C<Subscript>18</Subscript>-Free Organic–Inorganic Hybrid Silica Particles Derived from Sole Silsesquioxane for Reversed-Phase HPLC

Ethyl-bridged organic–inorganic hybrid silica particles were prepared via a sol–gel and hydrothermal synthesis approach using 1,2-bis(triethoxysilyl)ethane (BTESE) as the sole precursor, and triblock copolymer poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (P123) and dodecyltrimethylammonium bromide (DTAB) as combined templates. The morphology, pore structure, chemical composition and liquid chromatographic performance of the obtained materials were investigated in detail. The particles exhibit a high surface area of 1136.40 m²/g, together with a pore volume of 0.39 cm³/g and an average pore size of 2.30 nm. Used as stationary phase for high-performance liquid chromatography (HPLC), the particles without extra bonding either C₁₈ or C₈ can successfully separate a mixture of uracil, phenol, pyridine, methylbenzene, ethylbenzene and tert-butylbenzene. The obtained materials also show practical application in the separation of phthalate acid esters (PAEs), which are harmful to environment and human health. Although the columns packed with ethyl-bridged organic–inorganic hybrid silica show lower column efficiency and peak symmetry compared to commercial column, they have considerably higher chemical stability in alkaline mobile phase. The HSS column also possesses high mechanical stability which is similar to that of the commercial column.     

Synthesis of monodisperse silica microspheres and modification with diazoresin for mixed‐mode ultra high performance liquid chromatography separations

Monodisperse silica particles with average diameters of 1.9–2.9 μm were synthesized by a modified Stöber method, in which tetraethyl orthosilicate was continuously supplied to the reaction mixture containing KCl electrolyte, water, ethanol, and ammonia. The obtained silica particles were modified by self‐assembly with positively charged photosensitive diazoresin on the surface. After treatment with ultraviolet light, the ionic bonding between silica and diazoresin was converted into covalent bonding through a unique photochemistry reaction of diazoresin. Depending on the chemical structure of diazoresin and mobile phase composition, the diazoresin‐modified silica stationary phase showed different separation mechanisms, including reversed phase and hydrophilic interactions. Therefore, a variety of baseline separation of benzene analogues and organic acids was achieved by using the diazoresin‐modified silica particles as packing materials in ultra high performance liquid chromatography. According to the π–π interactional difference between carbon rings of fullerenes and benzene rings of diazoresin, C₆₀ and C₇₀ were also well separated by ultra‐high performance liquid chromatography. Because it has a small size, the ∼2.5 μm monodisperse diazoresin‐modified silica stationary phase shows ultra‐high efficiency compared with the commercial C₁₈‐silica high‐performance liquid chromatography stationary phase with average diameters of ∼5 μm.     

Hydrophilic interaction chromatography: A promising alternative to reversed‐phase liquid chromatography systems for the purification of small protonated bases

The overloaded band profiles of the protonated species of propranolol and amitriptyline were recorded under acidic conditions on four classes of stationary phases including a conventional silica/organic hybrid material in reversed‐phase liquid chromatography mode (BEH‐C₁₈), an electrostatic repulsion reversed‐phase liquid chromatography C₁₈ column (BEH‐C₁₈+), a poly(styrene‐divinylbenzene) monolithic column, and a hydrophilic interaction chromatography stationary phase (underivatized BEH). The same amounts of protonated bases per unit volume of stationary phase were injected in each column (16, 47, and 141 μg/cm³). The performance of the propranolol/amitriptyline purification was assessed on the basis of the asymmetry of the recorded band profiles and on the selectivity factor achieved. The results show that the separation performed under reversed‐phase liquid chromatography like conditions (with BEH‐C₁₈, BEH‐C₁₈+, and polymer monolith materials) provide the largest selectivity factors due to the difference in the hydrophobic character of the two compounds. However, they also provide the most distorted overloaded band profiles due to a too small loading capacity. Remarkably, symmetric band profiles were observed with the hydrophilic interaction chromatography column. The larger loading capacity of the hydrophilic interaction chromatography column is due to the accumulation of the protonated bases into the diffuse water layer formed at the surface of the polar adsorbent. This work encourages purifying ionizable compounds on hydrophilic interaction chromatography columns rather than on reversed‐phase liquid chromatography columns.     

C18‐bound porous silica monolith particles as a low‐cost high‐performance liquid chromatography stationary phase with an excellent chromatographic performance

Ground porous silica monolith particles with an average particle size of 2.34 μm and large pores (363 Å) exhibiting excellent chromatographic performance have been synthesized on a relatively large scale by a sophisticated sol–gel procedure. The particle size distribution was rather broad, and the d(0.1)/d(0.9) ratio was 0.14. The resultant silica monolith particles were chemically modified with chlorodimethyloctadecylsilane and end‐capped with a mixture of hexamethyldisilazane and chlorotrimethylsilane. Very good separation efficiency (185 000/m) and chromatographic resolution were achieved when the C₁₈‐bound phase was evaluated for a test mixture of five benzene derivatives after packing in a stainless‐steel column (1.0 mm × 150 mm). The optimized elution conditions were found to be 70:30 v/v acetonitrile/water with 0.1% trifluoroacetic acid at a flow rate of 25 μL/min. The column was also evaluated for fast analysis at a flow rate of 100 μL/min, and all the five analytes were eluted within 3.5 min with reasonable efficiency (ca. 60 000/m) and resolution. The strategy of using particles with reduced particle size and large pores (363 Å) combined with C₁₈ modification in addition to partial‐monolithic architecture has resulted in a useful stationary phase (C₁₈‐bound silica monolith particles) of low production cost showing excellent chromatographic performance.     

Separation of cannabinoids on three different mixed‐mode columns containing carbon/nanodiamond/amine‐polymer superficially porous particles

Three mixed‐mode high‐performance liquid chromatography columns packed with superficially porous carbon/nanodiamond/amine‐polymer particles were used to separate mixtures of cannabinoids. Columns evaluated included: (i) reversed phase (C₁₈), weak anion exchange, 4.6 × 33 mm, 3.6 μm, and 4.6 × 100 mm, 3.6 μm, (ii) reversed phase, strong anion exchange (quaternary amine), 4.6×33 mm, 3.6 μm, and (iii) hydrophilic interaction liquid chromatography, 4.6 × 150 mm, 3.6 μm. Different selectivities were achieved under various mobile phase and stationary phase conditions. Efficiencies and peak capacities were as high as 54 000 N/m and 56, respectively. The reversed phase mixed‐mode column (C₁₈) retained tetrahydrocannabinolic acid strongly under acidic conditions and weakly under basic conditions. Tetrahydrocannabinolic acid was retained strongly on the reversed phase, strong anion exchange mixed‐mode column under basic polar organic mobile phase conditions. The hydrophilic interaction liquid chromatography column retained polar cannabinoids better than the (more) neutral ones under basic conditions. A longer reversed phase (C₁₈) mixed‐mode column (4.6 × 100 mm) showed better resolution for analytes (and a contaminant) than a shorter column. Fast separations were achieved in less than 5 min and sometimes 2 min. A real world sample (bubble hash extract) was also analyzed by gradient elution.     

Silica particles encapsulated poly(styrene-divinylbenzene) monolithic stationary phases for micro-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 (micro-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 A 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 microm ProntoSIL C-18 particles with 300 A pore size, separation efficiencies in the range of 120,000-200,000 plates/m were obtained for protein separation, in a 6 cm x 200 microm i.d. capillary column. Using encapsulated silica C-18 with 1000 A 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.     

Chromatographic behavior of small organic compounds in low‐temperature high‐performance liquid chromatography using liquid carbon dioxide as the mobile phase

Low‐temperature high‐performance liquid chromatography, in which a loop injector, column, and detection cell were refrigerated at –35ºC, using liquid carbon dioxide as the mobile phase was developed. Small organic compounds (polyaromatic hydrocarbons, alkylbenzenes, and quinones) were separated by low‐temperature high‐performance liquid chromatography at temperatures from –35 to –5ºC. The combination of liquid carbon dioxide mobile phase with an octadecyl‐silica (C₁₈) column provided reversed phase mode separation, and a bare silica‐gel column resulted in normal phase mode separation. In both the cases, nonlinear behavior at approximately –15ºC was found in the relationship between the temperature and the retention factors of the analytes (van't Hoff plots). In contrast to general trends in high‐performance liquid chromatography, the decrease in temperature enhanced the separation efficiency of both the columns.     

Rapid determination of parabens in seafood sauces by high‐performance liquid chromatography: A practical comparison of core–shell particles and sub‐2 μm fully porous particles

In this work, the chromatographic performance of superficially porous particles (Halo core–shell C₁₈ column, 50 mm × 2.1 mm, 2.7 μm) was compared with that of sub‐2 μm fully porous particles (Acquity BEH C₁₈, 50 mm × 2.1 mm, 1.7 μm). Four parabens, methylparaben, ethylparaben, propylparaben, and butylparaben, were used as representative compounds for calculating the plate heights in a wide flow rate range and analyzed on the basis of the Van Deemter and Knox equations. Theoretical Poppe plots were constructed for each column to compare their kinetic performance. Both phases gave similar minimum plate heights when using nonreduced coordinates. Meanwhile, the flat C‐term of the core–shell column provided the possibilities for applying high flow rates without significant loss in efficiency. The low backpressure of core–shell particles allowed this kind of column, especially compatible with conventional high‐performance liquid chromatography systems. Based on these factors, a simple high‐performance liquid chromatography method was established and validated for the determination of parabens in various seafood sauces using the Halo core–shell C₁₈ column for separation.     

Factors affecting the reversed-phase liquid chromatographic separation of polycyclic aromatic hydrocarbon isomers

Reversed-phase liquid chromatography (LC) on C₁₈ stationary phases provides excellent selectivity for the separation of polycyclic aromatic hydrocarbons (PAH). Recent studies have shown that several factors affect selectivity for the LC separation of PAH including phase type (monomeric or polymeric), pore diameter and surface area of the silica substrate, and surface density of the C₁₈ ligands. In this paper the separation of eleven PAH isomers of molecular weight 278 is used to further illustrate the effect of stationary phase characteristics and shape of the solute (length-to-breadth ratio, L/B) on retention and selectivity. Only polymeric C₁₈ phases with a high C₁₈ surface coverage provided separation of all eleven isomers and the elution order of these isomers generally followed increasing L/B values. The effect of solute nonplanarity on reversed-phase LC retention was investigated on both monomeric and polymeric phases using a series of planar and nonplanar PAH pairs. For each solute pair, the nonplanar solute eluted earlier than the planar solute, the largest selectivity factors being observed on the C₁₈ phase with the highest percent carbon load. Based on these studies, a model is proposed to describe the retention of PAH on polymeric C₁₈ phases.     

Highly Robust Microporous Metal-Organic Frameworks for Efficient Ethylene Purification under Dry and Humid Conditions

Two C₂H₆-selective metal-organic framework (MOF) adsorbents with ultrahigh stability, high surface areas, and suitable pore size have been designed and synthesized for one-step separation of ethane/ethylene (C₂H₆/C₂H₄) under humid conditions to produce polymer-grade pure C₂H₄. Experimental results reveal that these two MOFs not only adsorb a high amount of C₂H₆ but also display good C₂H₆/C₂H₄ selectivity verified by fixed bed column breakthrough experiments. Most importantly, the good water stability and hydrophobic pore environments make these two MOFs capable of efficiently separating C₂H₆/C₂H₄ under humid conditions, exhibiting the benchmark performance among all reported adsorbents for separation of C₂H₆/C₂H₄ under humid conditions. Moreover, the affinity sites and their static adsorption energies were successfully revealed by single crystal data and computation studies. Adsorbents described in this work can be used to address major chemical industrial challenges.     

Automated screening of reversed‐phase stationary phases for small‐molecule separations using liquid chromatography with mass spectrometry

There are various reversed‐phase stationary phases that offer significant differences in selectivity and retention. To investigate different reversed‐phase stationary phases (aqueous stable C₁₈, biphenyl, pentafluorophenyl propyl, and polar‐embedded alkyl) in an automated fashion, commercial software and associated hardware for mobile phase and column selection were used in conjunction with liquid chromatography and a triple quadrupole mass spectrometer detector. A model analyte mixture was prepared using a combination of standards from varying classes of analytes (including drugs, drugs of abuse, amino acids, nicotine, and nicotine‐like compounds). Chromatographic results revealed diverse variations in selectivity and peak shape. Differences in the elution order of analytes on the polar‐embedded alkyl phase for several analytes showed distinct selectivity differences compared to the aqueous C₁₈ phase. The electron‐rich pentafluorophenyl propyl phase showed unique selectivity toward protonated amines. The biphenyl phase provided further changes in selectivity relative to C₁₈ with a methanolic phase, but it behaved very similarly to a C₁₈ when an acetonitrile‐based mobile phase was evaluated. This study shows the value of rapid column screening as an alternative to excessive mobile phase variation to obtain suitable chromatographic settings for analyte separation.     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.     

氮氧化硅反相色谱固定相材料的制备及色谱分离性能评价

硅胶是目前高效液相色谱(HPLC)固定相中应用最为广泛的基质材料,其流动相的最佳使用范围为pH 2～8。在高pH(pH＞8)的流动相条件下,流动相中的氢氧根会进攻硅胶基质表面残余的硅醇基,导致硅胶基质固定相骨架溶解。在前期的研究中,我们将高温氨气处理多孔硅胶微球得到的氮氧化硅材料用作HPLC固定相,氮氧化硅在高pH流动相条件下表现出了较硅胶更高的稳定性。本文利用元素分析对氮氧化硅材料的氮化程度及含氮量进行系统的表征,并考察了氮氧化硅材料在不同pH条件下的静态稳定性。利用十八烷基二甲基氯硅烷试剂对氮氧化硅材料表面进行疏水性修饰,并以元素分析和核磁共振波谱对表面键合氮氧化硅材料进行了表征。考察了不同碳链的烷基苯、酸性化合物、碱性化合物在疏水改性的C₁₈氮氧化硅反相色谱固定相上的色谱分离性能。进一步分别以酸、碱和中性化合物为分析对象,比较了C₁₈-SiON1050(10)与C₁₈-SiO₂色谱保留的差异。     

Base‐deactivated and alkaline‐resistant chromatographic stationary phase based on functionalized polymethylsilsesquioxane microspheres

Vinyl, chloropropyl, and mercaptopropyl functionalized particles were prepared by a two‐step acidic/alkaline catalyzed co‐hydrolysis/condensation of methyltrimethoxysilane with a different silane precursor that carries chemically reactive functional group including vinyl, chloropropyl, and mercaptopropyl, respectively. The morphology, pore structure, and functional groups of the synthesized packings were studied by SEM, nitrogen adsorption‐desorption measurements, and solid‐state ¹³C ²⁹Si NMR spectroscopy, respectively. The particles show ordered sphere, narrow particle size distribution, and mesoporous structure. The carbon contents of the microspheres are in the range of 17–19%, comparable to those of octadecyl‐bonded silica packings. The three‐kind of microspheres were directly used as packing materials for high‐performance liquid chromatography without size classification. The chromatographic performance of the columns was evaluated and compared with a commercially available C₁₈ phase. The results revealed that these columns possess typical reversed‐phase chromatographic properties with increased hydrophobicity than polymethylsilsesquioxane and symmetric peaks for basic compounds. They were applied to the simultaneous separation of combination bendazol hydrochlorothiazide capsules containing polar and basic drugs with peaks identified by tandem with mass spectrometry. In general, a novel method is provided for the synthesis of different methyltrimethoxysilane‐derived microspheres for high‐performance liquid chromatography, which are advantageous for separating basic compounds.     

A Rapid Isocratic Reversed-Phase Separation and Determination of Some Barbiturates by High-Performance Liquid Chromatography

Abstract

A high performance liquid chromatographic method for the rapid separation and quantitative determination of five barbiturates in pharmaceutical preparations and body fluids has been developed. A C₁₈ reversed phase column was used and the barbiturates were detected at 235nm.

A number of eluting systems were examined, the most suitable of them being, Ethanol:Propanol:Methanol:Water (26:5:29:40) at pH = 8.8.     

Evaluation of efficiency and trapping capacity of restricted access media trap columns for the online trapping of small molecules

Restricted access media are generally composed from multi‐modal particles that combine a size excluding outer surface and an inner‐pore retention mechanism for small molecules. Such materials can be used for either online isolation and pre‐concentration of target small molecules or removal of small molecule interferences from large macromolecules, such as proteins in complex biological matrices. Thus, they are considered as enhanced online solid‐phase extraction materials. We evaluated the efficiency and trapping capacity of different semi‐permeable surface restricted access media columns (C₁₈, C₈, and C₄ inner pores) for four model small molecule compounds (dopamine hydrochloride, acetaminophen, 4‐hydroxybenzoic acid, and diethyl phthalate) having variable physicochemical properties. We further studied the effect of mobile phase flow rate (0.25, 0.5, 1, and 2 mL/min) and pH, using 98:2 0.5% acetic acid in water/ methanol (pH 2.88) and 5 mM ammonium acetate in 98:2 water/methanol (pH 6.61) as mobile phases. Breakthrough curves generated using frontal analysis were analyzed to determine important chromatographic parameters specific for each of the studied compounds. Experimental determination of these parameters allowed selection of the most efficient trap column and the best loading mobile phase conditions for maximal solute enrichment and pre‐concentration on restricted access media trap columns.     

Synthesis and Characterization of Ordered Mesoporous Silica Spheres Using Anionic and Nonionic Mixed Surfactants for Their Potential Application in LC

Ordered mesoporous silicas (OMSs) with spherical morphology were synthesized by using mixed surfactants of anionic sodium dodecyl sulfate and nonionic block copolymer EO₂₀PO₇₀EO₂₀ (P123) as template through an acid-catalyzed silica sol?Cgel process. A variety of characterizations demonstrated that the silica products exhibited well-formed spherical morphology, ordered mesostructure, narrow pore size distribution and large surface area (~700 m² g^?1). It was found that the synthesized OMSs had high adsorption capacity by using oxymatrine as model solute. The column packed with the silica spheres exhibited low back pressure and baseline separation of aromatic compounds such as benzene and nitrobenzene could be achieved. These results demonstrated the synthesized OMSs as a potential stationary phase for liquid chromatography.     

Pore expansion of highly monodisperse phenylene-bridged organosilica spheres for chromatographic application

Monodisperse phenylene-bridged organosilica spheres show great potential as chromatographic stationary phase. In this paper, the tunable particle size of monodisperse phenylene-bridged organosilica spheres were prepared by co-condensing different proportion of 1,4-bis(triethoxysilyl)benzene (1,4-BTEB) and tetraethylorthosilicate (TEOS), and then pore size was expanded by two-step post-synthesis hydrothermal treatments using N,N-dimethyldecylamine (DMDA)/dodecylamine (DDA) and tris-(hydroxymethyl)-aminomethane (TRIS) in turn. Phenylene-bridged organosilica spheres with particle size of 3.0-3.5 μm and pore size of 85 Å were further surface modified by C₁₈ group and tested in reversed-phase high performance liquid chromatography (RP-HPLC). The primary chromatographic results demonstrated that C₁₈ bonded phenylene-bridged organosilica stationary phase has high retention and good chemical stability in the high pH mobile phase, which indicated that the phenylene-bridged organosilica can be used for HPLC packing supports.     

Comparison of various types of stationary phases in non-aqueous reversed-phase high-performance liquid chromatography–mass spectrometry of glycerolipids in blackcurrant oil and its enzymatic hydrolysis mixture

The selection of column packing during the development of high-performance liquid chromatography method is a crucial step to achieve sufficient chromatographic resolution of analyzed species in complex mixtures. Various stationary phases are tested in this paper for the analysis of complex mixture of triacylglycerols (TGs) in blackcurrant oil using non-aqueous reversed-phase (NARP) system with acetonitrile–2-propanol mobile phase. Conventional C₁₈ column in the total length of 45 cm is used for the separation of TGs according to their equivalent carbon number, the number and positions of double bonds and acyl chain lengths. The separation of TGs and their more polar hydrolysis products after the partial enzymatic hydrolysis of blackcurrant oil in one chromatographic run is achieved using conventional C₁₈ column. Retention times of TGs are reduced almost 10 times without the loss of the chromatographic resolution using ultra high-performance liquid chromatography with 1.7 μm C₁₈ particles. The separation in NARP system on C₃₀ column shows an unusual phenomenon, because the retention order of TGs changes depending on the column temperature, which is reported for the first time. The commercial monolithic column modified with C₁₈ is used for the fast analysis of TGs to increase the sample throughput but at cost of low resolution.     

Capillary electrochromatography with bare silicas of different pore sizes as stationary phases

Bare silica can be used with reversed phase eluents for the chromatographic separation of basic analytes. It provides high surface charge density within a certain pH range, thus generating a high electroosmotic flow (EOF) when applied in electrochromatography. The influence of pore size on EOF velocity and mass transport is demonstrated. High EOF and fast mass transfer were encountered with 100 nm and 200 nm material and related to a pore perfusion mechanism. On a silica with 200 nm average pore size at pH 7, an EOF velocity of 2 mm/s was obtained at 600 V/cm. Silicas with pore diameters between 6 nm and 200 nm, corresponding to surface areas between 500 m/g and 10 m/g (data calculated from inverse size exclusion chromatography experiments), were used for CEC and HPLC separation of strongly basic solutes. On separation of tricyclic antidepressants by CEC, “normal” and “abnormal” efficiencies were achieved and were found to vary with the charge density within the separation column.