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。     

羧基甜菜碱型亲水作用色谱柱的制备及性能研究

以甲基丙烯酰氧乙基二甲基乙酸铵(CBMA)为功能单体,利用表面引发原子转移自由基聚合(Surface-initiated atom transfer radical polymerization, SI-ATRP)技术,将CBMA接枝到硅胶表面,得到接枝聚合物CBMA的亲水作用色谱固定相(Silica-CBMA).通过改变SI-ATRP反应体系中单体的浓度,制备了3种不同接枝量的亲水作用色谱固定相.考察了Silica-CBMA固定相对有机酸类化合物的分离性能以及流动相中pH值、盐浓度、水含量等因素对溶质保留行为的影响.结果表明,在亲水作用色谱模式下,Silica-CBMA固定相对有机酸类化合物的分离是离子交换作用与亲水作用的混合色谱模式.流动相中盐浓度增大,溶质保留减弱,符合离子交换作用特征;固定相和溶质的离子化程度受流动相pH值影响较大,pH值增大,溶质保留增强;而溶质的保留时间随流动相水含量增加而降低则是典型的亲水作用色谱特征.使用自制Silica-CBMA柱,建立了芦丁片中维生素C、芦丁含量的亲水作用色谱测定方法,操作方法简单,为强极性样品的分离测定提供了新方法.     

糖类化合物亲水作用色谱保留行为评价

以糖类化合物为研究对象,系统评价了其在亲水模式下的色谱保留行为。分别考察了流动相、固定相和缓冲盐等对糖类化合物保留的影响,建立了糖类化合物在亲水模式下的保留方程。结果表明,糖类化合物随着流动相中乙腈比例的降低,保留时间减小;随着缓冲盐浓度的增加,保留时间增加;同时,糖类化合物的保留行为还会受到有机溶剂种类和固定相类型的影响;其保留行为可使用顶替吸附-液相相互作用模型定量描述。将该模型进一步用于实际样品中糖类化合物保留行为的预测,获得了较好的实验结果,预测保留时间与实测保留时间的相对误差小于0.3%。对糖类化合物亲水模式下的保留行为进行了系统的评价和定量描述,该研究结果将有助于糖类化合物亲水作用色谱分离方法的发展。     

用氨基色谱柱的亲水作用色谱法分析碘离子、碘酸根、硝酸根和亚硝酸根

本文发展了用氨基色谱柱的亲水作用色谱-紫外检测法测定I~-、IO~-_3、NO~-_3和NO~-_2的方法。研究了流动相中盐的种类和浓度、有机溶剂种类和浓度等因素对分离检测4种目标阴离子的影响,探讨了NO~-_2保留规律。采用Agilent Zorbax NH_2色谱柱,流动相为乙腈-NaCl溶液,实现4种目标阴离子的分离检测。I~-、IO~-_3、NO~-_3和NO~-_2的检出限分别为1.7、16.9、1.9和2.9μmol/L。方法的线性和重现性良好,应用于聚维酮碘药品和饮水机中烧开的饮用水的测定,加标回收率在91%～105%之间,相对标准偏差小于3.5%,符合定量分析要求。     

某些酚类的高温液相色谱行为

建立了高温液相色谱系统,在高温条件下,采用甲醇-水作为流动相,在Polymerx RP-1聚合物(PSDVB)色谱柱上考察了6种酚类样品的色谱行为.实验条件:温度40～160 ℃,流速0.2～5.5 mL/min,流动相中甲醇浓度范围40%～80%.考察了温度、流速和流动相组成对酚类样品的保留、分辨、柱效和系统压力的影响,探讨了酚类样品在聚合物柱上的热力学行为.温度升高2.35℃大约相当于流动相中甲醇浓度增加1%,可以通过改变色谱柱温度调节样品保留和改变选择性.柱温升高,降低了流动相的粘度,允许在高温条件下使用较高的流速实现快速分离.在160℃、V(甲醇):V(水)=40:60,为流动相和3 mL/min流速条件下,可于2.5 min内实现6种酚类的完全分离.     

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.     

反相液相色谱中同系物收敛的热力学表征

以液相色谱中的溶质计量置换保留模型(SDM-R)为理论基础,从热力学角度进一步对反相液相色谱中同系物的收敛性进行了研究。建立了溶质平均收敛点坐标的计算方程,并从自由能变的角度表征了收敛点坐标的物理意义,阐明了收敛点的纵坐标相等的原因是溶质在收敛点处的自由能变为零。浓度收敛点的横坐标是1mol纯溶剂的解吸附自由能;而碳数收敛点的横坐标为当流动相中有机溶剂的浓度为纯有机溶剂浓度的十分之一时的同系物端基的保留自由能的负值。并以实验数据对该方程进行了验证,两者符合程度甚佳。     

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。     

反相/亲水/阳离子交换混合模式色谱固定相的制备及在药物分离中的应用

通过巯基-烯点击化学方法,合成了一种新型双配体苯磺酸混合模式固定相(Ph/BS),通过元素分析和13C固体核磁共振进行表征。根据溶质结构和流动相组成不同,该混合模式固定相可提供反相(RP)、亲水(HILIC)和强阳离子交换(SCX)多种作用模式。反相条件下分离苯同系物,体现了较好的亚甲基选择性。使用小分子核苷和碱基对亲水色谱行为进行了评价;溶质的保留因子随流动相中水相含量的变化呈现典型的U型曲线,说明Ph/BS固定相具有RP/HILIC双重保留机理。胺类化合物在该固定相上的保留体现了RP/SCX混合色谱的特征。另外,应用Ph/BS固定相在SCX模式下高效分离了二甲双胍及其杂质,在RP/SCX模式下同时分离了酸性、中性和碱性药物。结果显示,该混合模式固定相在复杂样品的分离分析方面具有潜在的优势。     

表阿霉素及其相关物质在反相离子对色谱中的色谱行为

以表阿霉素及其6种相关物质为研究对象,系统评价了其在反相离子对色谱模式下的色谱行为.分别考察了流动相中有机相种类、有机相比例、水相中离子对试剂浓度、pH值对表阿霉素及其相关物质的影响.结果表明,使用乙腈作为有机相洗脱能力及分离效果优于甲醇,保留时间随乙腈比例增大而减小;随着离子对试剂十二烷基硫酸钠浓度增加,杂质阿霉素酮及柔红霉酮几乎无影响,其他5种物质保留时间增加.同时,表阿霉素及其杂质的保留行为受流动相pH值影响较大,当pH不高于4时可获得较好的分离效果.通过对表阿霉素及其相关物质反相离子对模式下的保留行为进行了系统的评价和定量描述,研究结果将有助于该类化合物液相色谱分离方法的发展.     

Hydrophilic interaction liquid chromatography for the separation of acidic agricultural compounds

Organic acids with very low pK_a require extremely low pH conditions to achieve adequate retention in reversed‐phase liquid chromatography, but an extremely low pH mobile phase can cause instrument reliability problems and limit the choice of columns. Hydrophilic interaction chromatography is a potential alternative to reversed‐phase liquid chromatography for the separation of organic acids using more moderate conditions. However, the hydrophilic interaction chromatography separation mechanism is known to be very complex and involves multiple competing mechanisms. In the present study, a hydrophilic interaction chromatography column packed with bare silica core–shell particles was used as the separation column and six agricultural organic acids were used as model analytes to evaluate the effects of buffer concentration, buffer pH, and temperature on sample loading capacity, selectivity, retention, and repeatability. It was found that using a higher concentration of buffer can lead to a significant improvement in the overall performance and reproducibility of the separation. Investigation of column equilibration time revealed that a very long equilibration time is needed when changing mobile phase conditions in between runs. This limitation needs to be acknowledged in hydrophilic interaction chromatography method development and sufficient equilibration time needs to be allowed in method scouting.     

Retention of opioids and their glucuronides on a combined zwitterion and hydrophilic interaction stationary phase

A stationary phase combining zwitterionic ion chromatography and hydrophilic interaction chromatography (ZIC-HILIC) from SeQuant was evaluated for the chromatography of some opiates and their polar metabolites. The effects of mobile phase constitution on retention and resolution were extensively evaluated. Different aspects of mobile phase constitution such as ion strength and type of buffer, type and amount of organic modifier and pH were examined. The selectivity and retention of the opiates compared to their glucuronides could be substantially altered with small changes of the mobile phase, especially when the type of buffer, i.e., formate or acetate and organic modifier, i.e., acetonitrile or methanol were changed. The retention on the ZIC-HILIC was dominated by hydrophilic interaction chromatography (HILIC) but considerable effects on the selectivity was observed, possibly caused by an ion exchange mechanism due to interactions with the charges on the stationary phase.     

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.     

Imidazoline type stationary phase for hydrophilic interaction chromatography and reversed-phase liquid chromatography

An imidazoline was prepared by solvent-free microwave-assisted organic synthesis and immobilized on porous silica particles by polymerization. The resulting material was composed of both hydrophobic alkyl ester chains and hydrophilic imidazoline rings, which gave it both hydrophilic interaction and reversed-phase characteristics. The titration curve suggests that the new material has buffering capacity and acquires increasing positive charge over the pH range 9-4, and is "zwitterionic" in the upper part of this pH range. Through investigating the effect of column temperature, the water content, pH and ion strength of mobile phase on the retention time of polar compounds in highly organic eluents, it was found that the new material could be used as a hydrophilic interaction liquid chromatography (HILIC) stationary phase which involved a complex retention process consisting of partitioning, surface adsorption and electrostatic interactions. In addition, the retention behavior of aromatic compounds in different mobile phase conditions was also studied, which showed the new material mainly exhibited a partitioning mechanism in the reversed-phase liquid chromatography (RPLC) mode. The separation of six water-soluble vitamins and five aromatic compounds were achieved by using the new material in the HILIC and RPLC modes, respectively.     

Preparation and application of novel zwitterionic monolithic column for hydrophilic interaction chromatography

A novel zwitterionic hydrophilic porous monolithic stationary phase was prepared based on the thermal‐initiated copolymerization of N,N‐dimethyl‐N‐(3‐methacryl‐amidopropyl)‐N‐(3‐(sulfopropyl)ammonium betaine and ethylene glycol dimethacrylate. A typical hydrophilic separation mechanism was observed at a highly organic mobile phase (ACN >60%) on this optimized zwitterionic hydrophilic interaction chromatography (HILIC) monolithic stationary phase. Good permeability, stability, and column efficiency were observed on the final monolithic column. Additionally, a weak electrostatic interaction for charged analytes was confirmed in analysis of six benzoic acids by studying the influence of mobile phase pH and salt concentration on their retention behaviors on the obtained zwitterionic HILIC monolithic column. The optimized zwitterionic HILIC monolith exhibited good selectivity for a range of polar test analytes.     

Evaluation of a silicon oxynitride hydrophilic interaction liquid chromatography column in saccharide and glycoside separations

The retention characteristics of a silicon oxynitride stationary phase for carbohydrate separation were studied in hydrophilic interaction chromatography mode. Four saccharides including mono‐, di‐, and trisaccharides were employed to investigate the effects of water content and buffer concentration in the mobile phase on hydrophilic interaction liquid chromatography retention. For the tested saccharides, the silicon oxynitride column demonstrated excellent performance in terms of separation efficiency, hydrophilicity, and interesting separation selectivity for carbohydrates compared to the bare silica stationary phase. Finally, the silicon oxynitride hydrophilic interaction liquid chromatography column was employed in the separation of complex samples of fructooligosaccharides, saponins, and steviol glycoside from natural products. The resulting chromatograms demonstrated good separation efficiency and longer retention compared with silica, which further confirmed the advantages and potential application of silicon oxynitride stationary phase for hydrophilic interaction liquid chromatography separation.     

Mobile phase effects on the retention on polar columns with special attention to the dual hydrophilic interaction–reversed‐phase liquid chromatography mechanism,a review

Hydrophilic interaction liquid chromatography on polar columns in aqueous–organic mobile phases has become increasingly popular for the separation of many biologically important compounds in chemical, environmental, food, toxicological, and other samples. In spite of many new applications appearing in literature, the retention mechanism is still controversial. This review addresses recent progress in understanding of the retention models in hydrophilic interaction liquid chromatography. The main attention is focused on the role of water, both adsorbed by the column and contained in the bulk mobile phase. Further, the theoretical retention models in the isocratic and gradient elution modes are discussed. The dual hydrophilic interaction liquid chromatography reversed‐phase retention mechanism on polar columns is treated in detail, especially with respect to the practical use in one‐ and two‐dimensional liquid chromatography separations.