多糖类物质的离子色谱检测技术研究
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摘要
离子色谱(IC)是高效液相色谱(HPLC)的一种,是分析阴离子和阳离子的一种液相色谱方法。对难以用其他仪器和方法分析的常见阴离子、阳离子、有机酸和有机胺类等组分的分析,离子色谱法具有选择性好,灵敏度高,快速,简便,并可同时测定多组分等的突出优点。基于上述优点,离子色谱法自1975年问世以来发展迅速,已经在环境监测、电力、半导体工业、食品、石油化工、医疗卫生和生化等领域得到广泛应用,已有数十项成为有关权威机构的标准方法。鉴于糖类化合物分子具有电化学活性及在强碱溶液中呈离子化状态,Roxklin等于1983年首先报道了用阴离子交换色谱柱分离,脉冲安培检测器测定糖的新方法(简称HPAEC)。用离子色谱法检测糖类,寻求操作简单,结果准确的检测体系,值得每一位分析化学学者进行深入研究。
本文包括两个部分。第一部分是文献综述,这部分结合多糖的利用价值及提取方法,评述了近十年离子色谱检测技术的发展以及其它相关领域在多糖类物质检测中的研究和应用。第二部分是研究报告。首先研究了绿茶中茶叶多糖的最佳提取方法和离子色谱检测茶叶多糖中的2种单糖的技术,在建立了高效,便捷的分析方法的同时选择了最佳的提取条件。利用温水浸提绿茶茶叶,超声辅助乙酸水解绿茶粗多糖,以METROSEPCARB 1(150×4.0 mm)阴离子交换柱为分离柱,以6.0 mmol/L NaOH溶液为洗脱液,在32℃的条件下对绿茶粗多糖水解液进行检测,实现了对绿茶粗多糖中的葡萄糖和甘露糖两种单糖的完全分离和定量检测。在此研究基础上,对其他不同种类茶叶多糖进行了分析研究,进一步优化实验条件,由对单纯两种单糖的分析拓展到同时分析检测四种单糖,取得了良好的试验效果,并且打破了多种单糖需要进行梯度淋洗才可实现分离的复杂方法,实现了固定浓度淋洗液直接分离多种单糖的高效方法。基于此,将该法的应用延伸到真菌类和中药领域,分别检测了香菇多糖和山药多糖,取得了良好的实验效果,从而进一步验证了该方法的应用价值。但在以上研究过程中,存在着对多糖水解液果糖检测方面的不足,突出表现为灵敏度低、分析时间过长等缺点。为了完善检测方法,继续优化实验条件,对果糖的检测方法进行改进,建立快速、高效检测多糖中的果糖的离子色谱检测方法,实验取得了很好的效果,得到了高的灵敏度和准确度,以及低的检测限。
一.绿茶中茶多糖的提取与离子色谱检测方法研究
本文在不同浸提条件下提取绿茶粗多糖的基础上,建立了用高效阴离子交换分离-脉冲安培检测(HPAE-PAD),在32℃的条件下分离测定绿茶茶叶多糖中的葡萄糖和甘露糖2种单糖的分析方法。以METROSEP CARB 1(150×4.0 mm)阴离子交换柱为分离柱,6.0 mmol/L NaOH溶液为淋洗液,淋洗液流速为1.0 mL/min,总分析时间为35 min。在优化的分离条件下,葡萄糖和甘露糖的检出限分别为0.125 mg/L和2.0 mg/L(进样体积20μL,峰高定量)。用20 mg/L葡萄糖和甘露糖的混标溶液连续7次进样,两种单糖峰高的相对标准偏差分别为5.79%和14.15%,保留时间的相对标准偏差分别为2.08%和2.19%。样品测定的回收率为94.9%-97.5%。用所建立的方法快速、有效分离了绿茶茶叶多糖中的葡萄糖和甘露糖,同时确定了茶叶多糖的最佳提取条件。
二.离子色谱法测定各种茶叶中的茶多糖研究
建立了用高效阴离子交换分离-脉冲安培检测分离测定不同种茶叶多糖中的半乳糖、葡萄糖、甘露糖和果糖的分析方法。以METROSEP CARB 1(150×4.0 mm)阴离子交换柱为分离柱,以6.0 mmol/L NaOH溶液为淋洗液在32℃的条件下将4种单糖从分离柱上洗脱,淋洗液流速为1.0 mL/min,总分析时间为45 min。在优化的分离条件下4种单糖的检出限为0.125 mg/L～2.0mg/L(进样体积20μL,峰高定量)。用20mg/L的半乳糖、葡萄糖、甘露糖和30mg/L的果糖混标溶液连续7次进样,峰高的相对标准偏差为5.82%～14.19%,保留时间的相对标准偏差为2.03%～2.26%。样品测定的回收率为91.8%～99.3%。用所建立的方法快速、有效分析了4种常见茶叶多糖,得到了满意的检测结果。
三.香菇多糖和山药多糖的离子色谱检测方法研究
建立了离子色谱法分离测定香菇多糖和山药多糖的分析方法。以METROSEP CARB 1(150×4.0 mm)阴离子交换柱为分离柱,脉冲安培检测,以7.0 mmol/L NaOH溶液为淋洗液,淋洗液流速1.0 mL/min,总分析时间为35 min。在优化的分析条件下,3种单糖的检测限分别为半乳糖0.125 mg/L,葡萄糖0.5 mg/L,甘露糖1.0mg/L(进样体积20μL,峰高定量),用30 mg/L的标准样品混合溶液连续进样6次,峰高的相对标准偏差为1.79%～3.52%,保留时间的RSD为3.07%～3.29%,样品测定的回收率为96.3%～99.5%。运用上述方法分离检测香菇多糖和山药多糖,得到了良好的分离效果和较高的检测灵敏度,分析结果令人满意。
四.离子色谱法检测多糖水解液中果糖的研究
建立了阴离子交换分离-脉冲安培检测分析检测多糖水解液中的果糖的离子色谱方法,并且分别讨论了峰高和峰面积两种不同分析定量方法的准确度。用METROSEP CARB1(150×4.0mm)阴离子交换柱为分离柱,以100 mmol/LNaOH溶液为淋洗液,流速为1.0mL/min,总分析时间为13 min。在优化的色谱条件下,果糖的最低检出限为0.4 mg/L(进样体积20μL)。用10mg/L果糖标准溶液连续进样7次,峰高的相对标准偏差为1.64%。峰面积的RSD为3.43%,保留时间的RSD为0.36%。样品的加标回收率为94.6%～96.1%(峰高定量)和93.2%～95.6(峰面积定量)。用所建立的方法快速、准确的分析了三种茶叶多糖水解液,分析结果令人满意。
Ion Chromatography is a branch of the High Performance Liquid Chromatography and it is a liquid chromatography method for the determination of the anions and cations.IC has good selectivity, high sensitivity,rapidity,simplicity and can detect various components simultaneously for the determination of the common cations and anions,the organic acids and amines which were difficult to detected by other analytical methods.Based on these advantages,since 1975 IC has a rapid development and has been widly used in many fields,such as environmental monitoring,electric power,semiconductor,foods,petroleum and chemical industry,medical and health,biochemistry and so on.At the meantime there are already in dozens of IC methods become the standard method for the authority.Ground on the state of ionization in alkali and the electrochemical activity of the sugars,Roxklin had reported the method that use the anion exchange column and pulsed amperemetric detection to determined the polysaccharides in 1983.Due to it's simply manipulate and accuratly results,using IC for determining sugars deserves people's concern and study.
The thesis consists of two parts.One is a review related to the application and the extraction of the polysaccharides,and makes an evaluation on the development of the IC detection technology and the other fields'detectation in recent ten years.The other is a research report which is composed of four components as follows.First of all,do the research on the extraction and the determination of glucose and mannose which are the two kinds of tea polysaccharides in green tea with IC technology. A fast,simple method was established and the best extracte condition was chosen at the same time. Using the warm water to distill the green tea and ultrasound compnent with the HAc to hydrolyze the tea polysaccharides,take the METROSEP CARB1(150×4.0 mm) anion exchange column as the chromatograph IC separation,at 32℃using the 6.0mmol/L NaOH as the mobile phase.The glucose and the mannose were totally separated and determined.Consideration of the top research,applying this system to the four kinds of teas,improving the condition,inlarge the application from the only two monosaccharides to four kinds of monosaccharides,after that good results were be gotten.This method broke the usual method that need gradient mobile phase,only need one fixed flow liquid which can easily separated the polysaccharides.Then apply this method to determine the polysaccharides which are distracted from the mushroom and the CommonYan Rhizome,good results were gotten and the method was be optimized.But in these researches,there are also some disadvantages,such as the determination of the fructose,clearly manifest as the low sensitivity and the long determine time that cost.For the development of the method,go on to improve the condition,and a fast,accurate method for determining fructose by IC was established with a high accuracy and low detection limite.
1.The studies of the extractionand and the Ion Chromatography determination of tea polysaccharides in Green Tea
A method for determine tea polysaccharides of glucose and mannose in green tea by high performance anion exchange with pulsed amperemetric detection was established,based on the different distill methods.The chromatograph separation was established on METROSEP CARB 1 (150×4.0 mm) anion exchange column,using 6.0 mmol/L NaOH at 1.0 mL/min in flow rate.The total analysis time is 35 min.The detection limits of 0.125 mg/L～2.0 mg/L(20μL injection,using the peak height for the quantitative analysis) were achieved under the optimal conditions.The RSDs of peak height for the seven consecutive determinations of 20 mg/L glucose and mannose were 5.79%and 14.15%respectively,and the RSDs of reserve time were 2.08%and 2.19%.The recovery percentages of the sample detection were 94.9%～97.5%.Glucose and mannose were be determined fast and effectively,at the meantime,the best distill condition was be chosen.
2.The determination of tea polysaccharides with Ion Chromatography
A method for the determination of tea polysaccharides of galactose,glucose,mannose and fructose by high performance anion exchange with pulsed amperemetric detection was established.The chromatograph separation was established on METROSEP CARB 1(150×4.0 mm) anion exchange column,using 6.0 mmol/L NaOH at 1.0 mL/min in flow rate and at 32℃.The total analysis time is 45 min.The detection limits of 0.125 mg/L～2.0 mg/L(20μL injection,using the peak height for the quantitative analysis) were achieved under the optimal conditions.The RSDs of peak height for the seven consecutive determinations of 20 mg/L galactose,glucose,mannose and 30 mg/L fructose were 5.82%～14.19%,and the RSDs of reserve time were 2.03%～2.26%.The recovery percentages of the sample detection were 91.8%～99.3%.Four kinds of tea polysaccharides were fast and effectively determined,and the results of the determination were satisfying.
3.Studies of the determination of the lentinan and the dioscoreaopposita polysaccharides by IC
This article applies the used of the IC technology to the fungus and Chinese traditional medicines field and a fast determining method has been established.The chromatograph separation was established METROSEP CARB 1(150×4.0 mm) anion exchange column,with pulsed amperemetric detection.Using 7.0 mmol/L NaOH at 1.0 mL/min in flow rate.The total analysis time is 35min.The detection limits was 0.125～1.0 mg/L(20μL injection,using the peak height for the quantitative analysis) were achieved under the optimal conditions.The RSDs of peak height for six consecutive determinations of 30 mg/L standard sample were 1.79%～3.52%,and the RSDs of reserve time were 3.07%～3.29%.The recovery percentages of the sample detection were 96.3%～99.5%.The system was applied for the determination of lentinan and dioscoreaopposita,good separation and high detection sensitivity were achieved,and the results of the determination were satisfying.
4.The determination of the fructose by IC
A method for determination of fructose in hydrolyzing polysaccharides by high performance anion exchange with pulsed amperemetric detection was established.And the accuracy that use the peak height and peak area for quantitative analysis were discussed.The chromatograph separation was established on METROSEP CAR 1(150×4.0 mm) anion exchange column,using 100 mmol/L NaOH at 1.0 mL/min in flow rate.The total analysis time is 13min.The detection limits of 0.4 mg/L(20μL injection) was achieved under the optimal conditions.The RSDs of peak height for seven consecutive determination of 10 mg/L fructose was 1.64%,the RSDs of peak area 3.43%,and the RSDs of reserve time 0.36%.The recovery percentages of sample detection were 94.6%～96.1%(using peak height) and 93.2%～95.6%(using peak area).Three kinds of tea polysaccharides were fast and effectively determined,and the results of the determination were satisfying.
本文包括两个部分。第一部分是文献综述,这部分结合多糖的利用价值及提取方法,评述了近十年离子色谱检测技术的发展以及其它相关领域在多糖类物质检测中的研究和应用。第二部分是研究报告。首先研究了绿茶中茶叶多糖的最佳提取方法和离子色谱检测茶叶多糖中的2种单糖的技术,在建立了高效,便捷的分析方法的同时选择了最佳的提取条件。利用温水浸提绿茶茶叶,超声辅助乙酸水解绿茶粗多糖,以METROSEPCARB 1(150×4.0 mm)阴离子交换柱为分离柱,以6.0 mmol/L NaOH溶液为洗脱液,在32℃的条件下对绿茶粗多糖水解液进行检测,实现了对绿茶粗多糖中的葡萄糖和甘露糖两种单糖的完全分离和定量检测。在此研究基础上,对其他不同种类茶叶多糖进行了分析研究,进一步优化实验条件,由对单纯两种单糖的分析拓展到同时分析检测四种单糖,取得了良好的试验效果,并且打破了多种单糖需要进行梯度淋洗才可实现分离的复杂方法,实现了固定浓度淋洗液直接分离多种单糖的高效方法。基于此,将该法的应用延伸到真菌类和中药领域,分别检测了香菇多糖和山药多糖,取得了良好的实验效果,从而进一步验证了该方法的应用价值。但在以上研究过程中,存在着对多糖水解液果糖检测方面的不足,突出表现为灵敏度低、分析时间过长等缺点。为了完善检测方法,继续优化实验条件,对果糖的检测方法进行改进,建立快速、高效检测多糖中的果糖的离子色谱检测方法,实验取得了很好的效果,得到了高的灵敏度和准确度,以及低的检测限。
一.绿茶中茶多糖的提取与离子色谱检测方法研究
本文在不同浸提条件下提取绿茶粗多糖的基础上,建立了用高效阴离子交换分离-脉冲安培检测(HPAE-PAD),在32℃的条件下分离测定绿茶茶叶多糖中的葡萄糖和甘露糖2种单糖的分析方法。以METROSEP CARB 1(150×4.0 mm)阴离子交换柱为分离柱,6.0 mmol/L NaOH溶液为淋洗液,淋洗液流速为1.0 mL/min,总分析时间为35 min。在优化的分离条件下,葡萄糖和甘露糖的检出限分别为0.125 mg/L和2.0 mg/L(进样体积20μL,峰高定量)。用20 mg/L葡萄糖和甘露糖的混标溶液连续7次进样,两种单糖峰高的相对标准偏差分别为5.79%和14.15%,保留时间的相对标准偏差分别为2.08%和2.19%。样品测定的回收率为94.9%-97.5%。用所建立的方法快速、有效分离了绿茶茶叶多糖中的葡萄糖和甘露糖,同时确定了茶叶多糖的最佳提取条件。
二.离子色谱法测定各种茶叶中的茶多糖研究
建立了用高效阴离子交换分离-脉冲安培检测分离测定不同种茶叶多糖中的半乳糖、葡萄糖、甘露糖和果糖的分析方法。以METROSEP CARB 1(150×4.0 mm)阴离子交换柱为分离柱,以6.0 mmol/L NaOH溶液为淋洗液在32℃的条件下将4种单糖从分离柱上洗脱,淋洗液流速为1.0 mL/min,总分析时间为45 min。在优化的分离条件下4种单糖的检出限为0.125 mg/L～2.0mg/L(进样体积20μL,峰高定量)。用20mg/L的半乳糖、葡萄糖、甘露糖和30mg/L的果糖混标溶液连续7次进样,峰高的相对标准偏差为5.82%～14.19%,保留时间的相对标准偏差为2.03%～2.26%。样品测定的回收率为91.8%～99.3%。用所建立的方法快速、有效分析了4种常见茶叶多糖,得到了满意的检测结果。
三.香菇多糖和山药多糖的离子色谱检测方法研究
建立了离子色谱法分离测定香菇多糖和山药多糖的分析方法。以METROSEP CARB 1(150×4.0 mm)阴离子交换柱为分离柱,脉冲安培检测,以7.0 mmol/L NaOH溶液为淋洗液,淋洗液流速1.0 mL/min,总分析时间为35 min。在优化的分析条件下,3种单糖的检测限分别为半乳糖0.125 mg/L,葡萄糖0.5 mg/L,甘露糖1.0mg/L(进样体积20μL,峰高定量),用30 mg/L的标准样品混合溶液连续进样6次,峰高的相对标准偏差为1.79%～3.52%,保留时间的RSD为3.07%～3.29%,样品测定的回收率为96.3%～99.5%。运用上述方法分离检测香菇多糖和山药多糖,得到了良好的分离效果和较高的检测灵敏度,分析结果令人满意。
四.离子色谱法检测多糖水解液中果糖的研究
建立了阴离子交换分离-脉冲安培检测分析检测多糖水解液中的果糖的离子色谱方法,并且分别讨论了峰高和峰面积两种不同分析定量方法的准确度。用METROSEP CARB1(150×4.0mm)阴离子交换柱为分离柱,以100 mmol/LNaOH溶液为淋洗液,流速为1.0mL/min,总分析时间为13 min。在优化的色谱条件下,果糖的最低检出限为0.4 mg/L(进样体积20μL)。用10mg/L果糖标准溶液连续进样7次,峰高的相对标准偏差为1.64%。峰面积的RSD为3.43%,保留时间的RSD为0.36%。样品的加标回收率为94.6%～96.1%(峰高定量)和93.2%～95.6(峰面积定量)。用所建立的方法快速、准确的分析了三种茶叶多糖水解液,分析结果令人满意。
Ion Chromatography is a branch of the High Performance Liquid Chromatography and it is a liquid chromatography method for the determination of the anions and cations.IC has good selectivity, high sensitivity,rapidity,simplicity and can detect various components simultaneously for the determination of the common cations and anions,the organic acids and amines which were difficult to detected by other analytical methods.Based on these advantages,since 1975 IC has a rapid development and has been widly used in many fields,such as environmental monitoring,electric power,semiconductor,foods,petroleum and chemical industry,medical and health,biochemistry and so on.At the meantime there are already in dozens of IC methods become the standard method for the authority.Ground on the state of ionization in alkali and the electrochemical activity of the sugars,Roxklin had reported the method that use the anion exchange column and pulsed amperemetric detection to determined the polysaccharides in 1983.Due to it's simply manipulate and accuratly results,using IC for determining sugars deserves people's concern and study.
The thesis consists of two parts.One is a review related to the application and the extraction of the polysaccharides,and makes an evaluation on the development of the IC detection technology and the other fields'detectation in recent ten years.The other is a research report which is composed of four components as follows.First of all,do the research on the extraction and the determination of glucose and mannose which are the two kinds of tea polysaccharides in green tea with IC technology. A fast,simple method was established and the best extracte condition was chosen at the same time. Using the warm water to distill the green tea and ultrasound compnent with the HAc to hydrolyze the tea polysaccharides,take the METROSEP CARB1(150×4.0 mm) anion exchange column as the chromatograph IC separation,at 32℃using the 6.0mmol/L NaOH as the mobile phase.The glucose and the mannose were totally separated and determined.Consideration of the top research,applying this system to the four kinds of teas,improving the condition,inlarge the application from the only two monosaccharides to four kinds of monosaccharides,after that good results were be gotten.This method broke the usual method that need gradient mobile phase,only need one fixed flow liquid which can easily separated the polysaccharides.Then apply this method to determine the polysaccharides which are distracted from the mushroom and the CommonYan Rhizome,good results were gotten and the method was be optimized.But in these researches,there are also some disadvantages,such as the determination of the fructose,clearly manifest as the low sensitivity and the long determine time that cost.For the development of the method,go on to improve the condition,and a fast,accurate method for determining fructose by IC was established with a high accuracy and low detection limite.
1.The studies of the extractionand and the Ion Chromatography determination of tea polysaccharides in Green Tea
A method for determine tea polysaccharides of glucose and mannose in green tea by high performance anion exchange with pulsed amperemetric detection was established,based on the different distill methods.The chromatograph separation was established on METROSEP CARB 1 (150×4.0 mm) anion exchange column,using 6.0 mmol/L NaOH at 1.0 mL/min in flow rate.The total analysis time is 35 min.The detection limits of 0.125 mg/L～2.0 mg/L(20μL injection,using the peak height for the quantitative analysis) were achieved under the optimal conditions.The RSDs of peak height for the seven consecutive determinations of 20 mg/L glucose and mannose were 5.79%and 14.15%respectively,and the RSDs of reserve time were 2.08%and 2.19%.The recovery percentages of the sample detection were 94.9%～97.5%.Glucose and mannose were be determined fast and effectively,at the meantime,the best distill condition was be chosen.
2.The determination of tea polysaccharides with Ion Chromatography
A method for the determination of tea polysaccharides of galactose,glucose,mannose and fructose by high performance anion exchange with pulsed amperemetric detection was established.The chromatograph separation was established on METROSEP CARB 1(150×4.0 mm) anion exchange column,using 6.0 mmol/L NaOH at 1.0 mL/min in flow rate and at 32℃.The total analysis time is 45 min.The detection limits of 0.125 mg/L～2.0 mg/L(20μL injection,using the peak height for the quantitative analysis) were achieved under the optimal conditions.The RSDs of peak height for the seven consecutive determinations of 20 mg/L galactose,glucose,mannose and 30 mg/L fructose were 5.82%～14.19%,and the RSDs of reserve time were 2.03%～2.26%.The recovery percentages of the sample detection were 91.8%～99.3%.Four kinds of tea polysaccharides were fast and effectively determined,and the results of the determination were satisfying.
3.Studies of the determination of the lentinan and the dioscoreaopposita polysaccharides by IC
This article applies the used of the IC technology to the fungus and Chinese traditional medicines field and a fast determining method has been established.The chromatograph separation was established METROSEP CARB 1(150×4.0 mm) anion exchange column,with pulsed amperemetric detection.Using 7.0 mmol/L NaOH at 1.0 mL/min in flow rate.The total analysis time is 35min.The detection limits was 0.125～1.0 mg/L(20μL injection,using the peak height for the quantitative analysis) were achieved under the optimal conditions.The RSDs of peak height for six consecutive determinations of 30 mg/L standard sample were 1.79%～3.52%,and the RSDs of reserve time were 3.07%～3.29%.The recovery percentages of the sample detection were 96.3%～99.5%.The system was applied for the determination of lentinan and dioscoreaopposita,good separation and high detection sensitivity were achieved,and the results of the determination were satisfying.
4.The determination of the fructose by IC
A method for determination of fructose in hydrolyzing polysaccharides by high performance anion exchange with pulsed amperemetric detection was established.And the accuracy that use the peak height and peak area for quantitative analysis were discussed.The chromatograph separation was established on METROSEP CAR 1(150×4.0 mm) anion exchange column,using 100 mmol/L NaOH at 1.0 mL/min in flow rate.The total analysis time is 13min.The detection limits of 0.4 mg/L(20μL injection) was achieved under the optimal conditions.The RSDs of peak height for seven consecutive determination of 10 mg/L fructose was 1.64%,the RSDs of peak area 3.43%,and the RSDs of reserve time 0.36%.The recovery percentages of sample detection were 94.6%～96.1%(using peak height) and 93.2%～95.6%(using peak area).Three kinds of tea polysaccharides were fast and effectively determined,and the results of the determination were satisfying.
引文
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