加压毛细管电色谱—微流蒸发光散射检测联用系统研究及其在食品安全检测中的应用研究
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摘要
加压毛细管电色谱(Pressurized Capillary Electrochromatography, pCEC)是近年来新兴的一种电动毛细管微分离技术。它基于毛细管电色谱的理论和技术发展,具备毛细管区带电泳和高效液相色谱双重分离特征,是一种组合式创新的分离技术。与传统电泳技术相比,pCEC可同时分离带电物质和中性物质,具备更加优异的选择性和分辨率,更好的重复性和准确度；与传统高效液相色谱技术相比,pCEC具备更高的柱效率和分离速度；而与传统毛细管电色谱(capillary electrochromatography, CEC)相比,pCEC由于采用压力驱动,改善了由于焦耳热效应所带来的气泡和柱子烧干问题,而且实现了CEC的梯度洗脱,从而有能力解决更加复杂体系的样品分离问题。且因为pCEC本身的微流特点,大大节省了分析成本,同时对环境保护起到积极作用。作为近十几年来色谱技术的重大突破之一,pCEC适应现代分析仪器微型化的发展趋势,已成功应用于生命科学、中医药研究、药物分析、环境监测以及食品安全等热点研究领域,受到广泛的关注。
蒸发光散射检测器(Evaporative light-scattering detector, ELSD),是一种自上世纪九十年代开始得到广泛应用的“通用型”和“质量型”检测器。相比目前液相色谱技术中使用最多的紫外检测器,ELSD不受物质本身分子结构的限制,不依赖物质本身的光学特性,对大多数物质的响应因子保持一致,只要物质的挥发性低于流动相溶剂的挥发性,ELSD就可用于绝大多数不挥发和半挥发性化合物的分析检测。近年来,随着ELSD的普及化程度不断提高,其应用领域也日益广泛,尤其在天然产物,中草药,脂类,表面活性剂及碳水化合物等物质的分析检测上表现出巨大的优势。
本论文的立意基于将ELSD"通用性”特征,与pCEC分离技术的高效、高选择性和高分辨度以及快速分离等优势相结合的目的,致力于ELSD的微型化研究,实现加压毛细管电色谱—微流蒸发光散射检测器的联用(pCEC-μELSD)以及应用技术开发。主要研究内容包括：通过对微流雾化、微型蒸发化及微型光散射检测等关键部件的设计与开发,实现ELSD的整体微型化,研制成功的μELSD适用于加压毛细管电色谱等毛细管微分离技术的联用检测；通过对ELSD的雾化,蒸发和光散射检测主要仪器部件的参数优化,并进行pCEC-μELSD接口技术研究,实现pCEC-μELSD的联用；对照常规HPLC-ELSD分离技术,基于pCEC-μELSD联用平台,建立合适的分析方法并通过方法学验证等手段,对pCEC-μELSD联用平台的适用性和稳定性做出初步评价；建立了食品中人工合成和天然甜味剂的pCEC-μELSD分离检测方法,验证了仪器的实用性和先进性。pCEC-μELSD联用平台的研制将为现代各前沿研究领域对分析复杂物质的需求提供了一种高效且经济的新技术。本论文主体部分共六章：
第一章系统综述了加压毛细管电色谱的发展历史,基本原理,联用检测技术,毛细管柱技术以及应用领域的情况；同样详细介绍了蒸发光散射检测器的发展史,基本原理,应用领域以及微型化研究现状；阐述了二者的特点和联用的可行性,并提出了本论文的研究目的和意义。
第二章基于蒸发光散射检测器的整机微型化目的,以及对ELSD检测机理的认识和研究,分别对ELSD的雾化,蒸发和光散射检测三大主要关键部件进行微型化结构设计,最终确定了由雾化喷嘴和雾化毛细管组成的微流雾化器结构；圆柱锥形管,表面分布连接加热电阻,外置保温套筒的微型蒸发管结构；用于连接蒸发管和光散射池,外侧面具备辅助载气进口的鞘流套结构；由激光作为光源,光电倍增管作为信号采集装置,并带有光阱结构设计的微型光散射检测池结构。而为有效控制各部件正常工作以及参数调整,配套设计的电路系统可对蒸发温度,载气流速进行实时设置与监控,可对检测信号数值,系统气压进行实时监控,并专门为采集的检测信号设计了放大电路,为后续硬件结构改进,参数优化提供了调整空间和手段。
第三章基于第二章ELSD的硬件结构设计,分别对μELSD的雾化,蒸发和光散射检测部件进行了参数优化。实验中研究了雾化喷嘴以及与之匹配的雾化毛细管的尺寸对雾化颗粒大小和均匀度的影响,确定了雾化喷嘴和雾化毛细管匹配的最佳尺寸为410μm和20μm I.D.×360μm O.D.;考察了雾化毛细管的位置对信噪比和重复性的影响,确定了雾化毛细管相对雾化喷嘴的位置状态；考察了不同长度、内径和形状的蒸发管,发现当锥形蒸发管的尺寸出口内径为1.0mm,入口内径为7.5mm,长10cm时,可获得最佳信噪比结果；实验中还制作了3种不同规格的光散射池进行考察,最终选择最大体积的光散射池(65mm i.d.×60mm高)以提高信噪比。通过上述优化实验,对μELSD整机性能进行了初步测试：其LOD是0.1ng(S/N=3,葡萄糖直接进样),基线噪声为8μV,基线漂移为0.182mV/h,峰面积重复性RSD6为0.4%,峰高重复性RSD6为0.3%。基于本文研制的μELSD,通过电隔离接口与零死体积三通两种接口技术,实现了pCEC-μELSD的联用。
第四章基于第三章pCEC-μELSD的整体优化和调试结果,为了验证pCEC-pELSD联用平台用于实际样品分析的适用性和准确性,对pCEC-μELSD的性能进行系统评价,本章实验建立了食品中果糖,葡萄糖和蔗糖的亲水作用色谱的pCEC-μELSD分析方法,并进行了方法学验证和准确度测试。实验结果表明,pCEC-μELSD平台上使用TSK Amide-80毛细管色谱柱(150μm I.D.×150mm,5μm),在乙腈：水(80：20,v/v),分离电压+5KV条件下,可实现果糖,葡萄糖和蔗糖的快速分离。其中线性范围为0.01-1.00μg；检出限4ng,回收率在88.1%-103.9%之间；RSD在0.9%-3.3%之间。对比参考文献中,常规HPLC-ELSD类似的实验数据,pCEC-μELSD的线性范围和检测限略好于前者,回收率和RSD数据则相近,且分析速度最快。通过本章实验结果的各项比照,说明本论文研制的pELSD以及搭建的pCEC-μELSD平台具备良好的系统适应性和稳定性,实验数据准确可靠。
第五章基于本论文研究的pCEC-μELSD平台,建立了一种无糖食品中赤藓糖醇、木糖醇、山梨糖醇、麦芽糖醇和乳糖醇的分离检测方法。实验中考察了流动相组成,分离电压对5种糖醇的分离影响,研究了不同酸碱体系下对检测信噪比的影响,并对微流蒸发光散射检测器的蒸发温度,载气流量的参数设置进行了优化。实验结果表明,赤藓糖醇的线性范围在0.01-1.0μg,检测限为4ng；木糖醇的线性范围在0.01-1.0μg,检测限也4ng；山梨糖醇的线性范围在0.015-1.5μg,检测限均为6ng；麦芽糖醇和乳糖醇的信噪比较低,线性范围分别为0.025-2.5μg,0.02-2.0μg,检测限分别为11ng和8ng。3个添加水平下的样品的平均回收率为82.4%-107.2%之间,相对标准偏差RSD6小于3.0%；方法最低检出限(LOD,S/N=3)在4-11ng之间；对比常规HPLC-ELSD分离5种糖醇的结果,分析速度可提高近30%。将该方法应用于两种实际样品：无糖饮料样品(产品标明含有木糖醇为主要甜味添加剂)和木糖醇口香糖样品(产品标明含有木糖醇为主要甜味添加剂,少量山梨糖醇和麦芽糖醇)的测定中,均准确检出了目标样品。该方法选择性好,分离速度快、灵敏度高,重现性好,经济环保,具有较高的实用性,为无糖食品中糖醇的定量检测提供了一种快速且行之有效的方法。
第六章基于本论文研究的pCEC-μELSD平台,针对目前食品中多类甜味剂复配添加的情况,建立了一种食品中人工合成甜昧剂和糖醇类甜味剂的分离检测方法,可同时分离检测安赛蜜,甜蜜素,阿斯巴甜,三氯蔗糖,赤藓糖醇,木糖醇,山梨糖醇,麦芽糖醇和乳糖醇9种常见甜味剂。实验中考察了流动相体系组成,流动相的酸碱pH环境,分离电压,ELSD蒸发温度,载气流速等实验参数并进行了优化。实验结果表明：9种甜味剂的线性范围为7ng..4.2μg(相关性系数大于0.992)之间；3个添加水平下的样品的平均回收率为85.4%-110.4%之间,相对标准偏差RSD6小于3.6%；方法检测限(LOD, S/N=3)在7ng-42ng之间。将该方法应用于三种实际样品：木糖醇无糖口香糖(柠檬味,产品标明含有木糖醇,麦芽糖醇,安赛蜜,阿斯巴甜),木糖醇无糖口香糖(苹果味,产品标明含有木糖醇,麦芽糖醇,安赛蜜,三氯蔗糖)和无糖薄荷糖(产品标明含有安赛蜜,三氯蔗糖,山梨糖醇)的测定,均准确检出了目标样品。该方法利用pCEC-μELSD的双重分离能力,通过调节电压参数,实现了两大类甜味剂的同时分离检测,有效应对了目前食品添加剂多类添加的复杂情况,数据稳定可靠,灵敏度满足常规要求。
Pressurized capillary electrochromatography (pCEC) based on the theory and technique of capillary electrochromatography (CEC) is emerging as an environmental friendly micro-separation technology in recent years. Compared with capillary zone electrophoresis (CE), where only charged compounds can be separated,bothcharged and neutral analytes can be separated simultaneously in pCEC. Moreover, pCEC can provide higher column efficiency than high performance chromatography (HPLC). Even more importantly, compared with traditional CEC, gradient elution mode can be achieved and some problem associated withJoule heating effect, like bubble formation and dryout of the column can bealsoovercome in pCEC due to the introduction of pump pressure. The appearance of pCEC followed the trend of high performance and miniaturization in the development of modern analytical instrumention. Thus, pCEC have been applied to the analysis of more complicated samples in life science, traditional Chinese medicine research, drug analysis, environment monitoring and food safety, as well as other popular research fields.
Evaporative light-scattering detector (ELSD) has been widely used as a universal detector for HPLC since the1990s. The application of ELSD doesn't depend on the molecular structure and optical properties of analytes. ELSD only requests the evaporitivity of analytes to be lower than that of the mobil phase. Recently, ELSD has displayed the huge advantage for the detection of natural products, Chinese herbal medicines, lipids, surfactants and carbohydrates.
Therefore, the aim of this dissertation focuses on the development of a platform based on pCEC coupledto micro-ELSD (μELSD)and its applications. Some key parts such as evaporation assembly, nebulization assembly, and micro light scattering module were designed. Subsequently, the interface technique of pCEC with μELSD was investigated through optimizing the operation conditions such as evaporation, nebulizer and light scattering parts. The performance of pCEC with μELSD was valuated in detail, and then a method based on pCEC-μELSD was applied successfully for the analysis of synthetic and natural sweeteners in food samples. pCEC-μELSD as an efficient and economical technology hasgreat potential for the analysis of complicated samples in various research fields。The content of this dissertation is as follows:
Chapter1:The research background of pCEC and ELSD including their advances history, basic principle and application is systematically summarized. According to their characteristics, the feasibility of the coupling of two techniques was proposed. The purpose and significanceof this dissertation wereput forward。
Chapter2:In order to investigate the miniaturization of the evaporative light-scattering detector and understand the mechanism of μELSD, the structure of evaporation assembly, nebulizer assembly and light scattering cell were designed in detail in this chapter. The finalized structure of micro-fluid nebulizer was composed of nebulization nozzle and nebulization capillary. The finalized mirco-evaporator was based on cylindrical conical tube which was connected to heating resistor and covered with insulation sleeve. Sheath liquid assembly equipped with supplementary carrier gas was used to connect the evaporative annular tube to light scattering detector cell. The laser and photomultiplier were used, respectively, as the light source and scattering light collector of μELSD. Light trap was also designed in the structure of μELSDin order to avoid the reflected light. The electronic circuit system that designed for μELSD can be used to adjust and monitorvarious parameters of μELSDin real time such as evaporation temperature, flow rate of carrier gas and system carrier gas pressure.
Chapter3:The parameters of evaporation assembly, nebulizer assembly and light scattering cell were optimized in this chapter. The influence of nebulization nozzle and matching nebulization capillary on atomizing effect was investigated. The finalized size of nebulization nozzle and matching nebulization capillary were410μm and20μm I.D.×360μm O.D,respectively. The influenceof the position of capillary on the signal to noise ratio and repeatability was also investigated. It is indicated that the best signal to noise ratio can be abtained under the condition of10cm length,7.5mm inlet diameter and1.0outlet diameter of cylindrical conical tube. Moreover, three different light scattering detector cells were made to evaluate the influence of detector cell size on the signal to noise ratio. Finally, the largestone(65mm i.d.x60mm high)was choosen to increase the signal to noise ratio. Therefore, the detection limit of μELSD was caculated as0.1ng (S/N=3, direct injection with glucose sample),the noise of baseline was0.8μV,the baseline drift was0.182mV/h, the reproducibility of peek aera (RSD6) was0.4%, and the reproducibility of peek height(RSD6) was0.3%. The platform of pCEC coupling toμELSD was built successfully.
Chapter4:Optimization and validation of pCEC with μELSD detector were processed. A method base on pCEC-μELSD was developed for the determination of a mixture of fructose, glucose and sucrose in food. TSK Amide-80capillary column (150mm x200μ m,5μm) was selected. The analytes can be separated within13min under-3kV of operation voltage. The mobile phase was chosen as80%acetonitrile in DI water. The linear range of the method was from0.01to1.0μg for each analytes. The detection limit was4ng. Recoveries within88.1%-103.9%and RSD within0.9%-3.3%(n=6) indicated that adaptability and stability of pCEC with μELSD are comparable with commercial HPLC-ELSD.
Chapter5:Based on the technique of the pCEC-μELSD, a method was established for analysis of a mixture of erythritol, xylitol, sorbitol, maltose and lactitol in the sugar-free food. Some operation conditions of ELSD such as evaporated temperature and flow rate of carrier gas and the chromatographic condition were optimized. The influence of pH value on the signal to noise ratio was also investigated. The linear range of each analyetes was from0.01to2.5μg (correlation coefficient greater than0.998).The average recovery was between82.4%-107.2%under three different level by using standard addition method and the relative standard deviation (RSD) was less than3.9%(n=6). The detection limit (LOD, S/N=3)was between4to11ng. Contrast to conventional method based on HPLC with ELSD, analysis speed was improved obviously. This method then was applied for analyses of two real samples. The proposed method provides a fast and effective method for the quantitative detection of sugar alcohol in sugar-free foods.
Chapter6:Based on the technique of the pCEC-μELSD, ananalytical method was established to detect simultaneously nine common sweetener. The operation conditions of ELSD and the separation condition were both optimized. The linear range of each sweetener was from7ng to4.2μg (correlation coefficient greater than0.998).The average recovery was between85.4%-110.4%under three different level by using standard addition method and the RSDwas less than3.6%(n=6). The detection limit (LOD, S/N=3)was between7ng to42ng. This method was applied for three reall xylitol samples and the targeted analytes were separated and detected accurately. The proposed method provides a sensitive and stable means for the determination of two kinds of sweetener simultaneously.
蒸发光散射检测器(Evaporative light-scattering detector, ELSD),是一种自上世纪九十年代开始得到广泛应用的“通用型”和“质量型”检测器。相比目前液相色谱技术中使用最多的紫外检测器,ELSD不受物质本身分子结构的限制,不依赖物质本身的光学特性,对大多数物质的响应因子保持一致,只要物质的挥发性低于流动相溶剂的挥发性,ELSD就可用于绝大多数不挥发和半挥发性化合物的分析检测。近年来,随着ELSD的普及化程度不断提高,其应用领域也日益广泛,尤其在天然产物,中草药,脂类,表面活性剂及碳水化合物等物质的分析检测上表现出巨大的优势。
本论文的立意基于将ELSD"通用性”特征,与pCEC分离技术的高效、高选择性和高分辨度以及快速分离等优势相结合的目的,致力于ELSD的微型化研究,实现加压毛细管电色谱—微流蒸发光散射检测器的联用(pCEC-μELSD)以及应用技术开发。主要研究内容包括：通过对微流雾化、微型蒸发化及微型光散射检测等关键部件的设计与开发,实现ELSD的整体微型化,研制成功的μELSD适用于加压毛细管电色谱等毛细管微分离技术的联用检测；通过对ELSD的雾化,蒸发和光散射检测主要仪器部件的参数优化,并进行pCEC-μELSD接口技术研究,实现pCEC-μELSD的联用；对照常规HPLC-ELSD分离技术,基于pCEC-μELSD联用平台,建立合适的分析方法并通过方法学验证等手段,对pCEC-μELSD联用平台的适用性和稳定性做出初步评价；建立了食品中人工合成和天然甜味剂的pCEC-μELSD分离检测方法,验证了仪器的实用性和先进性。pCEC-μELSD联用平台的研制将为现代各前沿研究领域对分析复杂物质的需求提供了一种高效且经济的新技术。本论文主体部分共六章：
第一章系统综述了加压毛细管电色谱的发展历史,基本原理,联用检测技术,毛细管柱技术以及应用领域的情况；同样详细介绍了蒸发光散射检测器的发展史,基本原理,应用领域以及微型化研究现状；阐述了二者的特点和联用的可行性,并提出了本论文的研究目的和意义。
第二章基于蒸发光散射检测器的整机微型化目的,以及对ELSD检测机理的认识和研究,分别对ELSD的雾化,蒸发和光散射检测三大主要关键部件进行微型化结构设计,最终确定了由雾化喷嘴和雾化毛细管组成的微流雾化器结构；圆柱锥形管,表面分布连接加热电阻,外置保温套筒的微型蒸发管结构；用于连接蒸发管和光散射池,外侧面具备辅助载气进口的鞘流套结构；由激光作为光源,光电倍增管作为信号采集装置,并带有光阱结构设计的微型光散射检测池结构。而为有效控制各部件正常工作以及参数调整,配套设计的电路系统可对蒸发温度,载气流速进行实时设置与监控,可对检测信号数值,系统气压进行实时监控,并专门为采集的检测信号设计了放大电路,为后续硬件结构改进,参数优化提供了调整空间和手段。
第三章基于第二章ELSD的硬件结构设计,分别对μELSD的雾化,蒸发和光散射检测部件进行了参数优化。实验中研究了雾化喷嘴以及与之匹配的雾化毛细管的尺寸对雾化颗粒大小和均匀度的影响,确定了雾化喷嘴和雾化毛细管匹配的最佳尺寸为410μm和20μm I.D.×360μm O.D.;考察了雾化毛细管的位置对信噪比和重复性的影响,确定了雾化毛细管相对雾化喷嘴的位置状态；考察了不同长度、内径和形状的蒸发管,发现当锥形蒸发管的尺寸出口内径为1.0mm,入口内径为7.5mm,长10cm时,可获得最佳信噪比结果；实验中还制作了3种不同规格的光散射池进行考察,最终选择最大体积的光散射池(65mm i.d.×60mm高)以提高信噪比。通过上述优化实验,对μELSD整机性能进行了初步测试：其LOD是0.1ng(S/N=3,葡萄糖直接进样),基线噪声为8μV,基线漂移为0.182mV/h,峰面积重复性RSD6为0.4%,峰高重复性RSD6为0.3%。基于本文研制的μELSD,通过电隔离接口与零死体积三通两种接口技术,实现了pCEC-μELSD的联用。
第四章基于第三章pCEC-μELSD的整体优化和调试结果,为了验证pCEC-pELSD联用平台用于实际样品分析的适用性和准确性,对pCEC-μELSD的性能进行系统评价,本章实验建立了食品中果糖,葡萄糖和蔗糖的亲水作用色谱的pCEC-μELSD分析方法,并进行了方法学验证和准确度测试。实验结果表明,pCEC-μELSD平台上使用TSK Amide-80毛细管色谱柱(150μm I.D.×150mm,5μm),在乙腈：水(80：20,v/v),分离电压+5KV条件下,可实现果糖,葡萄糖和蔗糖的快速分离。其中线性范围为0.01-1.00μg；检出限4ng,回收率在88.1%-103.9%之间；RSD在0.9%-3.3%之间。对比参考文献中,常规HPLC-ELSD类似的实验数据,pCEC-μELSD的线性范围和检测限略好于前者,回收率和RSD数据则相近,且分析速度最快。通过本章实验结果的各项比照,说明本论文研制的pELSD以及搭建的pCEC-μELSD平台具备良好的系统适应性和稳定性,实验数据准确可靠。
第五章基于本论文研究的pCEC-μELSD平台,建立了一种无糖食品中赤藓糖醇、木糖醇、山梨糖醇、麦芽糖醇和乳糖醇的分离检测方法。实验中考察了流动相组成,分离电压对5种糖醇的分离影响,研究了不同酸碱体系下对检测信噪比的影响,并对微流蒸发光散射检测器的蒸发温度,载气流量的参数设置进行了优化。实验结果表明,赤藓糖醇的线性范围在0.01-1.0μg,检测限为4ng；木糖醇的线性范围在0.01-1.0μg,检测限也4ng；山梨糖醇的线性范围在0.015-1.5μg,检测限均为6ng；麦芽糖醇和乳糖醇的信噪比较低,线性范围分别为0.025-2.5μg,0.02-2.0μg,检测限分别为11ng和8ng。3个添加水平下的样品的平均回收率为82.4%-107.2%之间,相对标准偏差RSD6小于3.0%；方法最低检出限(LOD,S/N=3)在4-11ng之间；对比常规HPLC-ELSD分离5种糖醇的结果,分析速度可提高近30%。将该方法应用于两种实际样品：无糖饮料样品(产品标明含有木糖醇为主要甜味添加剂)和木糖醇口香糖样品(产品标明含有木糖醇为主要甜味添加剂,少量山梨糖醇和麦芽糖醇)的测定中,均准确检出了目标样品。该方法选择性好,分离速度快、灵敏度高,重现性好,经济环保,具有较高的实用性,为无糖食品中糖醇的定量检测提供了一种快速且行之有效的方法。
第六章基于本论文研究的pCEC-μELSD平台,针对目前食品中多类甜味剂复配添加的情况,建立了一种食品中人工合成甜昧剂和糖醇类甜味剂的分离检测方法,可同时分离检测安赛蜜,甜蜜素,阿斯巴甜,三氯蔗糖,赤藓糖醇,木糖醇,山梨糖醇,麦芽糖醇和乳糖醇9种常见甜味剂。实验中考察了流动相体系组成,流动相的酸碱pH环境,分离电压,ELSD蒸发温度,载气流速等实验参数并进行了优化。实验结果表明：9种甜味剂的线性范围为7ng..4.2μg(相关性系数大于0.992)之间；3个添加水平下的样品的平均回收率为85.4%-110.4%之间,相对标准偏差RSD6小于3.6%；方法检测限(LOD, S/N=3)在7ng-42ng之间。将该方法应用于三种实际样品：木糖醇无糖口香糖(柠檬味,产品标明含有木糖醇,麦芽糖醇,安赛蜜,阿斯巴甜),木糖醇无糖口香糖(苹果味,产品标明含有木糖醇,麦芽糖醇,安赛蜜,三氯蔗糖)和无糖薄荷糖(产品标明含有安赛蜜,三氯蔗糖,山梨糖醇)的测定,均准确检出了目标样品。该方法利用pCEC-μELSD的双重分离能力,通过调节电压参数,实现了两大类甜味剂的同时分离检测,有效应对了目前食品添加剂多类添加的复杂情况,数据稳定可靠,灵敏度满足常规要求。
Pressurized capillary electrochromatography (pCEC) based on the theory and technique of capillary electrochromatography (CEC) is emerging as an environmental friendly micro-separation technology in recent years. Compared with capillary zone electrophoresis (CE), where only charged compounds can be separated,bothcharged and neutral analytes can be separated simultaneously in pCEC. Moreover, pCEC can provide higher column efficiency than high performance chromatography (HPLC). Even more importantly, compared with traditional CEC, gradient elution mode can be achieved and some problem associated withJoule heating effect, like bubble formation and dryout of the column can bealsoovercome in pCEC due to the introduction of pump pressure. The appearance of pCEC followed the trend of high performance and miniaturization in the development of modern analytical instrumention. Thus, pCEC have been applied to the analysis of more complicated samples in life science, traditional Chinese medicine research, drug analysis, environment monitoring and food safety, as well as other popular research fields.
Evaporative light-scattering detector (ELSD) has been widely used as a universal detector for HPLC since the1990s. The application of ELSD doesn't depend on the molecular structure and optical properties of analytes. ELSD only requests the evaporitivity of analytes to be lower than that of the mobil phase. Recently, ELSD has displayed the huge advantage for the detection of natural products, Chinese herbal medicines, lipids, surfactants and carbohydrates.
Therefore, the aim of this dissertation focuses on the development of a platform based on pCEC coupledto micro-ELSD (μELSD)and its applications. Some key parts such as evaporation assembly, nebulization assembly, and micro light scattering module were designed. Subsequently, the interface technique of pCEC with μELSD was investigated through optimizing the operation conditions such as evaporation, nebulizer and light scattering parts. The performance of pCEC with μELSD was valuated in detail, and then a method based on pCEC-μELSD was applied successfully for the analysis of synthetic and natural sweeteners in food samples. pCEC-μELSD as an efficient and economical technology hasgreat potential for the analysis of complicated samples in various research fields。The content of this dissertation is as follows:
Chapter1:The research background of pCEC and ELSD including their advances history, basic principle and application is systematically summarized. According to their characteristics, the feasibility of the coupling of two techniques was proposed. The purpose and significanceof this dissertation wereput forward。
Chapter2:In order to investigate the miniaturization of the evaporative light-scattering detector and understand the mechanism of μELSD, the structure of evaporation assembly, nebulizer assembly and light scattering cell were designed in detail in this chapter. The finalized structure of micro-fluid nebulizer was composed of nebulization nozzle and nebulization capillary. The finalized mirco-evaporator was based on cylindrical conical tube which was connected to heating resistor and covered with insulation sleeve. Sheath liquid assembly equipped with supplementary carrier gas was used to connect the evaporative annular tube to light scattering detector cell. The laser and photomultiplier were used, respectively, as the light source and scattering light collector of μELSD. Light trap was also designed in the structure of μELSDin order to avoid the reflected light. The electronic circuit system that designed for μELSD can be used to adjust and monitorvarious parameters of μELSDin real time such as evaporation temperature, flow rate of carrier gas and system carrier gas pressure.
Chapter3:The parameters of evaporation assembly, nebulizer assembly and light scattering cell were optimized in this chapter. The influence of nebulization nozzle and matching nebulization capillary on atomizing effect was investigated. The finalized size of nebulization nozzle and matching nebulization capillary were410μm and20μm I.D.×360μm O.D,respectively. The influenceof the position of capillary on the signal to noise ratio and repeatability was also investigated. It is indicated that the best signal to noise ratio can be abtained under the condition of10cm length,7.5mm inlet diameter and1.0outlet diameter of cylindrical conical tube. Moreover, three different light scattering detector cells were made to evaluate the influence of detector cell size on the signal to noise ratio. Finally, the largestone(65mm i.d.x60mm high)was choosen to increase the signal to noise ratio. Therefore, the detection limit of μELSD was caculated as0.1ng (S/N=3, direct injection with glucose sample),the noise of baseline was0.8μV,the baseline drift was0.182mV/h, the reproducibility of peek aera (RSD6) was0.4%, and the reproducibility of peek height(RSD6) was0.3%. The platform of pCEC coupling toμELSD was built successfully.
Chapter4:Optimization and validation of pCEC with μELSD detector were processed. A method base on pCEC-μELSD was developed for the determination of a mixture of fructose, glucose and sucrose in food. TSK Amide-80capillary column (150mm x200μ m,5μm) was selected. The analytes can be separated within13min under-3kV of operation voltage. The mobile phase was chosen as80%acetonitrile in DI water. The linear range of the method was from0.01to1.0μg for each analytes. The detection limit was4ng. Recoveries within88.1%-103.9%and RSD within0.9%-3.3%(n=6) indicated that adaptability and stability of pCEC with μELSD are comparable with commercial HPLC-ELSD.
Chapter5:Based on the technique of the pCEC-μELSD, a method was established for analysis of a mixture of erythritol, xylitol, sorbitol, maltose and lactitol in the sugar-free food. Some operation conditions of ELSD such as evaporated temperature and flow rate of carrier gas and the chromatographic condition were optimized. The influence of pH value on the signal to noise ratio was also investigated. The linear range of each analyetes was from0.01to2.5μg (correlation coefficient greater than0.998).The average recovery was between82.4%-107.2%under three different level by using standard addition method and the relative standard deviation (RSD) was less than3.9%(n=6). The detection limit (LOD, S/N=3)was between4to11ng. Contrast to conventional method based on HPLC with ELSD, analysis speed was improved obviously. This method then was applied for analyses of two real samples. The proposed method provides a fast and effective method for the quantitative detection of sugar alcohol in sugar-free foods.
Chapter6:Based on the technique of the pCEC-μELSD, ananalytical method was established to detect simultaneously nine common sweetener. The operation conditions of ELSD and the separation condition were both optimized. The linear range of each sweetener was from7ng to4.2μg (correlation coefficient greater than0.998).The average recovery was between85.4%-110.4%under three different level by using standard addition method and the RSDwas less than3.6%(n=6). The detection limit (LOD, S/N=3)was between7ng to42ng. This method was applied for three reall xylitol samples and the targeted analytes were separated and detected accurately. The proposed method provides a sensitive and stable means for the determination of two kinds of sweetener simultaneously.
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