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.     

强极性中药组分的亲水作用色谱保留行为

采用亲水作用色谱模式,以二醇基硅胶为固定相,水-有机溶剂为流动相,通过改变流动相中有机溶剂种类及浓度、缓冲盐、有机酸种类及其浓度、柱温等条件,研究了强极性中药组分在亲水作用色谱中的保留行为。结果表明:流动相中水的比例在0～100%(V/V)变化时,溶质保留呈U型曲线,属于亲水色谱和反相色谱的混合保留机理。水含量在0～50%(V/V)范围时,亲水作用控制溶质的保留。溶质保留随流动相中缓冲盐浓度的增大而增强。对于酸性溶质,其保留随有机酸三氟乙酸、甲酸、乙酸的酸性降低而增大。     

碱性药物在硫醚嵌入苯磺酸硅胶固定相上的色谱行为

混合模式色谱（MMC）在复杂样品的分离分析方面具有独到的优势,相比于单一模式色谱,MMC受到多种作用控制,保留机理更为复杂。利用巯基-烯点击化学方法分别制备了单配体和双配体两种硫醚嵌入苯磺酸硅胶固定相,通过改变pH、离子强度和有机溶剂强度等流动相条件,以4种碱性药物为模型,对其保留机理进行了探讨。结果表明,两种固定相都具有反相和离子交换的混合保留机理。通过改变流动相中盐浓度、考察溶质保留因子与盐浓度倒数的关系,证明了反相、单纯离子交换和反相协同离子交换三种作用形式的保留模型更为合理。定量研究表明,在两个固定相上,由单纯离子交换和反相协同离子交换构成的总离子交换作用占主导,各作用占比与溶质、流动相组成、固定相配体的类型及其比例等密切相关,并且协同作用对溶质的保留和分离选择性影响很大。混合模式色谱保留机理的研究对于新型固定相设计和复杂体系的分离优化具有重要理论指导意义。     

亲水作用电色谱用于吡啶类化合物的分离

 采用亲水电色谱对强极性吡啶类化合物进行了分离。以亲水性的强阳离子交换填料作固定相,乙腈-磷酸-三乙胺缓冲液作流动相,考察了流动相中有机改性剂含量、离子强度及流动相pH对吡啶类化合物保留行为的影响。结果表明,在亲水电色谱模式下,流动相中有机改性剂含量对吡啶类化合物保留有较大影响,随着流动相中乙腈含量的增加,吡啶类化合物的保留迅速增强;较高pH值有利于吡啶类化合物的峰形改善。实验证明,亲水电色谱是一种适用于极性化合物分离的新型微分离方法。     

磺酸甜菜碱型两性离子亲水作用色谱固定相的制备及色谱性能评价

以丙烯酸二甲氨基乙酯(DMAEA)和1,3-丙磺酸内酯为原料,合成了含磺酸甜菜碱型两性离子的N,N-二甲基-N-丙烯酰氧乙基-N-丙基磺酸铵(DMAEAPS)功能单体,通过原子转移自由基聚合(ATRP)技术将其接枝到硅胶表面,制备了磺酸甜菜碱型两性离子色谱固定相(Sil-DMAEAPS)。研究了该固定相对安息香、维生素B6、芸香叶苷、对香豆酸和咖啡酸5种极性溶质的亲水作用色谱分离性能。结果表明,在典型的亲水作用色谱条件下,极性溶质的保留主要由静电作用和亲水作用控制;而在典型的反相色谱条件下,极性溶质则表现出反相柱的分离特征。与ZIC-HILIC商品柱进行对比,自制色谱柱对5种极性溶质表现出不同的分离选择性。将自制色谱柱用于芦丁片中芸香叶苷含量的测定,操作方法简单,为极性样品的分离提供了新方法。     

多羟基化合物键合亲水色谱固定相的制备及其分离性能

用硅胶与氨丙基三甲氧基硅烷反应,再与δ-葡萄糖酸内酯反应,制备了一种多羟基化合物键合的新型亲水色谱固定相。以水-有机溶剂(乙醇、乙腈、四氢呋喃)为流动相,通过改变流动相中有机溶剂的种类及浓度、缓冲盐浓度和柱温,考察了该固定相对6种强极性中药组分的保留行为和保留机理。当水的比例在0～40%(v/v)范围时,溶质的保留随着流动相中水的比例的增大而减小,属于典型的亲水色谱分离模式;而当流动相中水的比例在0～100%(v/v)范围内变化时,溶质的保留随着水的比例变化呈“U”形曲线,属于亲水色谱和反相色谱的混合保留机理。缓冲盐的浓度和pH效应说明,所选用的中药组分与所制备的固定相间还存在弱的静电作用。该固定相对6种中药组分以及丹参注射液具有良好的分离性能,表明其在强极性中药有效成分的分离及其他强极性物质的分离分析中具有一定的应用前景。     

二甲双胍与吡格列酮两种2型糖尿病药物在亲水作用色谱上的保留机理研究（英文）

二甲双胍和吡格列酮是治疗2型糖尿病的常见药物,经常用于同一复方制剂。制药和临床领域常要求同时测定这两种药物。亲水作用色谱近来在医药分析领域展现出良好的前景。本文通过研究流动相的有机溶剂含量、pH值和缓冲盐浓度等参数对色谱保留的影响,从而尝试揭示两种药物在亲水性硅胶柱上的分离机理。流动相中甲醇的增加会导致二甲双胍的保留因子的增加。流动相pH值比较低时,二甲双胍的保留因子会随着pH值升高而增加,而当pH值6.09,其反而会随着pH值的升高而下降。吡格列酮的保留因子几乎不受甲醇比例和pH值的影响。缓冲盐浓度对两种药物的保留因子几乎都没有影响。因此,对于二甲双胍,亲水作用是主要的保留机理,静电作用和氢键也起到了重要作用。对于吡格列酮,亲水作用是支配性的。经过优化的亲水作用色谱方法可成功地应用到分离测定两种药物在2型糖尿病复方药物中的含量。     

新型咪唑嵌合氨基亲水作用色谱固定相的制备及其色谱性能

发展新型高效的亲水作用色谱分离材料对于极性化合物的分离分析具有重要的意义。本文设计合成了一种新型咪唑嵌合的氨基亲水作用色谱固定相（Sil-IEASP）,并分别采用傅里叶变换红外光谱仪、热重分析仪和元素分析仪对该固定相进行了表征,结果表明该固定相制备成功。以核苷和核酸碱基为样品,分别考察了流动相中的水含量、盐浓度和pH对其保留的影响,结果表明所发展的固定相具有良好的亲水作用特性;此外,缓冲盐浓度和pH几乎不影响上述物质的保留。进一步将该固定相应用于分离尿嘧啶、腺嘌呤、胞嘧啶、尿苷和3种位置异构体（邻三联苯、间三联苯和苯并菲）,与常用的氨基固定相相比,本文所发展的固定相具有更好的分离效果,有望在亲水作用色谱分离领域发挥潜在的应用价值。     

生物碱在十八烷基膦酸改性锆-镁复合氧化物固定相上的色谱行为

研究了某些生物碱在十八烷基膦酸改性锆-镁复合氧化物固定相(C18PZM)上的色谱行为。通过考察流动相参数如甲醇含量、缓冲液pH值和离子强度对生物碱保留的影响,对这类化合物在该固定相上的保留机理进行了探讨。结果表明,在实验色谱条件下,生物碱在C18PZM上表现出反相和阳离子交换的混合保留模式机理。锆-镁基质上化学吸附的十八烷基膦酸和其对流动相中路易斯碱的吸附以及锆羟基本身均有可能是该固定相的离子交换作用位点的来源。高pH值流动相,溶质大部分以分子状态使用形式存在,因此其保留以疏水作用为主。在甲醇-pH 10.1 Tris缓冲液,生物碱的分离取得了满意的结果。与传统的烷基键合硅胶反相固定相相比,C18PZM表现出了更优越的化学稳定性,对于碱性化合物,尤其是具有高pKa值的碱性化合物的分离分析有着广泛的应用前景,有望发展为与硅胶键合固定相互补的一类反相HPLC的固定相。     

万古霉素及其杂质的亲水作用色谱分析

目前,万古霉素色谱分析方法主要为反相色谱法,该法分离万古霉素及其杂质时,存在极性选择性不足以及所使用的流动相体系与质谱兼容性差等问题。基于亲水作用色谱(HILIC)对糖肽类物质的色谱保留和极性选择性,本文选取万古霉素及其常见杂质为对象,考察了HILIC固定相、流动相组成比例、缓冲盐添加剂浓度和pH值等色谱条件,对万古霉素及其主要杂质进行了HILIC分离方法研究。确立了以Click XIon色谱柱为固定相,以甲酸铵为流动相添加剂的亲水作用色谱条件,实现了万古霉素及主要杂质的分离,为万古霉素类化合物的分离提供了新的途径。     

Preparation of hydrophilic polymer-grafted polystyrene beads for hydrophilic interaction chromatography via surface-initiated atom transfer radical polymerization

A one-step procedure based on surface-initiated atom transfer radical polymerization (SI-ATRP) to hydrophilize monodisperse poly(chloromethylstyrene-co-divinylbenzene) beads has been presented in this work, using 2-hydroxyl-3-[4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl]propyl 2-methylacrylate (HTMA) as a monomer. The chain length of the grafted poly(HTMA) was controlled via varying the ratio of HTMA to initiator on the surface of the beads. When using the grafted beads as a stationary phase in hydrophilic interaction chromatography (HILIC), good resolution for nucleobases/nucleosides was obtained with acetonitrile aqueous solution as an eluent; while for phenolic acids and glycosides, they could be eluted and separated in the presence of TFA. The retention time of the solutes increased with the amount of the grafted HTMA. The retention mechanisms of solutes were investigated by the effects of mobile phase composition and buffer pH on the retention of solutes. The results illustrated that the retention behaviors of the tested solutes were dominated by hydrogen bonding interaction and electrostatic interaction. From the chemical structure of the ligands, the modified beads could not only be used as a stationary phase in HILIC, but also act as a useful building block to develop new stationary phases for other chromatographic modes such as affinity media.     

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.     

Investigating the Effect of Chromatographic Conditions on Retention of Organic Acids in Hydrophilic Interaction Chromatography Using a Design of Experiment

Small organic acids have shown significant retention on various stationary phases, such as amide, amino, aspartamide, silica and sulfobetaine phase commonly used in hydrophilic interaction chromatography (HILIC). This study investigated the effect of chromatographic conditions on the retention behavior of organic acids in HILIC using the tool of design of experiment (DOE). The results of the DOE study indicated that both the content of organic solvent (i.e., acetonitrile) and salt concentration in the mobile phase had significant effects on the retention of organic acids. Higher content of organic solvent in the mobile phase led to a significant increase in retention on all types of stationary phases. Increasing salt concentration also resulted in a moderate increase in retention; however, the effect of salt concentration varied with the type of stationary phase. The study also revealed that column temperature had less impact on retention than organic solvent content and salt concentration in HILIC.     

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.     

Controllable preparation of a hydrophilic/ion‐exchange mixed‐mode stationary phase by surface‐initiated atom transfer radical polymerization using a mixture of two functional monomers

Mixed‐mode chromatographic stationary phases require functionalization with at least two functional groups to yield multiple interactions with analytes. Departing from reported methods, a mixture of two different monomers, glycidyl methacrylate and 2‐dimethylaminoethylmethacrylate, was grafted onto the surface of silica by a one‐step surface‐initiated atom transfer radical polymerization to prepare a novel hydrophilic interaction/anion‐exchange mixed‐mode chromatographic stationary phase. The grafted amounts of functional groups were controlled via varying the ratio of monomers in the polymerization system. The influences of water content, salt concentration and pH in the mobile phase were investigated to illustrate the mixed interaction between the stationary phase and analytes. The retention of various solutes on three columns, especially acidic and basic solutes, showed an obvious dependence on the ratio of the two monomers in the polymerization system. The results indicated that the strategy proposed in this work was beneficial to develop various types of mixed‐mode chromatographic stationary phases with adjustable selectivity to meet the needs of complex samples. Finally, the column was successfully employed in the isolation of melamine in liquid milk.     

Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique

Hydrophilic interaction liquid chromatography (HILIC) provides an alternative approach to effectively separate small polar compounds on polar stationary phases. The purpose of this work was to review the options for the characterization of HILIC stationary phases and their applications for separations of polar compounds in complex matrices. The characteristics of the hydrophilic stationary phase may affect and in some cases limit the choices of mobile phase composition, ion strength or buffer pH value available, since mechanisms other than hydrophilic partitioning could potentially occur. Enhancing our understanding of retention behavior in HILIC increases the scope of possible applications of liquid chromatography. One interesting option may also be to use HILIC in orthogonal and/or two-dimensional separations. Bioapplications of HILIC systems are also presented.     

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。