一种聚甲基丙烯酸酯类亲水作用毛细管电色谱整体柱的制备与评价

以2-羟基乙基甲基丙烯酸酯(HEMA)为单体,乙二醇二甲基丙烯酸酯(EDMA)为交联剂,制备了亲水分离模式的聚HEMA-co-EDMA新型毛细管电色谱(CEC)整体柱。考察了整体柱的结构特征及其CEC性能,研究了极性物质的保留行为,并对其可能的保留机理进行了探讨。所制备的整体柱稳定性好,固定相表面带有极性羟基功能团,不仅能提供亲水相互作用位点,且能吸附流动相中的阴离子产生阳极电渗流(EOF)。在流动相中乙腈含量较高(＞62%,体积分数)的条件下,整体柱表现出典型的亲水作用,实现了对核苷、碱基和苯胺类带有碱性的强极性化合物的高效分离,并成功分离了苯酚类、苯甲酸类等中性或酸性的极性化合物。     

木糖醇和麦芽糖醇型亲水作用色谱固定相的制备及其分离性能的评价

利用-NCO和-OH的加成反应,通过简单的两步反应将木糖醇和麦芽糖醇成功地键合于硅胶表面,制备了两种新型糖醇类亲水作用色谱固定相。流动相中乙腈含量对保留的影响曲线表明,这两种糖醇固定相具有典型的亲水作用色谱固定相性质,对极性和亲水性化合物有很强的保留作用。利用这两种固定相成功分离了水溶性维生素、水杨酸及其类似物、碱基及其相应的核苷和淫羊藿苷类似物等模型混合物,同时糖醇固定相展现了新颖的选择性,特别是相对于线形的木糖醇键合固定相,非线形的麦芽糖醇键合固定相表现出了对糖基的独特保留能力。此外,缓冲盐的pH和浓度对保留的影响表明静电作用在这两种糖醇固定相的保留机理中也发挥着一定的作用。本文所发展的糖醇类固定相具有良好的分离性能,有望在亲水作用色谱分离领域发挥潜在的应用价值。     

奎宁功能化聚乙烯咪唑修饰硅胶亲水相互作用色谱固定相的制备及应用

通过表面自由基链转移聚合和亲核取代反应制备了一种新型奎宁功能化聚乙烯咪唑修饰硅胶亲水色谱固定相(Sil-PIm-Qn)。通过元素分析和红外光谱对该固定相进行表征,并在亲水相互作用色谱(HILIC)模式下对其进行了色谱性能评价。结果表明,该固定相对5种氨基酸、9种磺胺以及10种碱基核苷有较好的分离选择性。实验考察了流动相中有机相乙腈体积分数和水相中乙酸铵浓度对待分离物质保留行为的影响,并进一步对固定相分离的重复性进行了考察,其保留时间的相对标准偏差(RSD)为0.08%~2.30%(n=10)。该亲水色谱固定相制备方法简单,并且表现出了优异的亲水色谱分离性能,有望在磺胺类药物及生物样品中碱基核苷等亲水性物质的分离分析中有一定应用。     

核苷与碱基的苯胺甲基键合硅胶固定相高效液相色谱分离

建立苯胺甲基键合硅胶固定相(PAMS)高效液相色谱分离核苷与碱基的方法；研究流动相有机溶剂浓度、磷酸缓冲液pH值、离子强度对核苷和碱基在该键合固定相上的色谱保留及分离选择性的影响，用磷酸缓冲液(pH＝4)为流动相快速分离了部分核苷与碱基。     

L-异亮氨酸键合色谱固定相的制备及其性能

利用异氰酸丙基三乙氧基硅烷与L-异亮氨酸反应合成了一种新型的硅烷偶联剂,并进一步将其与硅胶反应制得键合有L-异亮氨酸的亲水色谱固定相。 通过核磁共振氢谱表明亮氨酸功能化硅烷偶联剂的成功合成、元素分析表征证明亮氨酸已成功键合到硅胶表面。 将其作为亲水模式下的固定相填料填装在150 mm×4.6 mm不锈钢色谱柱中,以一系列经典的极性小分子作为探针,考察了这些探针分子在固定相上的色谱行为。 极性化合物的保留时间随着流动相中有机溶剂含量提高而逐渐增大,表现出典型的亲水保留特征。 进一步研究了流动相中乙腈含量、缓冲盐pH值及缓冲盐浓度等因素对分析物在固定相上的保留的影响。 在优化了相关参数后,将固定相应用于碱性化合物、水溶性维生素以及核苷类极性物质的分离当中。 在等度洗脱下,5种碱性化合物、6种水溶性维生素和8种核苷类物质分别在8、18及25 min内被成功分离。 分离结果表明了合成的L-异亮氨酸键合亲水色谱固定相具有较好的色谱性能,在极性化合物的分离上具有良好的应用前景。     

姜黄素键合硅胶固定相HPLC分离水溶性维生素和核苷

以ODS为参比,研究了5种水溶性维生素和5种核苷在姜黄素键合硅胶固定相(CCSP)上的色谱行为,考察了甲醇、流动相pH和离子强度对CCSP分离这类极性化合物的影响,探讨了该新固定相的色谱保留机理。结果表明,在V(甲醇)∶V(0.01 mol/L NaH2PO4)=40∶60(pH 3.5)流动相体系中,5种水溶性维生素在CCSP上实现较好分离;核苷的分离则是在以纯水为流动相的条件下实现的。与ODS柱相比,在相同条件下,CCSP对5种水溶性维生素具有较高的选择性,洗脱顺序与ODS具有显著差异,且保留明显比ODS强(B6除外);与此相反,相同色谱条件下,CCSP对核苷的保留比ODS弱,CCSP可实现核苷的简便有效分离,同时胸苷与黄苷的洗脱顺序也与ODS不同。以上说明不同保留机理的存在,CCSP具有典型的反相色谱特征,但疏水性比ODS弱;极性的配体使CCSP在对溶质的分离分析中除疏水作用外,还存在n-π和π-π作用、氢键作用、偶极-偶极等作用;协同作用的结果使CCSP在水溶性极性化合物的分离分析中显示出优势。     

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。     

原子转移自由基聚合制备PDMAEMA亲水作用色谱固定相及其性能评价

以甲基丙烯酸二甲氨乙酯为单体,2-溴异丁酰溴为引发剂,CuBr/五甲基二乙烯基三胺(PMDETA)为催化剂,通过原子转移自由基聚合(ATRP)反应,将甲基丙烯酸二甲氨乙酯(DMAEMA)接枝到5μm大孔硅胶表面上,得到了接枝聚合物(PDMAEMA)亲水作用色谱固定相.通过改变反应体系中单体的量,制备了三种不同接枝量的亲水作用色谱固定相,利用元素分析对所制备的固定相进行了表征.详细考察了该固定相的分离性能以及流动相中盐浓度、水含量对溶质保留行为的影响,并将该固定相用于宁心宝胶囊中核苷类化合物的分离和测定.在亲水模式下,该固定相可以基线分离7种核苷类化合物,保留时间随着接枝量的增加而增大,与氨基亲水作用色谱柱相比,该合成柱的分离效率高,溶质在该填料上的保留符合分配作用保留机理.实验结果表明,该填料具有良好的分离性能.     

基于氧化石墨烯的环糊精型色谱固定相的制备及在对映体分离与亲水色谱中的应用

将氧化石墨烯(GO)通过酰胺键键合到氨基硅胶上,进一步将β-环糊精(β-CD)化学键合于GO上,制备了一种新型手性固定相材料,采用IR、SEM、TEM、元素分析和热重分析进行了表征。7种对映体得到不同程度的拆分,同时此固定相表现出典型的亲水作用色谱(HILIC)特征,4种核苷小分子在流动相为甲醇-乙腈-水(45∶45∶10,V/V)时得到分离。通过考察流动相组成、温度、pH值对分析物保留的影响,进一步探讨了该固定相的分离机理。在20~60℃时,温度越低越有利于分析物质分离;加入质子化溶剂甲醇,4种分析物的保留时间显著减弱;pH值的改变影响分析物的质子化程度进而改变与固定相作用力和保留时间。结果表明,此色谱固定相中GO和β-CD在手性拆分中具有协同作用,而在HILIC中分析物的保留归因于混合模式的保留机理,包括亲水作用、静电作用、氢键、π-π堆积作用等。     

γ-氨基丁酸键合硅胶毛细管色谱柱填料的绿色制备及其分析性能研究

该文在水相溶剂中绿色合成了γ-氨基丁酸键合硅胶(Sil-GABA)色谱固定相,采用红外光谱、元素分析等对其进行了表征,并将制备的Sil-GABA材料作为毛细管色谱柱的填料研究了其色谱分离模式和分析性能。结果显示,在以乙酸铵水溶液为流动相的条件下,该填料可有效分离硝基氯苯3种位置异构体和7种磺胺,表明Sil-GABA固定相具有富水作用色谱的特性;在高浓度有机流动相条件下可有效分离4种苯甲酸类化合物,表明Sil-GABA具有亲水作用色谱的特性;此外,该Sil-GABA固定相在反相模式下对硝基苯胺异构体也有一定的分离能力。在优化条件下,7种磺胺保留时间的相对标准偏差小于3.2%,表明制得的色谱柱具有良好的重复性。该Sil-GABA固定相具有多种保留机理,在毛细管液相色谱分离领域具有潜在的应用价值。     

Tetrazole-Functionalized Silica for Hydrophilic Interaction Chromatography of Polar Solutes

In this work, tetrazole-functionalized stationary phase was prepared with nitrile-modified silica by an ammonium-catalyzed (3 + 2) azide-nitrile cycloaddition reaction. The prepared stationary phase was used for separation of nucleobases and nucleosides by hydrophilic interaction chromatography (HILIC) mode. A typical HILIC mechanism was observed at higher content of acetonitrile (>85%, v/v) in the mobile phase. The retention mechanism of the column was investigated by the models used for describing partitioning and surface adsorption through adjustment ratio of water in the mobile phase, and by the influence of salt concentration, buffer pH, and temperature on the retention of solutes. The results illustrated that the surface adsorption through hydrogen bonding dominated the retention behavior of nucleobases/nucleosides under HILIC mode. From the separation ability, the tetrazole-functionalized stationary phase could become a valuable alternative for the separation of the compounds concerned.     

Preparation of imidazole-functionalized silica by surface-initiated atom transfer radical polymerization and its application for hydrophilic interaction chromatography

A novel imidazole-functionalized stationary phase for hydrophilic interaction chromatography (HILIC) was prepared via surface-initiated atom transfer radical polymerization (SI-ATRP). 1-Vinylimidazole as a monomer was polymerized on the surface of initiator-immobilized silica by SI-ATRP using CuCl and 2,2'-bipyridyl as a catalyst. The graft chain length and polymer grafting density were controlled by varying the ratio of monomer to initiator. The resulting materials were characterized by elemental analysis and thermogravimetric analysis. Then, high-performance liquid chromatography separation of eight nucleobases/nucleosides was performed on the imidazole-functionalized chromatographic column in HILIC mode. The effects of mobile phase composition, buffer pH, and column temperature on the separation of nucleobases/nucleosides were investigated, and the retention mechanisms were studied. Chromatographic parameters were calculated, and the results showed that surface adsorption through hydrogen bonding and electrostatic interaction dominated the retention behavior of the solutes in HILIC mode. Lastly, the stationary phase was successfully used to determine the nucleobases and nucleosides from Cordyceps militaris.     

Study of retention behaviour and mass spectrometry compatibility in zwitterionic hydrophilic interaction chromatography for the separation of modified nucleosides and nucleobases

A study has been made of the chromatographic behaviour of modified nucleosides and nucleobases using different stationary phases with functional groups of polar nature, all of them compatible with aquoorganic mobile phases. The stationary phases assayed were a pentafluorophenylpropyl (PFP) column for reverse phase separation, and another two for hydrophilic interaction chromatography (HILIC) separation. Six modified nucleosides and nucleobases (hydroxylated and methylated derivatives) were chosen as the target analytes. In the study, chromatographic resolution as well as the sensitivity in detection by mass spectrometry were taken into account. The results obtained showed that the zwitterionic (ZIC-HILIC) column was the most suitable one for the separation of these analytes. From the study of the different parameters affecting separation it may be concluded that in the ZIC-HILIC column separation is based on a mechanism of partition and interaction through weak electrostatic forces.     

Preparation of a Novel Amino-Phosphate Zwitterionic Stationary Phase for Hydrophilic Interaction Chromatography

A new stationary phase which contains both negatively charged phosphate groups and positively charged amino groups was successfully synthesized by modification of amino-functionalized silica particles with trichlorophosphine oxide (POCl₃) for hydrophilic interaction chromatography (HILIC). The composition of the surface grafts was determined by Fourier transform infrared spectroscopy and elemental analysis. Various parameters, such as column temperature, water content, pH values and ionic strength of the mobile phase were investigated to study the retention mechanism. The results demonstrated that the stationary phase involved a complex retention process including surface adsorption, partitioning and electrostatic interactions. Under optimized conditions, the separation of nucleobases and nucleosides, water-soluble vitamins, organic acids on the novel stationary phase could be achieved in the HILIC mode.     

硅胶色谱柱的亲水作用保留机理及其影响因素

亲水作用色谱（HILIC）是替代反相色谱（RPLC）分离强极性及亲水性化合物的另一色谱模式,其分离机理与RPLC有很大不同,具有和RPLC互补的选择性。在HILIC模式中,采用正相色谱（NPLC）中的极性固定相及含高浓度有机溶剂（通常为乙腈）的水溶液为流动相。硅胶是开发最早、研究最为深入及应用最为广泛的HILIC固定相,本文介绍了硅胶色谱柱的HILIC保留机理,详细概述了操作条件如硅胶柱类型、流动相组成及柱温对HILIC分离的影响,并对硅胶填料色谱柱的HILIC模式的发展方向与应用前景进行了展望。     

Preparation of a Novel Amino-Phosphate Zwitterionic Stationary Phase for Hydrophilic Interaction Chromatography

A new stationary phase which contains both negatively charged phosphate groups and positively charged amino groups was successfully synthesized by modification of amino-functionalized silica particles with trichlorophosphine oxide (POCl₃) for hydrophilic interaction chromatography (HILIC). The composition of the surface grafts was determined by Fourier transform infrared spectroscopy and elemental analysis. Various parameters, such as column temperature, water content, pH values and ionic strength of the mobile phase were investigated to study the retention mechanism. The results demonstrated that the stationary phase involved a complex retention process including surface adsorption, partitioning and electrostatic interactions. Under optimized conditions, the separation of nucleobases and nucleosides, water-soluble vitamins, organic acids on the novel stationary phase could be achieved in the HILIC mode.

     

Simultaneous determination nucleosides in marine organisms using ultrasound-assisted extraction followed by hydrophilic interaction liquid chromatography-electrospray ionization time-of-flight mass spectrometry

A new method has been developed based on ultrasound-assisted extraction (UAE) followed by hydrophilic interaction chromatography (HILIC) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF/MS) for the simultaneous determination of 16 nucleosides and nucleobases in medicinal extracts of various marine organisms. The separation was achieved on a Venusil HILIC column (250×4.6 mm id, 5 μm) and gradient elution using a solution of acetonitrile and buffer (0.20% formic acid and 20 mmol/L ammonium acetate) as the mobile phase. Identification of the 16 target nucleosides and nucleobases was based on the retention time, UV spectra, and mass measurements of the protonated molecules ([M+H](+)) and main fragment ions (ESI-TOF/MS). In addition, non-target compounds of 2'-deoxyinosine and four other amino acids were also tentatively identified by ESI-TOF/MS. The 16 target compounds were quantified by HILIC-ESI-TOF/MS under optimized mass conditions. All calibration curves showed good linearity (r(2)>0.9951). The recoveries were 84.72-124.10%, and the limits of detection of the 16 target compounds were 0.6-130.0 ng/mL. The developed method was applied to quantify the target compounds in 15 batches of various marine organisms. The method has potential applicability for the identification and determination of highly polar and low-concentration active compounds in marine organisms.     

Dual Retention Mechanism in Two-Dimensional LC Separations of Barbiturates,Sulfonamides, Nucleic Bases and Nucleosides on Polymethacrylate Zwitterionic Monolithic Micro-Columns

In-house prepared zwitterionic polymethacrylate micro-columns using in situ polymerization of N,N-dimethyl-N-metacryloxyethyl-N-(3-sulfopropyl) ammonium betaine (MEDSA) functional monomer with bisphenol A glycerolate dimethacrylate (BIGDMA) cross-linker provided excellent stability and reproducibility of preparation and separation efficiency of 60,000–70,000 theoretical plates m⁻¹ for small molecules under isocratic conditions. The column showed a dual retention mechanism, reversed-phase (RP) in highly aqueous mobile phases and aqueous normal-phase (HILIC) in acetonitrile-rich mobile phases. This property can be used to obtain complementary separation and combined information on the sample from repeated injections of a sample on a single column, in different mobile phases characteristic for the HILIC and for the RP modes, which is in fact a form of offline two-dimensional chromatography on a single column. The dual retention mechanism has been observed with a variety of columns, however, often with impractically narrow retention range in one of the two modes. To take full advantage from the combined single-column RP–HILIC experiments, the column should provide a sufficiently broad mobile phase interval both in the RP and in the HILIC mode. The BIGDMA-MEDSA micro-columns proved suitable earlier for the combined RP–HILIC separations of some phenolic compounds and flavonoids. In the present work, we investigated the effects of the mobile phase composition on the retention of a variety of polar compounds over full retention range of buffered aqueous acetonitrile mobile phases, to find potentially useful HILIC and RP retention ranges for barbiturates, sulfonamides, nucleosides and nucleic bases. In the HILIC mode, proton donor–acceptor interactions show a major effect on retention and selectivity of separation, whereas the size of the non-polar hydrocarbon part of the sample molecule is the most important factor in the water-rich mobile phases. The sample structure strongly affects the composition of aqueous–organic mobile phases at which the transition between the two retention modes occurs. Of the investigated sample types, barbiturates show better separation under reversed-phase conditions, whereas nucleosides and nucleic bases in the HILIC mode. Aromatic carboxylic acids and sulfonamides can be separated either in the reversed phase or under HILIC conditions, the two separation modes showing complementary selectivity of separation.

     

Highly hydrophilic and nonionic poly(2-vinyloxazoline)-grafted silica: a novel organic phase for high-selectivity hydrophilic interaction chromatography

A new hydrophilic and nonionic poly(2-vinyloxazoline)-grafted silica (Sil-VOX _n ) phase was synthesized and applied for the separation of nucleosides and nucleobases in hydrophilic interaction chromatography (HILIC). Polymerization and immobilization onto silica were confirmed by using characterization techniques including ¹H NMR spectroscopy, elemental analysis, and diffuse reflectance infrared Fourier transform spectroscopy. The hydrophilicity or wettability of Sil-VOX _n was observed by measuring the contact angle (59.9°). The chromatographic results were compared with those obtained with a conventional HILIC silica column. The Sil-VOX _n phase showed much better separation of polar test analytes than the silica column, and the elution order was different. Differences in selectivity between these two columns indicate that the stationary phase cannot function merely as an inert support for a water layer into which the solutes are partitioned from the bulk mobile phase. To elucidate the interaction mechanism, the separation of dihydroxybenzene isomers was performed on both columns in normal-phase liquid chromatography. Sil-VOX _n was very sensitive to the dipole moments of the positional isomers of polycyclic aromatic compounds in normal-phase liquid chromatography. The interaction mechanism for Sil-VOX _n in HILIC separation is also described.