Synthesis of a stationary phase based on silica modified with branched octadecyl groups by Michael addition and photoinduced thiol–yne click chemistry for the separation of basic compounds

A novel silica‐based stationary phase with branched octadecyl groups was prepared by the sequential employment of the Michael addition reaction and photoinduced thiol–yne click chemistry with 3‐aminopropyl‐functionalized silica microspheres as the initial material. The resulting stationary phase denoted as SiO₂‐N(C18)₄ was characterized by elemental analysis, FTIR spectroscopy and Raman spectroscopy, demonstrating the existence of branched octadecyl groups in silica microspheres. The separations of benzene homologous compounds, acid compounds and amine analogues were conducted, demonstrating mixed‐mode separation mechanism on SiO₂‐N(C18)₄. Baseline separation of basic drugs mixture was acquired with the mobile phase of acetonitrile/H₂O (5%, v/v). SiO₂‐N(C18)₄ was further applied to separate Corydalis yanhusuo Wang water extracts, and more baseline separation peaks were obtained for SiO₂‐N(C18)₄ than those on Atlantis dC18 column. It can be expected that this new silica‐based stationary phase will exhibit great potential in the analysis of basic compounds.     

Preparation and evaluation of monodispersed,submicron, non‐porous silica particles functionalized with β‐CD derivatives for chiral‐pressurized capillary electrochromatography

Submicron, non‐porous, chiral silica stationary phase has been prepared by the immobilization of functionalized β‐CD derivatives to isocyanate‐modified silica via chemical reaction and applied to the pressurized capillary electrochromatography (pCEC) enantio‐separation of various chiral compounds. The submicron, non‐porous, cyclodextrin‐based chiral stationary phases (sub_μm‐CSP2) exhibited excellent chiral recognition of a wide range of analytes including clenbuterol hydrochloride, mexiletine hydrochloride, chlorpheniramine maleate, esmolol hydrochloride, and metoprolol tartrate. The synthesized submicron particles were regularly spherical and uniformly non‐porous with an average diameter of around 800 nm and a mean pore size of less than 2 nm. The synthesized chiral stationary phase was packed into 10 cm × 100 μm id capillary columns. The sub_μm‐CSP2 column used in the pCEC system showed better separation of the racemates and at a higher rate compared to those used in the capillary liquid chromatography mode (cLC) system. The sub_μm‐CSP2 possessed high mechanical strength, high stereoselectivity, and long lifespan, demonstrating rapid enantio‐separation and good resolution of samples. The column provided an efficiency of up to 170 000 plates/m for n‐propylbenzene.     

The flow-through silica as the matrix to immobilize gold nanoparticles for HPLC applications

In the present study, the flow-through silica, featured with hierarchical pores, i.e., tunable mesopores and penetrable macropores, was attempted as the chromatographic stationary phase matrix to immobilize gold nanoparticles (AuNPs). It was first modified by mercapto groups (named as SiO₂-SH), and then by AuNPs (named as SiO₂-S-Au). Thanks to the characteristic macropores, the column backpressure of SiO₂-S-Au was comparable to SiO₂-SH, which effectively overcame the difficulty of high column backpressure upon the nanoparticles were introduced to the chromatographic matrix. Both the reversed-phase and hydrophilic interaction liquid chromatographic performance were observed on these two columns but with different selectivities. Hydrophobic, hydrophilic, hydrogen bond and electrostatic interactions between the SiO₂-S-Au stationary phase and analytes could contribute to the retention. The SiO₂-S-Au column showed excellent aqueous compatibility by “Stop-flow” test with the relative standard deviations (RSD) of analyte’s k (capacity factor) values from 0.59% to 2.88%. The reproducibility of SiO₂-S-Au was acceptable with RSDs of analyte’s k values in the range of 3.13%-5.03%. In addition, compared with the SiO₂-SH column, the SiO₂-S-Au column had better separation performance and selectivity. The results demonstrated that the flow-through silica was a promising matrix for nanoparticles with low backpressure and different selectivities.     

Basic Indole Ring Enantiomer Separation on Cellulose Tris(3,5‐dimethylphenylcarbamate) Coated TiO2/SiO2 Chiral Stationary Phase

Abstract

Cellulose tris(3,5‐dimethylphenylcarbamate) (CDMPC) coated TiO₂/SiO₂ has been prepared by coating CDMPC on TiO₂/SiO₂ which consists of micrometer‐sized silica spheres as core and nanometer‐sized titania particles as surface coating. Eight basic indole ring derivative enantiomers were separated on this CDMPC coated CSP and symmetrical peaks were obtained using hexane as the mobile phase and various alcohols as modifiers. The influence of the mobile phase composition and structural variation of the solutes on the enantioseparation was investigated and discussed.     

Preparation and evaluation of C18-bonded 1-microm silica particles for pressurized capillary electrochromatography

Nonporous silica spheres (1 microm) were synthesized and bonded with octadecylsilane functionality. These stationary phase particles were packed electrokinetically into fused-silica capillaries with 100 microm id for a length of 20 cm, which was evaluated by using pressurized CEC (pCEC). The efficiency of the C18 RP column was characterized through the theoretical plates of thiourea, benzyl alcohol, toluene, styrene, and naphthalene. The effects of experimental parameters such as the applied voltage, sample size, pump flow rate, pH value and the concentration of the buffer solution, and the content of methanol in the mobile phase, on-column efficiency were evaluated. Column efficiency as high as 200 000 theoretical plates per meter for naphthalene was obtained with the optimal condition of 70% v/v methanol and 30% v/v of 10 mmol/L phosphate buffer (pH 7.8) at an applied voltage of 10 kV and a supplementary pressure of 500 psi.     

Graphene oxide‐SiO2 hybrid nanostructure as coating material for capillary electrochromatography

Graphene oxide (GO) has been considered as a promising stationary phase for chromatographic separation. However, the very strong adsorption of the analytes on the GO surface lead to the severe peak tailing, which in turn resulting in decreased separation performance. In this work, GO and silica nanoparticles hybrid nanostructures (GO/SiO₂ NPs@column) were coated onto the capillary inner wall by passing the mixture of GO and silica sol through the capillary column. The successful of coating of GO/SiO₂ NPs onto the capillary wall was confirmed by SEM and electroosmotic flow mobilities test. By partially covering the GO surface with silica nanoparticles, the peak tailing was decreased greatly while the unique high shape selectivity arises from the surface of remained GO was kept. Consequently, compared with the column modified with GO (GO@column), the column modified with GO and silica nanoparticles through layer‐by‐layer method (GO‐SiO₂ NPs@column), or the column modified with silica nanoparticles (SiO₂ NPs@column), GO/SiO₂ NPs@column possessed highest resolutions. The GO/SiO₂ NPs@column was applied to separate egg white and both acidic and basic proteins as well as three glycoisoforms of ovalbumin were separated in a single run within 36 min. The intra‐day, inter‐day, and column‐to‐column reproducibilities were evaluated by calculating the RSDs of the retention of naphthalene and biphenyl in open‐tubular capillary electrochromatography. The RSD values were found to be less than 7.1%.     

A strong inorganic acid‐initiated methacrylate polymerization strategy for room temperature preparation of monolithic columns for capillary electrochromatography

A facile strong inorganic acid‐initiated methacrylate polymerization strategy was developed for fabricating monolithic columns at room temperature. The prepared monoliths were characterized by FTIR spectrometry, mercury intrusion porosimeter and SEM, while their performance was evaluated by CEC for the separation of various types of compounds including alkyl benzenes, polycyclic aromatic hydrocarbons, nonsteroidal anti‐inflammatory drugs, anilines, and nitrophenol isomers. The column‐to‐column and batch‐to‐batch reproducibility for the prepared monoliths in terms of the RSD of EOF flow velocity, retention factor, and the minimum plate height of naphthalene ranged from 3.4 to 12.4%. The fabricated monoliths gave excellent performance for the separation of the test neutral compounds with the theoretical plates of 170 000–232 000 plates per meter for thiourea, and 77 400–112 300 plates per meter for naphthalene. The proposed strong inorganic acid‐initiated methacrylate polymerization strategy is a promising alternative for fabricating organic polymer‐based monoliths.     

Preparation and Evaluation of a Novel Cellulose Tris(N‐3,5‐dimethylphenylcarbamate) Chiral Stationary Phase

A novel cellulose tris(N‐3,5‐dimethylphenylcarbamate) (CDMPC) chiral stationary phase (CSP) was prepared by coating CDMPC on TiO₂/SiO₂, which was prepared by coating titania nanoparticles on silica through a self‐assemble technique. At first, 2‐hydroxyl‐phenyl acetonitrile and α‐phenylethanol were separated on this new CSP to evaluate the chiral separation ability. Then, two pesticides, matalaxyl and diclofop‐methyl were separated. The influence of the mobile phase composition on the enantioselectivity was discussed, and the repeatability and stability of the CSP were studied too.     

亚微米C18键合硅胶固定相加压毛细管电色谱法同时拆分3种手性三唑类农药

采用改良Stöber法制备420 nm亚微米单分散二氧化硅微球,采用C18硅烷化修饰后装填成毛细管色谱柱。采用该色谱柱,在加压毛细管电色谱平台上成功地实现了3对手性三唑类农药烯效唑、烯唑醇和丙环唑的同时拆分和分离。考察了各因素对手性分离效果的影响,优化后的色谱条件为：流动相为乙腈-20 mmol/L磷酸盐缓冲液（pH=6.8）（45：55,v/v）,其中缓冲液中含20 mmol/L羟丙基-γ-环糊精（HP-γ-CD）;泵流速为0.04 mL/min;施加电压-9.4 kV;检测波长220 nm。在上述条件下,烯效唑、烯唑醇和丙环唑3种对映体同时得到拆分和分离,相邻两峰之间的分离度依次为4.20、12.9、4.41、4.09、1.70,分离时间仅为12 min,柱效最高达到310000 plates/m。该研究为手性三唑类农药的同时分离提供了新的分离分析思路。     

Cucurbit(6)uril immobilized on silica: A novel high‐performance liquid chromatographic stationary phase

In the present study, one of the new generation of host molecules, cucurbit(6)uril (CB(6)), was immobilized onto silica (CB(6)/SiO₂) by a sol–gel approach. CB(6)/SiO₂ was characterized by NMR spectroscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, and elemental analysis. It was used as a high‐performance liquid chromatographic stationary phase and its chromatographic performance was systematically investigated with different types of analytes as probes. The results revealed that the CB(6)/SiO₂ stationary phase exhibited weak hydrophobic and strong hydrophilic properties. Hence, the variables for hydrophilic interaction liquid chromatography, including components and pH of the mobile phase, were further investigated to explore the retention mechanism of this CB(6)/SiO₂ stationary phase. For less polar analytes, both hydrophobic and hydrophilic interactions could contribute to the retention, while for polar analytes, hydrophilic interaction may be predominant. Compared to the tetraethoxylsilane‐coated SiO₂ stationary phases, the CB(6)/SiO₂ stationary phase exhibited a different retention behavior toward basic analytes with excellent stability. It is a novel promising hydrophilic interaction liquid chromatography stationary phase.     

Fabrication of ZIF‐8@SiO2 Core–Shell Microspheres as the Stationary Phase for High‐Performance Liquid Chromatography

The unique features of high porosity, shape selectivity, and multiple active sites make metal–organic frameworks (MOFs) promising as novel stationary phases for high‐performance liquid chromatography (HPLC). However, the wide particle size distribution and irregular shape of conventional MOFs lead to lower column efficiency of such MOF‐packed columns. Herein, the fabrication of monodisperse MOF@SiO₂ core–shell microspheres as the stationary phase for HPLC to overcome the above‐mentioned problems is reported. Zeolitic imidazolate framework 8 (ZIF‐8) was used as an example of MOFs due to its permanent porosity, uniform pore size, and exceptional chemical stability. Unique carboxyl‐modified silica spheres were used as the support to grow the ZIF‐8 shell. The fabricated monodisperse ZIF‐8@SiO₂ packed columns (5 cm long × 4.6 mm i.d.) show high column efficiency (23 000 plates m^?1 for bisphenol A) for the HPLC separation of endocrine‐disrupting chemicals (bisphenol A, β‐estradiol, and p‐(tert‐octyl)phenol) and pesticides (thiamethoxam, hexaflumuron, chlorantraniliprole, and pymetrozine) within 7 min with good relative standard deviations for 11 replicate separations of the analytes (0.01–0.39, 0.65–1.7, 0.70–1.3, and 0.17–0.91 % for retention time, peak area, peak height, and half peak width, respectively). The ZIF‐8@SiO₂ microspheres combine the advantages of the good column packing properties of the uniform monodisperse silica microspheres and the separation ability of the ZIF‐8 crystals.     

Separation behavior of basic compounds on unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases with reversed‐phase eluents

Unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases have been evaluated and compared for the separation of basic compounds of differing molecular weight, pK_a, and log D using aqueous/organic mobile phases. The influences of percentage of organic modifier, buffer pH, and concentration in the mobile phase on base retention were investigated on unbonded silicon oxynitride and silica phases. The results confirmed that unbonded silicon oxynitride and silica phases demonstrated excellent separation performance for model basic compounds and both the unbonded phases examined possessed a hydrophobic/adsorption and ion‐exchange character. The silicon oxynitride stationary phase exhibited high hydrophilicity compared with silica with a reversed‐phase mobile phase. An ion‐exclusion‐type mechanism becomes predominant for the separation of three aimed bases on the silicon oxynitride column at pH 2.8. Different from silicon oxynitride stationary phase, no obvious change for the retention time of three model bases on silica stationary phase at pH 2.8 can be observed.     

Pressurized capillary electrochromatographic assay of trimethoprim impurities using 1 microm particle-based columns

One micrometre silica particles, derivatized with C18, were electrokinetically packed into a 75-microm-i.d. capillary. The resulting column was evaluated for the separation of trimethoprim (TMP) and its impurities using pressurized capillary electrochromatography (pCEC), starting from a capillary liquid chromatographic (CLC) separation. These samples require gradient elution when separated by high performance liquid chromatography (HPLC), but with the new columns isocratic elution suffices for their separation by CLC or pCEC. Only 70,000 theoretical plates/m for impurity C were achieved using CLC mode at relative low pressure (78 bar) although very small particles were utilized. When a voltage above 2 kV (50 V/cm) was applied, unknown peaks appeared, which was assumed due to an electrophoretic effect with the unknown peaks resolving as a result of the applied voltage. In order to minimize these unfavorable contributions, only a low voltage was applied, still leading to higher separation performances and shorter separation times than in CLC. The optimal analyzing conditions in pCEC included a pressure of 78 bar, an applied voltage of 1 kV, and a mobile phase consisting of 80 mM sodium perchlorate (pH 3.1)/methanol (60/40, v/v). These conditions were used to separate and quantify four major impurities in TMP within 22 min. The obtained calibration curves were linear (r>0.9980) in concentration ranges between 0.005 and 0.1 mg/mL for impurities A and C; 0.02 and 0.10 mg/mL for impurity F; and 0.01 and 0.10 mg/mL for impurity H. The detection limits (S/N=3) for impurities A, C, F, and H were 0.52, 0.84, 3.18, and 2.41 microg/mL, respectively. The calibration curves were successfully applied to analyze spiked bulk samples, with mean recoveries ranging from 92% to 110%. The developed method can therefore be considered simple, rapid, and repeatable.     

Silica‐based zwitterionic monolithic stationary phase for separation of neutral and ionized solutes using pressurized CEC

A porous zwitterionic monolith was prepared by in situ covalent attachment of lysine to a γ‐glycidoxypropyltrimethosysilane‐modified silica monolith. The prepared column was used to perform neutral and ionized solutes separations by pressurized (pCEC). Due to the zwitterionic nature of the resulting stationary phase, the monolithic column provided both electrostatic attraction and repulsion sites for electrochromatographic retention for ionized solutes. Separation of several nucleotides was investigated on the monolithic column. It was shown that the nucleotides could be separated based on hydrophilic and electrostatic interactions between the stationary phase and analyte. Besides, the separation property of the zwitterionic silica monolith was compared with the use of diamine‐bonded silica monolith as stationary phase. As expected, the lysine monolith exhibited a lower retention for the five nucleotides, which was due to the dissociation of the external carboxylic acid groups, leading to electrostatic repulsion with negatively charged solutes. Under the same experimental conditions, separation of the five nucleotides on the zwitterionic column was in less than 8 min, while that on the diamine column was in approximately 60 min.     

Focusing and stabilization of bis‐intercalating dye–DNA complexes for high‐sensitive CE‐LIF DNA analysis

Multiple labeling of nucleic acids by intercalative dyes is a promising method for ultrasensitive nucleic acid assays. The properties of the fast dissociation and instability of dye–DNA complexes may prevent from their wide applications in CE‐LIF nucleic acid analysis. Here, we describe an optimum CE focusing method by using appropriately paired sample and separation buffers, Tris‐glycine buffer and Tris‐glycine‐acetic acid buffer. The developed method was applied in both uncoated and polyacrylamide coated fused‐silica capillary‐based CE‐LIF analysis while the sample and separation buffers were conversely used. The complexes of intercalative dye benzoxazolium‐4‐pyridinium dimer and dsDNA were greatly focused (separation efficiency: 1.8 million theoretical plates per meter) by transient isotachophoresis mechanism in uncoated capillary, and moderately focused by transient isotachophoresis in combination of field amplified sample stacking and further stabilized by the paired buffer in polyacrylamide coated capillary. Based on the developed focusing strategy, an ultrasensitive DNA assay was developed for quantitation of calf thymus dsDNA (from 0.02 to 2.14 pM). By the use of an excitation laser power as low as 1 mW, the detection limits of calf thymus dsDNA (3.5 kb) are 7.9 fM in concentration and 2.4×10^?22 mol (150 molecules) in mass. We further demonstrate that the non‐gel sieving CE‐LIF analysis of DNA fragments can be enhanced by the same strategy. Since the presented strategy can be applied to uncoated and coated capillaries and does not require special device, it is also reasonable to extend to the applications in chip‐based CE DNA analysis.     

A novel and facile preparation method of hollow silica spheres containing small SiO2 cores

This article presents a novel and facile preparation method of hollow silica spheres with loading small silica inside. In this approach, positively charged SiO₂/polystyrene core‐shell composite particles were synthesized first, when the silica shells from the sol‐gel process of tetraethoxysilane were then coated on the surfaces of composite particles via electrostatic interaction, the polystyrene was dissolved subsequently even synchronously in the same medium to form hollow silica spheres with small silica cores. TEM, SEM, and FTIR measurements were used to characterize these composite spheres. Based on this study, some inorganic or organic compounds could be loaded into these hollow silica spheres. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 3431–3439, 2007     

Pressure‐assisted CEC versus CEC using methacrylate‐based monolithic columns: Influence of the polymerization‐mixture composition

Pressure‐assisted CEC (pCEC) can either be performed on a CE instrument by adding pressure at the column inlet, or by applying voltage on a capillary liquid chromatography system. This study investigates the pressure's added value in pCEC using an LC instrument as well as the influence of the polymerization‐mixture composition on monolithic columns in such experimental circumstances. Two factors of the polymerization mixture, which is used to prepare the monolithic capillary columns, were varied according to an experimental design approach: the pore‐forming solvent/total monomer ratio and the pore‐forming solvents composition. Initially, the effect of the resulting stationary phase on the elution behavior of mainly pharmaceutical compounds was studied. Four responses were used to evaluate the effects on the chromatography: retention time, retention factor, peak asymmetry and number of theoretical plates. After processing the results, the stationary phase composition with the best chromatographic behavior was determined and tested. The advantageous properties of this stationary phase compared with the design center‐point column were demonstrated. Secondly, the results of these pCEC experiments were compared with those generated in an identical experimental setup previously performed using CEC. Chromatographic conditions were chosen so that the center‐point column showed similar retention in CEC and pCEC. The expected advantage (faster analysis) and drawback (decreased efficiency) of pCEC in the analysis of pharmaceuticals was evaluated. Analysis time and efficiency were both found to depend greatly on the porosity of the column. The conclusion of this comparison is that pCEC did not have a significant added value to CEC. However, this was mainly due to the instrument's limitation of the pressure‐driven flow over the column. A clear benefit of the pCEC setup was apparatus‐related, i.e. the presence of a loop injection system on the pCEC instrument, which avoids the injection problems that were occasionally observed in CEC.     

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.     

Monodispersed submicron porous silica particles functionalized with CD derivatives for chiral CEC

Rapid and efficient enantioseparation of halogen aryl alcohols and β‐blockers propranolol and pindolol in packed bed CEC (p‐CEC) using as‐prepared submicron porous silica chiral stationary phases (CSPs) has been achieved. Monodispersed 0.66 and 0.81 μm chiral submicron porous silica spheres were prepared using tetramethoxysilane and hexadecyltrimethylammonium bromide, followed by a hydrothermal treatment method with ammonia–ethanol to expand the pore of silica spheres without changing their spherical morphology. A proper specific surface of ca. 230 m²/g and pore sizes average of 6–8 nm were obtained by this method. The submicron porous silica spheres were modified with mono‐6‐phenylcarbamoylated β‐CD via thiol‐en radical addition. They were packed into 9 cm 50 μm id capillary columns with photopolymerized monolithic frits. These submicron CSPs showed greater column efficiency (about 476 000 plates/m for 4‐iodophenyl‐1‐ethanol) and higher resolution than the corresponding 3 μm CSP.     

End-functionalized polyethylene oxide coated silica particles for packed capillary column supercritical fluid chromatography

Summary Polyethylene oxide (PEO)-based polymers with hydroxy, methoxy, and aminopropoxy terminal groups were coated on diol functionalized and hexamethyldisilazane end-capped silica particles. Proton-donor and proton-acceptor test solutes, including carboxylic acids, hydroxy-containing compounds, arylamines, and alkylamines were used to evaluate the chromatographic performances of these polymer coated particles under SFC conditions with neat CO₂ as mobile phase. It was found that the particles coated with hydroxy-terminated PEO were suitable for the separation of proton-donor compounds such as hydroxy-containing compounds and carboxylic acids, and the particles coated with aminopropoxy-terminated PEO could be used for the separation of amines. That is, the proton-accepting stationary phase is suitable for the separation of proton accepting solutes, including strong basic alkylamines (pK_b4), using neat CO₂ as mobile phase, while the protondonating stationary phase is suitable for the separation of proton-donating compounds such as carboxylic acids (pK_a4). Hydrogen bond basicity was found to be a critical factor for the chromatography of basic amines. Low volatility acidic and basic drugs were chromatographed using the new stationary phases. The stability of the PEO coated particles was determined by measuring the loss of organic carbon under SFC conditions. It was found that approximately 18 % of the coating (average molecular weight of 15,000) was washed out of the particles by supercritical CO₂ after 7 h at 350 atm and 50°C