二维液相色谱兼容性解决方案及系统构建
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摘要
现代科学研究所涉及的样品越来越复杂,也对分析化学提出更高的要求。通常的—维色谱方法能够提供的峰容量有限,通过偶联两种不同分离机理的色谱柱,构建成二维液相色谱分离系统,可以极大地提高系统对复杂样品的分离分析能力。随着蛋白质组研究、环境科学、天然产物研究的发展,二维液相色谱也得到了较快的发展,多种分离模式结合的多维液相色谱分离系统被构建,并发挥重要作用。
正相(NP)、反相(RP)、体积排阻(SEC)、离子交换(IEX)等不同的液相色谱分离模式,依据的分离机理不同,分离选择性也有很大差异。采用不同分离模式,甚至同一种分离模式采用不同种类色谱分离介质构建的多维液相色谱分离系统,在正交性和分离能力方面皆可能存在很大差异。将不同的一维液相色谱分离系统结合,构建成多维分离系统的关键是选择性、分离速度、流动相和样品容量等兼容性的解决,只有进行合理的匹配,才可能实现最佳的分离效果。
论文将具有不同选择性的色谱柱串联,构建了无需特殊设计接口的IEX-RP、RP-RP和ZIC-HILIC-RP连续二维液相色谱系统。通过对流动相兼容性的系统考察,以不同流动相交替进行洗脱,实现了复杂样品的二维分离。IEX-RP连续二维分离系统分离多肽样品时,以进样盐梯度和有机相梯度交替洗脱,峰容量为675；RP-RP连续二维液相色谱系统分离六味地黄丸时,比一维分离时出峰数目提高了60%,识别出86个色谱峰；ZIC-HILIC-RP连续二维液相色谱系统,可以应用到多肽、蛋白质和中药的分离分析。
二维液相色谱分离中第二维的分离速度是影响整体分析时间的关键。快速梯度程序和高温是实现快速洗脱的有效方法。考察温度对反相色谱分离速度、柱效、选择性的影响,研究温度对保留时间和柱效的理论关系,并使用标准样品进行验证,进一步构建高温二维液相色谱系统。通过将第二维分离的柱温升高至80℃,样品的分离速度明显加快,峰形更尖锐,峰容量更高；相比较常温二维分离,峰容量提高了13%,达到了1840。
由于流动相不匹配,亲水相互作用色谱和反相色谱在线联用难以实现。亲水相互作用色谱法(HILIC)洗脱液中含有较高浓度的有机溶剂,无法直接导入反相色谱进行二维分离。通过构建稀释接口,将馏分中有机溶剂比例降低,进而构建了在线HILIC/RP二维液相色谱系统。使用三种标准品评价系统,证明该系统可以将第一维馏分中的大多数组份转移到第二维进行分离,强极性的组份不能导入到第二维进行分离时,由接口处的检测器获得其信息。分离多肽样品时识别出208个色谱峰,峰容量达2323。在线HILIC/RP二维液相色谱系统也被应用于天然产物、发酵液和蛋白质的分离分析。
第二维使用短柱可有效改善二维液相色谱系统分析速度的兼容性,但也会带来样品容量不兼容的问题。基于中心切割模式的二维液相色谱系统,可以灵活地对第一维分离中超柱容量馏分进行收集,有效降低导入到第二维中样品的复杂程度。采用构建的中心切割IEX/RP二维液相色谱系统分离多肽样品,分离度和检测灵敏度明显获得改善,多识别出21个色谱峰。提高第二维色谱柱的容量和分离能力是增加二维系统峰容量的有效方法。将平行柱接口后串联分析柱,有效增加第二维的分离空间,从而构建了IEX/RP二维液相色谱系统,分离多肽样品的峰容量达707,比常规平行柱接口提高了44%。该系统还可以应用于小鼠血清和天麻的分离分析。
Today scientists must deal with complex samples that either cannot be adequately separated using one-dimensional chromatography or that require an inordinate amount of time for separation. Two-dimensional liquid chromatography (2D-LC) which combining different separation mechanisms will generates extremely high peak capacity and ultra-high resolving power in separation of complex mixtures. It has received considerable attention in the separation complex samples from proteomics, environment, and natural products. Various 2D-LC system was developed through combining different separation modes.
In particular, LC techniques offer a wide variety of separation mechanisms, such as normal phase (NP), reversed phase (RP), size exclusion (SEC) or ion exchange (IEX), characterized by different selectivity. Consequently,2D-LC can be theoretically employed in many multidimensional combinations, generating increased peak capacity, selectivity and resolution, especially in the comprehensive LC mode. However, the combination of certain LC modes can present a series of compatibility difficulties, if not impossibilities, such as, for example, selectivity, separation speed, mobile phase immiscibility and sample capacity. Systematic study of the compatibility of the two dimensions is necessary to construct a successful 2D-LC system.
Directly couple of two columns with different separation selectivity is a simple way to construct 2D-LC. IEX-RP, RP-RP and HILIC-RP system was constructed through eluting by different mobile phase alternately. The compatibility of selectivity and mobile phase was investigated. Directly coupled columns IEX-RP system was developed for the separation of peptides. Step gradient of salt concentration for eluting components from weak anion exchange (SCX) column to RP column was followed by linear gradient of organic solvent proportion for eluting fractions on RP column. Total 2D-LC peak capacity of 675 was achieved through eluting of 11 fractions continuously. Directly coupled columns RP-RP system separated 60% more peaks than single column RP system, with 86 peaks was recognized in the separation of Liuwei Dihuang Pill. The developed HILIC-RP system was successful used in the separation of peptides, proteins and Traditional Chinese Medicine.
The second dimensional separation speed had an important influence on the peak capacity of 2D-LC system. Higher modulation frequency could significantly decrease the affection of under-sampling on peak capacity. Running with fast gradient and elevated temperature was an effective way to increase the separation speed and increase the compatibility with first dimension. Investigation of the effect of temperature on the separation speed and selectivity were carried out and verified by reference sample. A high temperature two dimensional IEX/RP liquid chromatography system was constructed through elevating the 2nd column temperature to 80℃. Faster separation and narrower peaks were observed. Compared with separation under room temperature,13% more peak capacity was got which was 1840.
The coupling of HILIC and NP is difficult to implement due to the mobile phase incompatibility. The high concentration of organic solvent in in HILIC disables the transfer between the two dimensions. On-line two-dimensional HILIC/RP liquid chromatography system was developed based on weakening of the mobile phase strength in the interface. Separation of three reference compounds shown that sample could be effectively introduced to the second dimension. A detector was used in the interface to detect the non-retained compounds. Mixture of tryptic digestion of four proteins was separated to test the performance of the system.208 peaks were recognized under the optimized condition and peak capacity reached 2323. Separations of rat liver protein, rhizome Gastrodiae and Cordyceps were also carried out to evaluate the performance of constructed on-line HILIC/RP system for analysis of real world sample.
The use of short second dimensional column will benefit the separation speed compatibility but may result in incompatibility of sample volume. Based on heart-cut interface, the components gushed from first dimension column can be removed and collected in another column. The removing of fraction which will overload on second dimension column greatly increases the resolving power and detection sensitivity. An IEX/RP 2D-LC system was developed by addition of heart-cut unit. Tryptic digest of four proteins were separated on this system. Compared with that of traditional 2D-LC, another 21 peaks were identified and some low abundance components were identified after weak retained components were removed.
The use of short second dimensional column will benefit the separation speed compatibility but may result in incompatibility of sample volume. Based on heart-cut interface, the components gushed from first dimension column can be removed and collected in another column. The removing of fraction which will overload on second dimension column greatly increases the resolving power and detection sensitivity. An IEX/RP 2D-LC system was developed by addition of heart-cut unit. Tryptic digest of four proteins were separated on this system. Compared with that of traditional 2D-LC, another 21 peaks were identified and some low abundance components were identified after weak retained components were removed. A tandem column connected to parallel second-dimensional separation column can help to improve the separation efficiency. An IEX/RP 2D-LC system has been developed based on a parallel-tandem column interface. The peak capacity of the 2D-LC system reached 707 under high temperature conditions. When compared with the traditional parallel column interface constructed in this paper, the new interface produced 44% higher peak capacity. Rat serum and rhizome gastrodiae were separated to evaluate the performance of constructed 2D-LC system for analysis of real world sample.
正相(NP)、反相(RP)、体积排阻(SEC)、离子交换(IEX)等不同的液相色谱分离模式,依据的分离机理不同,分离选择性也有很大差异。采用不同分离模式,甚至同一种分离模式采用不同种类色谱分离介质构建的多维液相色谱分离系统,在正交性和分离能力方面皆可能存在很大差异。将不同的一维液相色谱分离系统结合,构建成多维分离系统的关键是选择性、分离速度、流动相和样品容量等兼容性的解决,只有进行合理的匹配,才可能实现最佳的分离效果。
论文将具有不同选择性的色谱柱串联,构建了无需特殊设计接口的IEX-RP、RP-RP和ZIC-HILIC-RP连续二维液相色谱系统。通过对流动相兼容性的系统考察,以不同流动相交替进行洗脱,实现了复杂样品的二维分离。IEX-RP连续二维分离系统分离多肽样品时,以进样盐梯度和有机相梯度交替洗脱,峰容量为675；RP-RP连续二维液相色谱系统分离六味地黄丸时,比一维分离时出峰数目提高了60%,识别出86个色谱峰；ZIC-HILIC-RP连续二维液相色谱系统,可以应用到多肽、蛋白质和中药的分离分析。
二维液相色谱分离中第二维的分离速度是影响整体分析时间的关键。快速梯度程序和高温是实现快速洗脱的有效方法。考察温度对反相色谱分离速度、柱效、选择性的影响,研究温度对保留时间和柱效的理论关系,并使用标准样品进行验证,进一步构建高温二维液相色谱系统。通过将第二维分离的柱温升高至80℃,样品的分离速度明显加快,峰形更尖锐,峰容量更高；相比较常温二维分离,峰容量提高了13%,达到了1840。
由于流动相不匹配,亲水相互作用色谱和反相色谱在线联用难以实现。亲水相互作用色谱法(HILIC)洗脱液中含有较高浓度的有机溶剂,无法直接导入反相色谱进行二维分离。通过构建稀释接口,将馏分中有机溶剂比例降低,进而构建了在线HILIC/RP二维液相色谱系统。使用三种标准品评价系统,证明该系统可以将第一维馏分中的大多数组份转移到第二维进行分离,强极性的组份不能导入到第二维进行分离时,由接口处的检测器获得其信息。分离多肽样品时识别出208个色谱峰,峰容量达2323。在线HILIC/RP二维液相色谱系统也被应用于天然产物、发酵液和蛋白质的分离分析。
第二维使用短柱可有效改善二维液相色谱系统分析速度的兼容性,但也会带来样品容量不兼容的问题。基于中心切割模式的二维液相色谱系统,可以灵活地对第一维分离中超柱容量馏分进行收集,有效降低导入到第二维中样品的复杂程度。采用构建的中心切割IEX/RP二维液相色谱系统分离多肽样品,分离度和检测灵敏度明显获得改善,多识别出21个色谱峰。提高第二维色谱柱的容量和分离能力是增加二维系统峰容量的有效方法。将平行柱接口后串联分析柱,有效增加第二维的分离空间,从而构建了IEX/RP二维液相色谱系统,分离多肽样品的峰容量达707,比常规平行柱接口提高了44%。该系统还可以应用于小鼠血清和天麻的分离分析。
Today scientists must deal with complex samples that either cannot be adequately separated using one-dimensional chromatography or that require an inordinate amount of time for separation. Two-dimensional liquid chromatography (2D-LC) which combining different separation mechanisms will generates extremely high peak capacity and ultra-high resolving power in separation of complex mixtures. It has received considerable attention in the separation complex samples from proteomics, environment, and natural products. Various 2D-LC system was developed through combining different separation modes.
In particular, LC techniques offer a wide variety of separation mechanisms, such as normal phase (NP), reversed phase (RP), size exclusion (SEC) or ion exchange (IEX), characterized by different selectivity. Consequently,2D-LC can be theoretically employed in many multidimensional combinations, generating increased peak capacity, selectivity and resolution, especially in the comprehensive LC mode. However, the combination of certain LC modes can present a series of compatibility difficulties, if not impossibilities, such as, for example, selectivity, separation speed, mobile phase immiscibility and sample capacity. Systematic study of the compatibility of the two dimensions is necessary to construct a successful 2D-LC system.
Directly couple of two columns with different separation selectivity is a simple way to construct 2D-LC. IEX-RP, RP-RP and HILIC-RP system was constructed through eluting by different mobile phase alternately. The compatibility of selectivity and mobile phase was investigated. Directly coupled columns IEX-RP system was developed for the separation of peptides. Step gradient of salt concentration for eluting components from weak anion exchange (SCX) column to RP column was followed by linear gradient of organic solvent proportion for eluting fractions on RP column. Total 2D-LC peak capacity of 675 was achieved through eluting of 11 fractions continuously. Directly coupled columns RP-RP system separated 60% more peaks than single column RP system, with 86 peaks was recognized in the separation of Liuwei Dihuang Pill. The developed HILIC-RP system was successful used in the separation of peptides, proteins and Traditional Chinese Medicine.
The second dimensional separation speed had an important influence on the peak capacity of 2D-LC system. Higher modulation frequency could significantly decrease the affection of under-sampling on peak capacity. Running with fast gradient and elevated temperature was an effective way to increase the separation speed and increase the compatibility with first dimension. Investigation of the effect of temperature on the separation speed and selectivity were carried out and verified by reference sample. A high temperature two dimensional IEX/RP liquid chromatography system was constructed through elevating the 2nd column temperature to 80℃. Faster separation and narrower peaks were observed. Compared with separation under room temperature,13% more peak capacity was got which was 1840.
The coupling of HILIC and NP is difficult to implement due to the mobile phase incompatibility. The high concentration of organic solvent in in HILIC disables the transfer between the two dimensions. On-line two-dimensional HILIC/RP liquid chromatography system was developed based on weakening of the mobile phase strength in the interface. Separation of three reference compounds shown that sample could be effectively introduced to the second dimension. A detector was used in the interface to detect the non-retained compounds. Mixture of tryptic digestion of four proteins was separated to test the performance of the system.208 peaks were recognized under the optimized condition and peak capacity reached 2323. Separations of rat liver protein, rhizome Gastrodiae and Cordyceps were also carried out to evaluate the performance of constructed on-line HILIC/RP system for analysis of real world sample.
The use of short second dimensional column will benefit the separation speed compatibility but may result in incompatibility of sample volume. Based on heart-cut interface, the components gushed from first dimension column can be removed and collected in another column. The removing of fraction which will overload on second dimension column greatly increases the resolving power and detection sensitivity. An IEX/RP 2D-LC system was developed by addition of heart-cut unit. Tryptic digest of four proteins were separated on this system. Compared with that of traditional 2D-LC, another 21 peaks were identified and some low abundance components were identified after weak retained components were removed.
The use of short second dimensional column will benefit the separation speed compatibility but may result in incompatibility of sample volume. Based on heart-cut interface, the components gushed from first dimension column can be removed and collected in another column. The removing of fraction which will overload on second dimension column greatly increases the resolving power and detection sensitivity. An IEX/RP 2D-LC system was developed by addition of heart-cut unit. Tryptic digest of four proteins were separated on this system. Compared with that of traditional 2D-LC, another 21 peaks were identified and some low abundance components were identified after weak retained components were removed. A tandem column connected to parallel second-dimensional separation column can help to improve the separation efficiency. An IEX/RP 2D-LC system has been developed based on a parallel-tandem column interface. The peak capacity of the 2D-LC system reached 707 under high temperature conditions. When compared with the traditional parallel column interface constructed in this paper, the new interface produced 44% higher peak capacity. Rat serum and rhizome gastrodiae were separated to evaluate the performance of constructed 2D-LC system for analysis of real world sample.
引文
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