新型自动液相微萃取与高效液相色谱联用技术及应用研究
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摘要
样品前处理技术是样品分析过程中最关键的步骤，尤其是分析复杂样品基质中痕量组分时，它直接影响到样品的分析速度和整个测定结果的准确度和精密度。操作简便快速、有机溶剂用量少、装置微型化和自动化、易于与各种检测仪器联用的新型样品前处理技术已成为当前分析化学领域的重要发展趋势，其中预处理装置自动化是目前研究的热点之一。
液相微萃取技术集萃取、富集、净化于一体，是一种新型的溶剂微型化样品预处理技术，具有富集倍数高、操作简单、有机溶剂用量少、灵敏度高、样品净化功能突出、对环境友好、易于与各种检测仪器联用和自动化等特点。该技术可以根据目标物的性质和样品基质的特点来选择多样化的萃取模式进行高效和快速分析，在痕量有机污染物分析中具有广阔的应用前景。
本文设计和研制了一种新型自动液相微萃取装置，并与高效液相色谱联用，采用不同的萃取模式建立了环境水样、奶制品、饲料和土壤等复杂样品中多种有机污染物分析的新技术。主要研究内容包括：
（1）基于虚拟仪器控制技术，研制了一种新型自动液相微萃取装置，包括液流驱动体系、液流路径切换体系、管道体系等硬件部分的构建和控制软件的编写。本装置自动化程度高，有助于提高液相微萃取的准确度和精密度。
（2）将自动液-液-液三相微萃取与高效液相色谱联用，以苯酚、4-硝基酚、2,4-二氯酚为对象，研究建立了环境水样中酚类污染物分析的新技术。研究表明，最优萃取条件为：以正辛醇为有机溶剂，pH=5的水样为给体相，给体相中NaCl的加入量为50g/L，0.1mol/L NaOH溶液为受体相，搅拌速度为900rpm，萃取时间为20min，注射泵速度为8μL/s，停留时间为30s。在最优操作条件下，三种酚类化合物的富集倍数为85-101倍，方法检出限为0.9-2.2ng/mL （S/N=3），方法线性范围为20-1000ng/mL（R＞0.9981），相对标准偏差低于4.2%（n=6）。该技术成功应用于武汉东湖表层水中酚类化合物的测定，三种酚类化合物的加标回收率为102.2-134.3%，相对标准偏差低于4.5%。
（3）将自动液-液-液三相微萃取与高效液相色谱联用，以三聚氰胺为对象，研究建立了奶制品中三聚氰胺快速测定的新技术。研究表明，最优萃取条件为：超声处理15min，以正辛醇为有机溶剂，pH=8的水溶液为给体相，pH=5的0.02mol/L硫酸铵为受体相，搅拌速度为900rpm，萃取时间为15min，注射泵速度为6μL/s。在最优操作条件下，三聚氰胺的富集倍数为43倍，方法检出限为0.025μg/mL（S/N=3），方法线性范围为0.1-100μg/mL （R=0.9987），相对标准偏差为3.8%（n=6）。该技术成功应用于实际奶制品中三聚氰胺的分析，加标回收率为96.9-104.2%，相对标准偏差低于6.1%。
（4）将载体辅助自动液-液-液三相微萃取与高效液相色谱联用，以四环素、土霉素、金霉素、强力霉素为对象，研究建立了饲料中四环素类抗生素分析的新技术。研究表明，最优萃取条件为：以正辛醇为有机溶剂，Aliquat-336载体浓度为10%（w/v），pH=9的水溶液为给体相，pH=2的盐酸溶液为受体相，搅拌速度为800rpm，萃取时间为15min，注射泵速度为8μL/s，停留时间为30s。在最优操作条件下，四种抗生素的富集倍数为56-99倍，方法检出限为1.2-2.8ng/mL （S/N=3），方法线性范围为1-500ng/mL（R＞0.9930），相对标准偏差低于5.0%。该技术成功应用于市售鸡饲料中四环素类抗生素的分析，加标回收率为95.6-115.6%，相对标准偏差低于5.7%。
（5）将超声辅助自动液相微萃取技术与高效液相色谱联用，以萘、菲、蒽、苯并（a）蒽为对象，研究建立了土壤中多环芳烃类污染物分析的新技术。研究表明，最优萃取条件为：1g土壤样品加入15mL水，超声功率100W时超声10min，以正辛醇为有机溶剂和接收相，搅拌速度为1000rpm，萃取时间为20min，有机溶剂的运动速度为10μL/s，停留时间为60s。在最优操作条件下，四种多环芳烃的方法检出限为0.05-0.1μg/g （S/N=3），方法线性范围为0.5-50μg/g（R＞0.9945），相对标准偏差低于5.0%。该技术成功应用于武汉某染料厂旧址处土壤样品的测定，在三个不同的加标浓度，四种多环芳烃的加标回收率为78.4-127.3%，相对标准偏差低于8.5%。
Sample pretreatment technology is the most critical procedure in sample analysis,especially in trace analysis of organic pollutants in complex samples. Sample pretreatmentprocess directly influences the speed of sample analysis and accuracy and precision of theresults. Recently, the development of rapid, simple, less solvent consumption,miniaturization, automation, and easy combination to analytical instruments are theimportant trend of sample pretreatment technology. The automation of samplepretreatment devices is one of the hot points in analytical chemistry.
Liquid-phase microextraction has emerged as a new sample preparation techniquebased on solvent-miniaturization because of the obvious advantages of simple, low cost,efficiency, selectivity, high enrichment factor, excellent sample clean-up, easycombination to analytical instruments and automation. It combines extraction,concentration and clean-up in a single step and it is a sensitive and environmental friendlysample preparation technique. Based on properties of the target analytes and types of thereal samples, different extraction modes can be chosen for efficient and fast analysis. Ithas been widely used in trace analysis of organic pollutants in complex samples.
This work focuses on the development of a novel automated liquid-phasemicroextraction device combined with HPLC and its applications in environmental watersamples, dairy samples, feed samples and soil samples analysis based on differentextraction modes. The main contents are as follows：
(1) A novel automated liquid-phase microextraction device was developed based onvirtual instrument technology. Hardware systems of liquid flow-driven system, liquid flowpath switching system, and pipeline system were established. The control software waswritten. It will improve the accuracy and precision of liquid phase microextrction.
(2) A novel analytical method for determination of Phenol,4-NP and2,4-DCP inwater samples, based on liquid-liquid-liquid microextraction combined with HPLC wasdeveloped. The optimal conditions were as follows:1-Octanol as extraction solvent, pH=5water sample as donor phase (containing50g/L of sodium chloride), sodium hydroxide of0.1mol/L as acceptor phase, stirring speed900rpm, extraction time20min, pump speed8 μL/s, dwell time30s. Under the optimal conditions, for three phenolic compounds,enrichment factors were85-101fold, and linearity was obtained in the range of20-1000ng/mL(R＞0.9981). The limits of detection defined for a signal-to-noise ratio of3(S/N=3) were0.9-2.2ng/mL. The relative standard deviations were below4.2%(n=6).This technique was successfully applied in the determination of phenolic compounds insurface water of East Lake, Wuhan. The recoveries from the spiked water sample variedbetween102.2and134.3%. Good reproducibilities were obtained with relative standarddeviation (RSD) values below4.5%.
(3) A novel analytical method for determination of melamine in dairy samples, basedon liquid-liquid-liquid microextraction combined with HPLC was developed. The optimalconditions were as follows: Ultrasonic time15min,1-Octanol as extraction solvent, pH=8water sample as donor phase, pH=5ammonium sulphate of0.02mol/L as acceptor phase,stirring speed900rpm, extraction time15min, pump speed6μL/s. Under the optimalconditions, for melamine, enrichment factor was43fold, and linearity was obtained in therange of0.1-100μg/mL(R=0.9987). The limits of detection defined for a signal-to-noiseratio of3(S/N=3) were0.025μg/mL. The relative standard deviations were3.8%(n=6).This technique was successfully applied in the determination of melamine in real dairysamples. The recoveries from the spiked dairy samples varied between96.9and104.2%.Good reproducibilities were obtained with relative standard deviation (RSD) values below6.1%.
(4) A novel analytical method for determination of TCs in feed samples, based oncarrier mediated liquid-liquid-liquid microextraction microextraction combined withHPLC was developed. The optimal conditions were as follows:1-Octanol as extractionsolvent (containing10%(w/v)of Aliquat-336as carrier), pH=9water sample as donorphase, pH=2hydrochloric acid as acceptor phase, stirring speed800rpm, extraction time15min, pump speed8μL/s, dwell time30s. Under the optimal conditions, for fourtetracycline antibiotics, enrichment factors were56-99fold, and linearity was obtained inthe range of1-500ng/mL (R＞0.9930). The limits of detection defined for asignal-to-noise ratio of3(S/N=3) were1.2-2.8ng/mL. The relative standard deviationswere below5.0%(n=6). This technique was successfully applied in the determination of real chicken feed samples. The recoveries from the spiked feed samples varied between95.6and115.6%. Good reproducibilities were obtained with relative standard deviation(RSD) values below5.6%.
(5) A novel green analytical method for determination of PAHs in soil samples, basedon ultrasound-assisted extraction combined with liquid-phase microextraction technologyand HPLC was developed. The optimal ultrasonic conditions were as follows:1g soilsample adding15mL ultrapure water, in the room temperature, ultrasonic power100W,ultrasonic time10min. The optimal liquid-phase microextraction conditions were as follows:1-Octanol as acceptor phase and extraction solvent, stirring speed1000rpm, extraction time20min. Under the optimal conditions, the limits of detection of PAHs were in the range of0.05-0.1μg/g (S/N=3). Good linear range (0.5-50μg/g)(R＞0.9945)was obtained for allthe analytes. The relative standard deviations were below5.0%(n=6). This technique wassuccessfully applied in the determination of PAHs in soil samples of a dye factory inWuhan. The recoveries from the spiked soil samples varied between78.4and127.3%.Good reproducibilities were obtained with relative standard deviation (RSD) values below8.5%.
液相微萃取技术集萃取、富集、净化于一体，是一种新型的溶剂微型化样品预处理技术，具有富集倍数高、操作简单、有机溶剂用量少、灵敏度高、样品净化功能突出、对环境友好、易于与各种检测仪器联用和自动化等特点。该技术可以根据目标物的性质和样品基质的特点来选择多样化的萃取模式进行高效和快速分析，在痕量有机污染物分析中具有广阔的应用前景。
本文设计和研制了一种新型自动液相微萃取装置，并与高效液相色谱联用，采用不同的萃取模式建立了环境水样、奶制品、饲料和土壤等复杂样品中多种有机污染物分析的新技术。主要研究内容包括：
（1）基于虚拟仪器控制技术，研制了一种新型自动液相微萃取装置，包括液流驱动体系、液流路径切换体系、管道体系等硬件部分的构建和控制软件的编写。本装置自动化程度高，有助于提高液相微萃取的准确度和精密度。
（2）将自动液-液-液三相微萃取与高效液相色谱联用，以苯酚、4-硝基酚、2,4-二氯酚为对象，研究建立了环境水样中酚类污染物分析的新技术。研究表明，最优萃取条件为：以正辛醇为有机溶剂，pH=5的水样为给体相，给体相中NaCl的加入量为50g/L，0.1mol/L NaOH溶液为受体相，搅拌速度为900rpm，萃取时间为20min，注射泵速度为8μL/s，停留时间为30s。在最优操作条件下，三种酚类化合物的富集倍数为85-101倍，方法检出限为0.9-2.2ng/mL （S/N=3），方法线性范围为20-1000ng/mL（R＞0.9981），相对标准偏差低于4.2%（n=6）。该技术成功应用于武汉东湖表层水中酚类化合物的测定，三种酚类化合物的加标回收率为102.2-134.3%，相对标准偏差低于4.5%。
（3）将自动液-液-液三相微萃取与高效液相色谱联用，以三聚氰胺为对象，研究建立了奶制品中三聚氰胺快速测定的新技术。研究表明，最优萃取条件为：超声处理15min，以正辛醇为有机溶剂，pH=8的水溶液为给体相，pH=5的0.02mol/L硫酸铵为受体相，搅拌速度为900rpm，萃取时间为15min，注射泵速度为6μL/s。在最优操作条件下，三聚氰胺的富集倍数为43倍，方法检出限为0.025μg/mL（S/N=3），方法线性范围为0.1-100μg/mL （R=0.9987），相对标准偏差为3.8%（n=6）。该技术成功应用于实际奶制品中三聚氰胺的分析，加标回收率为96.9-104.2%，相对标准偏差低于6.1%。
（4）将载体辅助自动液-液-液三相微萃取与高效液相色谱联用，以四环素、土霉素、金霉素、强力霉素为对象，研究建立了饲料中四环素类抗生素分析的新技术。研究表明，最优萃取条件为：以正辛醇为有机溶剂，Aliquat-336载体浓度为10%（w/v），pH=9的水溶液为给体相，pH=2的盐酸溶液为受体相，搅拌速度为800rpm，萃取时间为15min，注射泵速度为8μL/s，停留时间为30s。在最优操作条件下，四种抗生素的富集倍数为56-99倍，方法检出限为1.2-2.8ng/mL （S/N=3），方法线性范围为1-500ng/mL（R＞0.9930），相对标准偏差低于5.0%。该技术成功应用于市售鸡饲料中四环素类抗生素的分析，加标回收率为95.6-115.6%，相对标准偏差低于5.7%。
（5）将超声辅助自动液相微萃取技术与高效液相色谱联用，以萘、菲、蒽、苯并（a）蒽为对象，研究建立了土壤中多环芳烃类污染物分析的新技术。研究表明，最优萃取条件为：1g土壤样品加入15mL水，超声功率100W时超声10min，以正辛醇为有机溶剂和接收相，搅拌速度为1000rpm，萃取时间为20min，有机溶剂的运动速度为10μL/s，停留时间为60s。在最优操作条件下，四种多环芳烃的方法检出限为0.05-0.1μg/g （S/N=3），方法线性范围为0.5-50μg/g（R＞0.9945），相对标准偏差低于5.0%。该技术成功应用于武汉某染料厂旧址处土壤样品的测定，在三个不同的加标浓度，四种多环芳烃的加标回收率为78.4-127.3%，相对标准偏差低于8.5%。
Sample pretreatment technology is the most critical procedure in sample analysis,especially in trace analysis of organic pollutants in complex samples. Sample pretreatmentprocess directly influences the speed of sample analysis and accuracy and precision of theresults. Recently, the development of rapid, simple, less solvent consumption,miniaturization, automation, and easy combination to analytical instruments are theimportant trend of sample pretreatment technology. The automation of samplepretreatment devices is one of the hot points in analytical chemistry.
Liquid-phase microextraction has emerged as a new sample preparation techniquebased on solvent-miniaturization because of the obvious advantages of simple, low cost,efficiency, selectivity, high enrichment factor, excellent sample clean-up, easycombination to analytical instruments and automation. It combines extraction,concentration and clean-up in a single step and it is a sensitive and environmental friendlysample preparation technique. Based on properties of the target analytes and types of thereal samples, different extraction modes can be chosen for efficient and fast analysis. Ithas been widely used in trace analysis of organic pollutants in complex samples.
This work focuses on the development of a novel automated liquid-phasemicroextraction device combined with HPLC and its applications in environmental watersamples, dairy samples, feed samples and soil samples analysis based on differentextraction modes. The main contents are as follows：
(1) A novel automated liquid-phase microextraction device was developed based onvirtual instrument technology. Hardware systems of liquid flow-driven system, liquid flowpath switching system, and pipeline system were established. The control software waswritten. It will improve the accuracy and precision of liquid phase microextrction.
(2) A novel analytical method for determination of Phenol,4-NP and2,4-DCP inwater samples, based on liquid-liquid-liquid microextraction combined with HPLC wasdeveloped. The optimal conditions were as follows:1-Octanol as extraction solvent, pH=5water sample as donor phase (containing50g/L of sodium chloride), sodium hydroxide of0.1mol/L as acceptor phase, stirring speed900rpm, extraction time20min, pump speed8 μL/s, dwell time30s. Under the optimal conditions, for three phenolic compounds,enrichment factors were85-101fold, and linearity was obtained in the range of20-1000ng/mL(R＞0.9981). The limits of detection defined for a signal-to-noise ratio of3(S/N=3) were0.9-2.2ng/mL. The relative standard deviations were below4.2%(n=6).This technique was successfully applied in the determination of phenolic compounds insurface water of East Lake, Wuhan. The recoveries from the spiked water sample variedbetween102.2and134.3%. Good reproducibilities were obtained with relative standarddeviation (RSD) values below4.5%.
(3) A novel analytical method for determination of melamine in dairy samples, basedon liquid-liquid-liquid microextraction combined with HPLC was developed. The optimalconditions were as follows: Ultrasonic time15min,1-Octanol as extraction solvent, pH=8water sample as donor phase, pH=5ammonium sulphate of0.02mol/L as acceptor phase,stirring speed900rpm, extraction time15min, pump speed6μL/s. Under the optimalconditions, for melamine, enrichment factor was43fold, and linearity was obtained in therange of0.1-100μg/mL(R=0.9987). The limits of detection defined for a signal-to-noiseratio of3(S/N=3) were0.025μg/mL. The relative standard deviations were3.8%(n=6).This technique was successfully applied in the determination of melamine in real dairysamples. The recoveries from the spiked dairy samples varied between96.9and104.2%.Good reproducibilities were obtained with relative standard deviation (RSD) values below6.1%.
(4) A novel analytical method for determination of TCs in feed samples, based oncarrier mediated liquid-liquid-liquid microextraction microextraction combined withHPLC was developed. The optimal conditions were as follows:1-Octanol as extractionsolvent (containing10%(w/v)of Aliquat-336as carrier), pH=9water sample as donorphase, pH=2hydrochloric acid as acceptor phase, stirring speed800rpm, extraction time15min, pump speed8μL/s, dwell time30s. Under the optimal conditions, for fourtetracycline antibiotics, enrichment factors were56-99fold, and linearity was obtained inthe range of1-500ng/mL (R＞0.9930). The limits of detection defined for asignal-to-noise ratio of3(S/N=3) were1.2-2.8ng/mL. The relative standard deviationswere below5.0%(n=6). This technique was successfully applied in the determination of real chicken feed samples. The recoveries from the spiked feed samples varied between95.6and115.6%. Good reproducibilities were obtained with relative standard deviation(RSD) values below5.6%.
(5) A novel green analytical method for determination of PAHs in soil samples, basedon ultrasound-assisted extraction combined with liquid-phase microextraction technologyand HPLC was developed. The optimal ultrasonic conditions were as follows:1g soilsample adding15mL ultrapure water, in the room temperature, ultrasonic power100W,ultrasonic time10min. The optimal liquid-phase microextraction conditions were as follows:1-Octanol as acceptor phase and extraction solvent, stirring speed1000rpm, extraction time20min. Under the optimal conditions, the limits of detection of PAHs were in the range of0.05-0.1μg/g (S/N=3). Good linear range (0.5-50μg/g)(R＞0.9945)was obtained for allthe analytes. The relative standard deviations were below5.0%(n=6). This technique wassuccessfully applied in the determination of PAHs in soil samples of a dye factory inWuhan. The recoveries from the spiked soil samples varied between78.4and127.3%.Good reproducibilities were obtained with relative standard deviation (RSD) values below8.5%.
引文
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