基于巯基-烯点击化学法的β-环糊精固定相多模式色谱保留行为研究

基于巯基硅胶与单取代-6A-烯丙氨基-β-环糊精的巯基-烯点击化学反应,制备了β-环糊精(Click TE-CD)共价键合固定相。元素分析结果表明β-环糊精被成功键合到硅胶表面。以黄酮苷类化合物为模型,考察了Click TE-CD固定相在亲水、反相和超临界流体色谱等分离模式下的色谱保留行为。黄酮苷类化合物保留时间随流动相中乙腈含量的变化呈现典型的U型曲线,表明Click TE-CD固定相具有亲水/反相的双重保留特性。应用几何学方法测得Click TE-CD固定相在反相/亲水、亲水/超临界、反相/超临界混合模式下的正交性分别为69.8%、50.8%、50.8%。对比复杂中药样品降香提取物在反相、亲水、超临界等模式下的分离情况,结果表明Click TE-CD固定相在分离中药复杂样品方面具有极大潜力,可以在一根色谱柱上通过分离模式的改变,实现二维液相色谱的分离。Click TE-CD固定相不同分离模式的分离性能和较好的正交性表明该固定相具有在液相色谱方法发展和二维液相色谱分离方面应用的潜力。     

Synthesis,chromatographic evaluation and hydrophilic interaction/reversed-phase mixed-mode behavior of a “Click β-cyclodextrin” stationary phase

A native β-cyclodextrin (β-CD) stationary phase was prepared by covalently bonding β-CD on silica particles via Huisgen [3 + 2] dipolar cycloaddition between the organic azide and terminal alkyne, the so-called Click chemistry. The resulting β-CD bonded silica (Click β-CD) was characterized by FT-IR, solid state ¹³C cross polarization/magic-angle spinning (CP/MAS) NMR and elemental analyses, which proved the successful immobilization of β-CD on the silica support with Click chemistry. The retentive properties of Click β-CD were investigated under hydrophilic interaction liquid chromatography (HILIC) mode in different mobile phase conditions with a set of polar compounds including nucleosides, organic acids and alkaloids. The effects of water content, concentration of the salt and pH of the buffer solution on the retention time were studied and the results demonstrated the typical retention behavior of HILIC on Click β-CD. Separation of very polar components, such as nucleosides and oligosaccharides, and chiral separation under HILIC mode were successfully achieved. In addition, Click β-CD was chromatographically evaluated with a set of flavone glycosides. The retention curves depending on the mobile phase of acetonitrile content were “U” curves, which is an indication of HILIC/RPLC mixed-mode retention behavior. The difference of the separation selectivity between HILIC and RPLC was described as orthogonality by using geometric approach and the orthogonality reached 69.4%. The mixed-mode HPLC properties and excellent orthogonality demonstrated the flexibility in HPLC methods development and great potential in two-dimensional liquid chromatography separation.     

Click novel glycosyl amino acid hydrophilic interaction chromatography stationary phase and its application in enrichment of glycopeptides

A novel glycosyl amino acid hydrophilic interaction chromatography (HILIC) stationary phase was prepared via click chemistry. The key intermediate N₃-glycosyl d-phenylglycine was prepared by a three steps procedure, including selective condensation of amino glucose with N-succinimidyl ester of Boc-d-phenylglycine, deprotection and transformation of amino group to azido group. The structure of all the intermediates and functionalized silica beads were confirmed by ¹H NMR, IR, elemental analysis and ¹³C CP-MAS. The chromatography test showed that this new type of separation material possessed good HILIC properties and glycopeptide enrichment characteristics. Nucleosides and bases could be separated in a simple eluent composition (only acetonitrile in combined with water), and with the same condition, these model compounds could not be separated on the commercial HILIC column (Atlantis). Click glycosyl amino acid thus prepared also showed longer retention and better separation ability in the separation of polar organic acids.     

Efficient purification of high‐purity compounds from the stem of Lonicera japonica Thunb using two‐dimensional preparative chromatography

Purification of high‐purity compounds from traditional Chinese medicines (TCMs) plays an important role in investigating their bioactivity. Nevertheless, it is often quite difficult to isolate compounds with high purity because of the complexity of TCMs in chemical composition. In this work, a two‐dimensional preparation method was successfully developed for the preparation of high‐purity compounds from the stem of Lonicera japonica Thunb, based on two novel polar copolymerized RP stationary phases, XAqua C3 and XAqua C18. An XAqua C3 prep column was used to separate the sample in the first‐dimensional preparation, and 14 g of sample was fractionated into eight fractions with a recovery of 82%. An XAqua C18 prep column was selected to prepare high‐purity compounds in the second‐dimensional preparation for its good orthogonality with the XAqua C3 stationary phase. As a result, major compounds in the sample were isolated with more than 99% purity. This method is a potent method to realize the efficient purification of compounds with high purity from the stem of L. japonica Thunb and it shows great potential in the separation of high‐purity compounds from complex samples.     

Tetra‐proline‐modified calix[4]arene‐bonded silica stationary phase for simultaneous reversed‐phase/hydrophilic interaction mixed‐mode chromatography

A new water‐soluble tetra‐proline‐modified calix[4]arene‐bonded silica stationary phase was prepared straightforwardly by an indirect method and characterized by elemental analysis, energy dispersive Spectrometry, solid‐state ¹³C NMR spectroscopy, Fourier‐transform infrared spectroscopy, and thermogravimetric analysis. Due to the simultaneous introduction of polar tetra‐proline and nonpolar calix[4]arene, the developed column possessing a double retention mode of reverse‐phase liquid chromatography and hydrophilic interaction liquid chromatography. A series of hydrophobic and hydrophilic test samples, including nucleosides and nucleotides, amines, monosubstituted benzenes, chiral compounds, and phenols, were used to evaluate the developed stationary phase. A rapid separation capability, high separation efficiency, and selectivity were achieved based on the multiple interactions between solutes and tetra‐proline‐modified calix[4]arene‐bonded silica stationary phase. Moreover, the developed stationary phase was further used to detect and separate hexamethylenetetramine in rice flour. All the results indicated the potential merits of the developed stationary phase for simultaneous separation of complex hydrophobic and hydrophilic samples with high selectivity.     

Highly selective separation of aminoglycoside antibiotics on a zwitterionic Click TE‐Cys column

Hydrophilic interaction liquid chromatography has emerged as a valuable alternative approach to ion‐pair chromatography for the separation of aminoglycoside antibiotics in recent years. However, the resolution of structurally related aminoglycosides is a great challenge owing to the limited selectivity. In this work, a cysteine‐based zwitterionic stationary phase (named Click TE‐Cys) was utilized and compared with five commonly used hydrophilic interaction liquid chromatography columns. Click TE‐Cys displayed much better selectivity for structurally similar aminoglycosides. The retention behaviors of aminoglycosides were investigated in detail, revealing that low pH (2.7 or 3.0) and high buffer concentration (≥50 mM) were preferable for achieving good peak shape and selectivity. Effective resolution of ten aminoglycosides including spectinomycin, dihydrostreptomycin, streptomycin, gentamicin C1, gentamicin C2/C2a, gentamicin C1a, kanamycin, paromonycin, tobramycin, and neomycin was realized at optimized conditions. Additionally, spectinomycin and its related impurities were successfully resolved. The results indicated the great potential of the Click TE‐Cys column in the separation of aminoglycoside mixtures and related impurities.     

Synthesis and evaluation of a maltose‐bonded silica gel stationary phase for hydrophilic interaction chromatography and its application in Ginkgo Biloba extract separation in two‐dimensional systems

Maltose covalently bonded to silica was prepared by using carbonyl diimidazole as a cross‐linker and employed as a stationary phase for hydrophilic interaction liquid chromatography. The column efficiency and the effect of water content, buffer concentration, and pH value influenced on retention were investigated. The separation or enrichment selectivity was also studied with nucleosides, saccharides, amino acids, peptides, and glycopeptides. The results indicated that the stationary phase processed good separation efficiency and separation selectivity in hydrophilic interaction liquid chromatography mode. Moreover, a two‐dimensional hydrophilic interaction liquid chromatography× reversed‐phase liquid chromatography method with high orthogonality was developed to analyze the Ginkgo Biloba extract fractions. The development of this two‐dimensional chromatographic system would be an effective tool for the separation of complex samples of different polarities and contents.     

亲水/反相二维制备液相色谱法分离纯化络石藤中的化学成分

建立了亲水/反相二维制备液相色谱(Pre-2D-HILIC/RPLC)分离纯化络石藤中化学成分的分析方法。络石藤药材经醇提、活性炭脱色后用反相固相萃取柱除去色素和强极性物质,最终得到干燥的浅黄色粉末。一维亲水色谱选择Click XIon色谱柱(250 mm×20 mm,10μm)作为固定相,水和乙腈作为流动相,进行梯度洗脱,以紫外触发模式收集馏分,共得到15个组分。二维反相色谱选择C18色谱柱(250 mm×20 mm,5μm)作为固定相,水和乙腈作为流动相,进行梯度洗脱,最终得到14个高纯度化合物,并通过质谱和核磁共振对其进行确认。实验结果表明,该法具有良好的正交选择性,可以有效提高分离度和峰容量,对于分离络石藤等复杂样品具有重要意义。     

Novel two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography, an excellent orthogonal system for practical analysis

A novel two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography (2D-RPLC/HILIC) system is developed with the introduction of the click beta-cyclodextrin (beta-CD) stationary phase. The offline system shows excellent abilities for the separation of polar and medium-polarity components in traditional Chinese medicine (TCM). It facilitates us not only to separate components that cannot be resolved by uni-dimensional chromatography, but also to achieve much more efficient detection of components with low abundance. The orthogonality of this new system is excellent. A simple geometric approach is developed to characterize the practical orthogonality of 2D-LC in the analysis of complex unknown samples. The mathematical characterization results are in good accordance with the experimental findings, and both demonstrate that the proposed 2D-RPLC/HILIC system is a powerful tool for the separation of polar and medium-polarity complex samples. This system may have a great potential for the separation of peptides and metabolomic compounds with similar polarity.     

Polar-copolymerized approach based on horizontal polymerization on silica surface for preparation of polar-modified stationary phases

A new approach for preparation of polar-modified reversed-phase liquid chromatography stationary phases was developed by using horizontal polymerization technique on silica surface, which was defined as “polar-copolymerized” approach. Based on this new approach, a representative polar-copolymerized stationary phase composed of mixed n-octadecyl and chloropropyl (C18–C3Cl) was synthesized. The resulting stationary phase named C18HCE was characterized with elemental analysis and solid phase ¹³C and ²⁹Si NMR, which proved the chemistry of polar-copolymerized stationary phases. Chromatographic evaluation and application of the C18HCE were also investigated. The results of preliminary chromatographic evaluation demonstrated that the C18HCE stationary phase exhibited 100% aqueous mobile phase compatibility, low silanol activity. In addition, the application results demonstrated that the C18HCE had superior separation performance in alkaloids separation at acidic conditions compared to some commercial stationary phases.     

Synthesis and characterization of novel polar-embedded silica stationary phases for use in reversed-phase high-performance liquid chromatography

We have developed a series of new C₁₀ dipeptide stationary phases via a simple and effective synthetic method. The preparation of the new phases involves the synthesis of silanes and the surface modification of silica. Chromatographic evaluations of these columns were performed using the Engelhardt, Tanaka, and Neue test mixtures. The applicability of these new stationary phases was also evaluated using a series of diagnostic probes including acids, bases or neutral compounds and several generic applications. These new C₁₀ dipeptide stationary phases showed excellent hydrolytic stability over a wide pH range. Like other existing amide-embedded columns, these new stationary phases exhibit higher retention for polar and hydrophilic compounds and different selectivity as compared to conventional C₁₈ columns. These new phases are compatible with 100% aqueous mobile phases, and also provide high column efficiency and good peak shapes for both acidic and basic compounds.     

Efficient separation of high‐purity compounds from Oxytropis falcata using two‐dimensional preparative chromatography

The separation of high‐purity compounds from traditional Tibetan medicines plays an important role in investigating their bioactivity. Nevertheless, it is often quite difficult to isolate compounds with high purity because of the complexity of traditional Tibetan medicines. In this work, an offline two‐dimensional reversed‐phase preparative method was successfully developed for the separation of high‐purity compounds from Oxytropis falcata . Based on the analysis results, an ODS C18 prep column was used for first‐dimensional preparation, and 14.8 g of the crude sample was separated into five fractions with a recovery of 74.6%. Then, an XAqua C18 prep column was used to isolate high‐purity compounds in the second‐dimensional preparation because its separation selectivity is different with the ODS C18 stationary phase. As a result, eight compounds in the crude sample were isolated in more than 98% purity. This is the first report of trans‐cinnamic acid ( 1 ) and trifolirhizin ( 2 ) from Oxytropis falcata . This method has the potential to be an efficient separation method of high‐purity compounds from Oxytropis falcata and it shows great promise for the separation of high‐purity compounds from complex samples.     

C18-磺酸基双改性液相色谱固定相的制备与评价

以十八烷基三氯硅烷和3-巯丙基三甲氧基硅烷为改性剂,采用一锅法对硅胶表面加以修饰,进一步将巯基氧化制备成C18-磺酸基双改性液相色谱固定相.在优化的反应条件下得到了十八烷基和磺酸基摩尔比为3∶7的固定相,分别采用扫描电镜、元素分析、红外光谱对固定相的形貌和特征加以表征.针对制备的固定相,在不同的分离模式下,系统考察其色谱分离性能.在反相色谱模式下,成功分离了5种烷基苯化合物;在亲水模式下,分离了3种核苷;进一步应用于牛血清白蛋白酶解产物的分离,得到了较好的结果.实验结果表明,制备的混合模式固定相同时具有多种分离机理,在分离复杂样品、调整选择性方面具有潜在的优势.     

A series of novel ferrocene‐based dipeptide receptors for electrochemistry and biological activity

Synthesis of ferrocene‐based dipeptide receptors FcL1 , FcL2 , FcL3 , FcL4 , FcL5 was carried out by the acylation reaction of ferrocenecarboxylic acid with dipeptide esters. Compounds were characterized by IR, ¹H NMR, ¹³C NMR and elemental analysis. Single‐crystal X‐ray analysis of FcL3 showed it has two independent chiral molecules and the two chiral atoms (C12 and C35) are in R configuration. Their cyclic voltammetric behaviors showed a pair of well‐defined and stable redox waves in the potential range 0.00–1.00 V. The configuration of the metals influenced the receptor's characteristics greatly, and at the same time the FcL compounds could selectively sense group IIB metal ions Zn²⁺, Cd²⁺ and Hg²⁺. The biological activities of FcL1 , FcL2 , FcL3 , FcL4 , FcL5 were studied against the tested bacteria; among them, FcL4 showed significant activity against all five bacterial strains tested. Copyright © 2013 John Wiley & Sons, Ltd.     

Off‐line 2‐D RPLC/RPLC method for separation of components in Dalbergia odorifera T. Chen

An off‐line 2‐D RPLC/RPLC system incorporating a β‐CD based column and a BEH C18 column was developed in the separation of components in Dalbergia odorifera T. Chen. (Jiang Xiang). Both orthogonality and van Deemter curves of the two columns were investigated. The orthogonality was about 57%, which was evaluated with 40 standard solutes. Optimized column efficiency could be achieved under optimal linear velocity (0.04 cm/s) on the CD column or under high linear velocity (0.32 cm/s) on the BEH C18 column. With the 2‐D LC system, totally 637 peaks were separated in 114 fractions from the extraction of Jiang Xiang. Meanwhile, 19 flavanoids were tentatively identified from 114 fractions with Q‐TOF MS. The results demonstrated the separation power of this 2‐D LC system and further proved the high orthogonality between CD and C18 columns.     

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 of a novel carboxyl stationary phase by “thiol‐ene” click chemistry for hydrophilic interaction chromatography

A novel carboxyl‐bonded silica stationary phase was prepared by “thiol‐ene” click chemistry. The resultant Thiol‐Click‐COOH phase was evaluated under hydrophilic interaction liquid chromatography (HILIC) mobile phase conditions. A comparison of the chromatographic performance of Thiol‐Click‐COOH and pure silica columns was performed according to the retention behaviors of analytes and the charged state of the stationary phases. The results indicated that the newly developed Thiol‐Click‐COOH column has a higher surface charge and stronger hydrophilicity than the pure silica column. Furthermore, the chromatographic behaviors of five nucleosides on the Thiol‐Click‐COOH phase were investigated in detail. Finally, a good separation of 13 nucleosides and bases, and four water‐soluble vitamins was achieved.     

离线二维液相色谱-串联质谱法检测姜黄中姜黄素类和倍半萜类化合物

采用二维高效液相色谱-电喷雾质谱法,以新型色谱填料ClickCD为第一维分离材料,WatersXTerraMSC18柱作为第二维,以乙腈和0.2%甲酸-水为梯度洗脱流动相,对姜黄中的姜黄素类和倍半萜类化合物进行分离鉴定.结果表明,基于不同分离机理的ClickCD和C18二维色谱系统具有分离度高、正交性好的优点,使待测化合物得到了有效分离.特别是与高灵敏度的质谱联用,可以大大提高化合物的鉴定能力.通过与对照品的色谱保留时间和质谱数据对比,并结合文献报道,共鉴定了20个姜黄素类化合物和19个倍半萜类化合物,其中27个为对照品的同分异构体.为中药化合物的表征和活性化合物的制备提供了有效方法.     

Preparation of a permethylated β‐cyclodextrin chiral stationary phase by one‐pot hydrosilylation and immobilization at the C2 position for chiral high‐performance liquid chromatography

A novel cyclodextrin intermediate, mono‐2^A‐allylcarbamido‐2^A‐deoxy‐permethylated β‐cyclodextrin, was synthesized by reacting allylamine and newly prepared mono‐2^A‐azido‐2^A‐deoxy‐permethylated β‐cyclodextrin by the Staudinger reaction and anchored onto porous silica beads by a one‐pot hydrosilylation and immobilization procedure to afford a novel chiral stationary phase. This stationary phase acts as a new member of the previous chiral stationary phase series immobilized on the cyclodextrin C2 position. This stationary phase depicted enantiomeric separation abilities toward a series of bicyclic and tricyclic racemates under reversed‐phase conditions. The resolutions for hesperetin and naringenin achieved on the current phase reached 3.91 and 1.11, respectively, much higher than the previous permethylated β‐cyclodextrin with the linkage at the C6 position.     

Silica spheres coated with C18‐modified gold nanoparticles for capillary LC and pressurized CEC separations

Nonporous monodispersed silica spheres of 1.3 μm were coated with gold nanoparticles (AuNPs) and subsequently coated with n‐octadecanethiol. By transmission electron microscopy analysis, the average diameter of the AuNPs on the silica spheres was determined to be 12 nm. The chromatographic and electrochromatographic properties of self‐assembled n‐octadecanethiol AuNP‐coated silica microspheres (C18‐AuNPs‐SiO₂) were investigated using a group of nonpolar PAHs. The stationary phase appears to display a characteristic reversed‐phase behavior. Higher separation efficiency and shorter separation times were obtained using pressurized CEC (pCEC) compared with capillary LC (CLC). A maximum column efficiency of about 2.5×10⁵ plates per meter and less than 18 min separation time for benzene were obtained in pCEC while only 66 507 plates per meter and an analysis time of nearly 100 min were observed in CLC mode. A chemical stability test of the C18‐AuNPs‐SiO₂ stationary phase under extremely high and low pH conditions demonstrated that it is stable at pH 12 and 1 for at least 60 h. The results confirm that C18‐AuNPs‐SiO₂ possesses a high rigidity to withstand high packing pressures and can be used as a good stationary phase for CLC and pCEC.