混合模式毛细管液相色谱柱的制备及其性能研究

发布时间：2018-05-03 17:29

本文选题：刚果红 + 牛磺酸　；参考：《华东师范大学》2017年博士论文

【摘要】：随着色谱学科的发展,色谱工作者更加注重于复杂样品的分离及分析。这就要求同一根色谱柱能同时分离极性和非极性混合物,离子形式的和中性的混合物或有机和无机混合物。通常,针对于复杂混合物的分离,高效液相色谱法中的梯度分析方法常常被人们首先使用。然而,一旦完成单次样品分析,系统必须返回到初始状态,这大大降低了多试样分析的吞吐量。混合模式色谱方法是基于改变流动相的选择而开发的一种色谱方法,它至少同时存在两种相互作用的机制,能对极性和非极性分子具有很强的保留。相比于反相或离子交换方法,该色谱方法由于使用了一个具有多个相互作用机制固定相,使其具有更大的应用范围。混合色谱法的一个主要优势是同一色谱柱可以使用一个单一的模式(如离子交换或反相)下使用或在组合的模式下使用。多维分离作为混合色谱方法中的一种,它是通过耦合两个或两个以上的具有相同或不同的分离机制类似柱或独立柱,使特殊的分析峰从第一个维度转移到第二个维度或更多维度。相比于简单的一维分离,这种简单的方法提供了更高的分辨率和灵敏度以及足够的吞吐量和再现性。因此,本论文主要采用化学改性或是物理封装技术制备了一系列混合色谱柱并对其色谱性能进行研究探讨。具体研究内容如下:第一章绪论首先介绍了毛细管液相色谱(CLC)的基本概念包括CLC的发展史、色谱柱的类型以及常见色谱固定相的应用领域;其次对亲水作用色谱(HILIC)进行了介绍;再次对混合模式色谱(MMC)固定相及其应用进行综述。最后提出本论文的研究目的和意义。第二章是刚果红衍生硅胶(Sil-CR)固定相的制备及其在毛细管液相色谱中的应用。刚果红(CR)作为磺酸偶氮苯酸碱指示剂,当环境pH值为3.0-5.2时,两个偶氮基团发生质子化,表现出两性离子特征,染料颜色变化从蓝色到红色。离子化的磺酸基团、质子化的偶氮基团和pi-pi-stacking行为使CR固定相具有多重作用机制。因此,我们将CR衍生硅胶用作固定相填充制备了 Sil-CR毛细管MMC柱,探讨其在不同模式下的分离机制。实验结果表明,CR毛细管MMC柱在反相液相色谱(RPLC),HILIC和富水色谱(PALC)三种模式下均能使用的多模色谱分离能力。苯甲酸衍生物和磺胺类药物的有效分离揭示出CR衍生硅胶具有混合模式保留行为。与ODS单模保留行为不同,该色谱柱除了亲疏水保留机理外还有离子交换作用机理。另外,醇羟基及残氨基的存在使得该色谱柱具有多个作用位点,能增强与分析物间相互作用,扩大了被分析物的测试范围。同时该材料制备的色谱柱对被分析物有很好保留,而且作为色谱柱具有良好重现性和稳定性。由于多个功能基团的存在,可预见的是这种材料不仅可以用作通用的色谱固定相而且在其他化学领域可作为一个固体萃取吸附剂。第三章是刚果红衍生硅胶与十八烷基硅胶混合模式色谱柱的制备和性能评价。十八烷基硅胶(ODS)作为经典的反相色谱材料针对于离非极性化合物和弱极性化合物具有优异的分离能力。因此,为了提升CR柱在非极性化合物的分离能力,本章分别通过化学方法和物理方法制备了四种经ODS改性的CR柱,它们分别是 Sil-CR-con-ODS 接枝柱、Sil-CR-bis-ODS 键合柱、Sil-CR-mix-ODS 混合柱和Sil-CR-tan-ODS两相柱。通过对苯系物的分离分析得到Sil-CR-tan-ODS两相柱不但在制备上能精确可控其固定相组成比例还具有与一次填充色谱柱柱效相当。通过对异构体,极性与非极性化合物组成的混合物的分离分析得出Sil-CR-tan-ODS两相柱的制备是非常成功的。第四章是牛磺酸衍生硅胶与十八烷基硅胶混合模式色谱柱的制备与性能评价。牛磺酸衍生硅胶(TDS)固定相用于HILIC分离各种极性和弱极性化合物表现出极强的保留。结合ODS和牛磺酸的两大优点,很容易实现分离极性和弱极性化合物的混合物。相比单一的固定相柱,该柱可以扩大范围的样本选择和减少样品预处理。更重要的是,多个作用机制能大大延长被分析物的保留时间,提高对样品的选择性和分离度。与一般的二维系统相比,这种毛细管连续装填色谱操作简单、稳定,具有较好的适用性。因此,在本章中,我们制备了一种TDS-ODS MMC两相柱,苯同系物、多环芳烃、核苷酸和生物胺分别在该色谱柱上进行分离测试。实验结果表明,该两相柱结合ODS和TDS的各自优势,不仅展示了对核苷酸具有良好的选择性,也展示对高疏水性和高亲水混合物的同时保留特性。该系统结合了多维分离的诸多优势和固定相组成的灵活调配优势,能更好增强对被分析物的选择性,适合于复杂样品的分析。第五章是氨丙基衍生硅胶和牛磺酸衍生硅胶混合模式色谱柱的制备和性能评价。氨丙基衍生硅胶(APS)被用于HILIC,特别是针对于碳水化合物的分离起到优越的分离性能,它对于分析芳香族化合物、有机酸及无机阴离子分离也具有较好选择效果。然而,它对于某些化合物特别是阳离子化合物则表现不佳或不具有选择保留特性。TDS为强阳离子交换材料在HILIC模式对核苷核碱以及生物胺表现出良好的分离特性。由于它们都能被运用于HILIC,故而选用TDS作为APS改性材料来弥补其在分离阳离子化合物上的不足。在本章中,我们制备了一种APS-TDSMMC两相柱,经其对生物胺类物质,有机酸类物质,核苷核碱和磺胺药物的分离试验,我们发现TDS对APS的改性是非常成功的。APS-TDS柱不仅兼具有单一 APS柱和单一 TDS柱的全部功能,同时两种固定相的共同作用使得分析物进行了一次等度的2D-LC(HILIC×HILIC)分离,其直接的结果是出现了超过于单一APS柱或单一TDS柱的分离性能。满意的结果表明,该连续填充柱能简单且完美实现APS和TDS的相互改性,这为任意多个色谱固定相的物理改性提供了一种依据和方法。第六章是磺基甜菜碱衍生硅胶和十八烷基硅胶混合模式色谱柱的制备及性能评价。磺基甜菜碱两性离子固定相(ZIC)是一种含有APS与TDS功能基团的化学改性衍生材料。它不但可以分离反相中保留很弱,或是根本没有保留的化合物甚至是高极性的水溶性化合物,而且由于存在阴阳离子基团,它能随着pH的改变而改变自身的电荷类型达到阴阳离子的交换。但是对于未能电离的化合物和LogP值较大的物质则出现弱的选择性保留或是几乎无选择性保留。然而,该类化合物在反相(RP)柱上却是极其容易被保留并被选择性分离。一旦能够将两者结合在一起,发挥各自的性能,那么这将大大扩大了分析样品的适用范围。因此,在本章中我们采用了商品化的ZIC制备了一种ZIC-ODS MMC两相柱,该色谱柱对由核苷、核碱和苯同系物组成的跨极性混合物具有同时保留分离的能力。此外,对于具有极性相近的磺胺类药物,ODS所具有的RP机制弥补了 ZIC分离上的不足。在对位置异构体的分离上,由于RP机制的引入,使得ZIC-ODS具有了很好的空间识别能力。
[Abstract]:With the development of the chromatography discipline, chromatograph workers pay more attention to the separation and analysis of complex samples. This requires the simultaneous separation of polar and nonpolar mixtures, ionic and neutral mixtures or organic and inorganic mixtures on the same chromatographic column. Generally, the ladder in the separation of complex mixtures, the ladder in high performance liquid chromatography The degree analysis method is often used first. However, once the single sample analysis is completed, the system must return to the initial state, which greatly reduces the throughput of the multi sample analysis. The mixed mode chromatography is a chromatographic method based on the choice of changing the mobile phase. At least two mechanisms of interaction exist at the same time, One of the main advantages of the mixed chromatography method is that the same chromatographic column can use a single mode (such as ion exchange). Use or use under a combined mode. Multidimensional separation is one of the mixed chromatography methods, which transfers special analysis peaks from the first dimension to second dimensions or more dimensions by coupling two or more of the same or different separation mechanisms that have the same or different separation mechanisms to second dimensions or more dimensions. One dimensional separation, this simple method provides higher resolution and sensitivity as well as sufficient throughput and reproducibility. Therefore, a series of mixed chromatographic columns are prepared by chemical modification or physical encapsulation technology. The content of the study is as follows: first introduction is introduced. The basic concepts of capillary liquid chromatography (CLC) include the history of the development of CLC, the type of chromatographic column and the application of common chromatographic stationary phase. Secondly, the hydrophilic interaction chromatography (HILIC) is introduced, and the fixed phase of the mixed mode chromatography (MMC) and its application are reviewed. Finally, the purpose and significance of this paper are put forward. Second chapters It is the preparation of Congo red derived silica gel (Sil-CR) fixed phase and its application in capillary liquid chromatography. Congo red (CR) is a sulfonated azobenzene acid base indicator. When the environmental pH value is 3.0-5.2, the two azo groups are protonated, showing the characteristics of the amphoteric ions, the color changes of the dye from blue to red. The sulfonated sulfonic group and the mass of the ions. The azo group and the pi-pi-stacking behavior of the azo groups make the CR stationary phase multiple mechanism. Therefore, we use CR derived silica gel as a stationary phase to prepare the Sil-CR capillary MMC column and discuss the separation mechanism under different modes. The experimental results show that the CR capillary MMC column is in the reverse phase liquid chromatography (RPLC), HILIC and water rich chromatography (PALC). The effective separation of multimode chromatography that can be used in the three modes. The effective separation of benzoic acid derivatives and sulfonamides reveals that the CR derived silica gel has a mixed mode retention behavior. Unlike the ODS single mode retention behavior, the chromatographic column has the mechanism of ion exchange in addition to the hydrophobic retention mechanism. At the same time, the chromatographic column can enhance the interaction between the analyte and expand the test range of the analyte. The chromatographic column prepared by this material is well preserved for the analyte and has good reproducibility and stability as a chromatographic column. The material can not only be used as a general chromatographic stationary phase but also as a solid extractive adsorbent in other chemical fields. The third chapter is the preparation and performance evaluation of the mixed mode chromatography column of Congo red derived silica gel and eighteen alkyl silica gel. The eighteen alkyl silica gel (ODS) is used as a classic anti phase chromatography material for the separation of non polar compounds and The weak polar compounds have excellent separation ability. Therefore, in order to improve the separation ability of CR columns in non polar compounds, four kinds of ODS modified CR columns were prepared by chemical and physical methods, respectively, Sil-CR-con-ODS grafting column, Sil-CR-bis-ODS bonding column, Sil-CR-mix-ODS column and Sil-CR-tan-ODS two respectively. Phase column. Through the separation and analysis of the benzene series, it is found that the Sil-CR-tan-ODS two phase column is not only able to accurately control the composition of its stationary phase, but also is equivalent to the column effect of a packed column. The preparation of the Sil-CR-tan-ODS two phase column is obtained by the separation and analysis of the mixture of isomers, polar and non polar compounds. Successful. The fourth chapter is the preparation and performance evaluation of the mixed mode chromatography column of taurine derived silica gel and eighteen alkyl silica gel. The immobilized phase of taurine derived silica gel (TDS) is used in HILIC separation of various polar and weak polar compounds with very strong retention. It is easy to achieve separation polarity and weak polarity by combining the two advantages of ODS and taurine. A mixture of compounds. Compared to a single stationary phase column, the column can expand the range of sample selection and reduce sample pretreatment. More importantly, multiple mechanisms can greatly prolong the retention time of the analyte, improve the selectivity and separation of the sample. Compared with the general two-dimensional system, this capillary continuous loading chromatography operation Therefore, in this chapter, we have prepared a TDS-ODS MMC two-phase column, benzene homologue, polycyclic aromatic hydrocarbons, nucleotides and biogenic amines separately on the column. The experimental results show that the two phase column combines the advantages of ODS and TDS, which not only shows the good selection of nucleotides. The fifth chapter is a mixture of ammonia and taurine derived silica gel and taurine derived silica gel. The system combines the advantages of multidimensional separation and the flexibility of the fixed phase, which can better enhance the selectivity of the analyte and be suitable for the analysis of complex samples. Preparation and performance evaluation of chromatographic columns. Ammonia propyl derived silica gel (APS) has been used in HILIC, especially for separation of carbohydrates. It has a better choice for the analysis of aromatic compounds, organic acids and inorganic anions. .TDS is a strong cationic exchange material that shows good separation characteristics for nucleoside nucleobase and biogenic amines in HILIC mode. Because they can all be used in HILIC, TDS is used as a APS modified material to make up for its deficiency in separation of cationic compounds. In this chapter, we A APS-TDSMMC two phase column was prepared by the separation test of biogenic amines, organic acids, nucleoside nucleobases and sulfonamides. We found that the modification of TDS to APS is a very successful.APS-TDS column not only with a single APS column and a single TDS column, but also the combined action of two stationary phases to make the analyte in the same time. The separation of an equal degree of 2D-LC (HILIC x HILIC) has been performed. The direct result is that the separation performance exceeds the single APS column or single TDS column. The satisfactory results show that the continuous filling column can easily and perfectly realize the mutual modification of APS and TDS, which provides a basis and method for the physical modification of any fixed phase fixed phase. The sixth chapter is the preparation and performance evaluation of the mixed mode chromatographic column with sulfonated betaine derived silica gel and eighteen alkyl silica gel. The sulfonated betaine amphoteric ion fixed phase (ZIC) is a chemically modified derivative containing APS and TDS functional groups. It can not only separate the compounds that are very weak in the reverse phase or are not retained at all or even the compounds that are not retained. The high polarity water soluble compound, and because of the existence of the anion group, it can change its charge type to exchange of yin and Yang with the change of pH, but there is a weak selective retention or few selective retention for the compounds which are not ionized and the material with larger LogP value. However, this kind of compound is in the opposite direction. The phase (RP) column is extremely easy to be retained and selectively separated. Once the two can be combined to perform their respective performance, this will greatly enlarge the scope of application of the analytical samples. Therefore, in this chapter, we use a commercialized ZIC to prepare a ZIC-ODS MMC two-phase column, which is composed of nucleosides, nuclear bases and benzene. The cross polar mixture composed of homologues has the ability to retain the separation at the same time. In addition, for the sulfonamides with very similar sulfonamides, the RP mechanism of ODS has made up for the shortage of ZIC separation. In the separation of the location isomers, the ZIC-ODS has a good spatial recognition ability due to the introduction of the RP mechanism.

【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.72

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