropy compensation point the elution volume of PEO was very different from the column void volume. These observations are quite different from the RPLC retention behavior of many organic polymers. The peculiar retention behavior of PEO in RPLC separation can be understood in terms of the hydrophobic interaction of this class of typical amphiphilic compounds with the non-polar stationary phase, on the one hand, and with the aqueous mobile phase, on the other. The entropy gain due to the release of the solvated water molecules from the PEO chain and the stationary phase is believed to be responsible for the entropy-driven separation process. On the other hand, in NPLC using an amino-bonded silica stationary phase and an acetonitrile–water mixture mobile phase, PEO showed normal enthalpy-driven retention behavior: Δ H°<0 and Δ S°<0, with the retention decreasing with increasing temperature and PEO eluting near the column void volume at the enthalpy–entropy compensation point. Therefore, high-resolution temperature gradient NPLC separation of high-molecular-mass PEO samples can be achieved with relative ease. The molecular mass distribution of high-molecular-mass PEO was found to be much narrower than that measured by size-exclusion chromatography.

Amino-bonded silica as stationary phase for liquid chromatographic determination of cyclopiazonic acid in fungal extracts

A new high-performance liquid (HPLC) chromatographic method is described for cyclopiazonic acid (CPA) determination in fungal cultures on a propylamino-bonded stationary phase with a CH 3CN/CH 3COONH 4 buffer as mobile phase. Retention of CPA on propylamino modified silica under acidic conditions (protonated amino groups and deprotonated CPA) is governed by a mixed ion-exchange-reversed-phase mechanism. In addition to non-polar (hydrophobic) interactions, polar interactions with the surface silanols are also possible and become important as the polarity of the mobile phase decreases. A detection limit of 25 pg of CPA standard is obtained that represents an improvement of more than two orders of magnitude compared to existing HPLC procedures. UV-detector response was linear to 200 ng of CPA. Fungal extracts can be analysed after a simple dilution step with UV diode array detection that provides peak identity/purity assessment. The suitability of the proposed method as a rapid confirmatory test to assess the toxigenic potential of different Aspergillus and Penicillium strains is demonstrated by the analysis of 54 fungal extracts.

Diversity of O-linked glycosylation patterns between species. Characterization of 25 carbohydrate chains from oviducal mucins of Rana ridibunda.

Amphibia egg jelly coats are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they pass along the different oviducal portions. Mucin type glycoproteins are the major constituents of the egg jelly coats. In this study, the O-linked carbohydrate chains of the jelly coats surrounding the eggs of Rana ridibunda were released by alkaline borohydride treatment. Fractionation of the mixture of O-linked oligosaccharide-alditols was achieved by a combination of chromatographic techniques including gel-permeation chromatography, ion-exchange chromatography and high-performance liquid chromatography using an amino-bonded silica column. The primary structures of these O-glycans were determined by one-dimensional and two-dimensional 1H-NMR spectroscopy and matrix-assisted laser-desorption-ionization-time-of flight mass spectrometry. 25 oligosaccharide structures, possessing a core consisting of Gal(beta1-3)GalNAc-ol with or without branching through a GlcNAc residue linked (beta1-6) to the GalNAc residue (core type 2 or core type 1, respectively) are described. The most representative antennae are: HSO3(6)[Fuc(alpha1-3)]GlcNAc; Gal(beta1-2)Gal; Gal(beta1-2)Gal(alpha1-3)[Fuc(alpha1-2)]Gal; GlcA(beta1-3)-Gal(beta1-3)[Fuc(alpha1-2)]Gal; GalNAc(alpha1-4)Gal(beta1-4)Gal; Gal(beta1-3)GalNAc(alpha1-4)Gal(beta1-4)Gal and GlcA(beta1-3)Gal(beta1-3)GalNAc. These results confirm the species-specific O-glycosylation of Amphibia oviducal mucins. The significance of this observation should be linked to a symbiotic role of carbohydrates involved in host-parasite interactions.

Determination of Mono-, Di-, and Oligosaccharides in Legumes by High-Performance Liquid Chromatography Using an Amino-Bonded Silica Column

A high-performance liquid chromatography (HPLC) method for soluble sugars analysis, using an [[(aminopropyl)methyl]silyl]-bonded amorphous silica column, was applied to several dry legumes: chickpeas, lentils, white beans, pinto beans, and peas. Monosaccharide (ribose, fructose, glucose, and galactose), disaccharide (sucrose, maltose, and melibiose), and oligosaccharide (raffinose, ciceritol, and stachyose) composition of these samples was analyzed, with special interest on α-galactosides, because of their physiological role in inducing flatulent phenomena in humans after ingestion of legumes. Ciceritol (an inositol digalactoside) was the main sugar in chickpea samples and it was present also in lentils. In all the samples stachyose was found at higher levels than raffinose, and the content of these two flatulence-inducing sugars is higher in beans and lentils and lower in peas and chickpeas. However, great variability has been found in the sugar content of the different samples analyzed, probably due to...

Structural analysis of oligosaccharide-alditols released by reductive beta-elimination from the jelly coats of the anuran Bufo arenarum.

Amphibian egg jelly coats are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they are transported toward the cloaca. Mucin type glycoproteins are the major constituents of the egg jelly coats. In this study, the O-linked carbohydrate chains of the jelly coat surrounding the eggs of Bufo arenarum were released by alkaline borohydride treatment. Fractionation of the mixture of O-linked oligosaccharide-alditols was achieved by a combination of chromatographic techniques comprising gel-permeation chromatography, ion-exchange chromatography and high-performance liquid chromatography using an amino-bonded silica column, afforded 11 fractions. The primary structures of these O-glycans were determined by one-dimensional and two-dimensional 1H-NMR spectroscopy in conjunction with matrix-assisted laser-desorption-ionization--time-of-flight mass spectrometry. 11 oligosaccharide structures, possessing a core consisting of Galbeta1-->3GalNAc-ol with or without branching through a GlcNAc residue linked beta1-->6 to the GalNAc residue (core type 2 or core type 1, respectively) are described. These oligosaccharide-alditols with these types of cores have been identified previously in mammalian mucins or in oviducal amphibian jellies. These glycans contain blood group determinants such as H, A or Cad antigens.

Isolation of the O-glycosidically linked oligosaccharides obtained by alkaline borohydride degradation from oviducal mucins of the toad Bufobufo

A combination of normal-phase high-performance liquid chromatography (HPLC) on amino-bonded silica and reversed-phase HPLC on octadecylsilica has been used to separate the reduced oligosaccharides produced by alkaline borohydride degradation of oviducal mucins obtained from the jelly coat of Bufo bufo. The former technique provides suitable separation on the basis of molecular size, while the latter method offers selectivity for stereoisomers. Thirty-four compounds, ranging in size from a trisaccharide to a dodecaoligosaccharide, have been isolated preparatively using a Supelcosyl LC-NH2 normal-phase column eluted with aqueous acetonitrile and a Zorbax ODS reversed-phase column eluted with water.

Liquid chromatographic analysis and isolation of diastereoisomers obtained by an aldolization reaction

A new route using an aldolization reaction has been investigated in order to synthesize a precursor of pilocarpin, an alkaloid having sympathomimetic properties. To study the stereoselectivity of this route and to propose a mechanism, liquid chromatography has been used to analyse and to isolate each diastereoisomer for a structural determination by NMR. To further investigate the reaction mechanism, seven different aldehydes (or ketones) were used in the aldolization reaction, producing seven different mixtures. This paper describes the improvement of LC conditions with diol- and amino-bonded silica and the nonaqueous mobile phases required for the isolation of each isomer. Keywords: diastereoisomers, pilocarpin precursor, normal phase liquid chromatography, diol- and amino-bonded silicas.