醇酸盐缓冲体系在毛细管区带电泳中的氢键分离效用研究
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摘要
选用醇酸(乳酸、苹果酸、酒石酸、柠檬酸)的盐溶液作为高效毛细管区带电泳的缓冲体系,针对可能与之形成氢键作用力的不同分离对象,如：烟草生物碱[降烟碱(Nornicotine)、烟碱(Nicotine)、麦斯明(Myosmine)、假木贼碱(Anabasine)]、DNA、氨基酸、氨基酸的同分异构体等,进行了分离研究。通过对醇酸盐缓冲体系、pH条件、醇酸(乳酸：pKal=3.86,酒石酸：pKa1＝2.96、 pKa2=4.16,苹果酸：pKa1＝3.40、 pKa2=5.05,柠檬酸：pKa1=3.13、 pKa2=4.76、 pKa3=6.40)盐缓冲液浓度、电泳运行电压、样品进样条件、电泳运行温度等进行考察,获得了最优条件；进而成功地应用到了不同的分析体系,学位论文主要成果和创新点有：
(1)在集中关注缓冲体系与被分离物之间的氢键作用力存在的同时,发现了一种在烟草生物碱麦斯明阳离子与柠檬酸盐阴离子子之间的类似络合物螯合作用方式,其作用保持了麦斯明电泳分离过程中环亚胺结构的稳定性,成功解决了烟草中麦斯明的毛细管区带电泳分离检测问题。
(2)以pH3.2、360mmol/L的酒石盐作缓冲溶液,利用阳离子选择性耗尽在柱富集技术,实现了烟草生物碱的毛细管区带电泳基线分离。与磷酸盐缓冲溶液进行比较,卷烟样品生物碱测定的数量多、检测限低。
(3)从手性异构体分子结构上的差异,依靠同样具有同分异构体的醇酸盐缓冲体系,通过相互间氢键的立体选择性作用,实现了对氨基酸手性异构体的分离,为今后手性异构体的拆分提供了新思路。
(4)设想醇酸盐与富含氢键作用源(碱基)的DNA片段作用,将导致DNA片段在醇酸盐溶液中定向运动时,由于分子链越长,形成的氢键越多,所受阻力越大,迁移速率越小,进而可以据此将不同长度的DNA片段一一分开。通过实验研究,证实以醇酸(柠檬酸)盐作缓冲体系的毛细管区带电泳可以成功地分离DN A Marker。
(5)借鉴DNA在较高电场强度下的无交联高分子溶液筛分理论之瞬态缠结偶合(Transient entanglement coupling,TEC)及其改进机理,特别是DNA和缓冲溶液分子之间的氢键特殊相互作用,充分利用当DNA分子在缓冲溶液分子中进行迁移时,DNA片段与缓冲溶液分子相互缠结或碰撞,且较大的DNA分子具有更大的可能性与更多的缓冲溶液分子相遇并缠结,导致淌度下降更多,从而可以按照大小对DNA进行分离的原理。创新地将柠檬酸盐既作缓冲溶液,又作筛分介质,应用于单链DNA的高效毛细管区带电泳分离；分析方法的精密度高,重现性好。
Selecting hydroxy acid (lactic acid, Malic acid, tartaric acid, citric acid) salt as a buffer system of high performance capillary zone electrophoresis, according to the possible different forces from the formation of all hydrogen bonds betweet objects such as nicotine nornicotine, myosmine, anabasine, DNA, amino acids, amino acid isomers and alcholic acid, a series separation studies were completed. The separation conditions such as buffer pH, alkyd concentration, electrophoresis run voltage, and sample injection lengh andelectrophoresis run temperature were investigated. The optimal conditions were applied in different appications successfully. The main results and innovation points are as follows:
(1) While concerning whether the hydrogen bond between the buffer and the separated object exist or not, an important similar complex formed by hydrogen bond between tobacco biological alkaloid myosmine cation and citric anion are found,which mantaining stable separation behavior of myosmine in CZE. Basing on the principle, myosmine can be analyzed successfully.
(2) Tobacco alkaloids were separated on baseline by capillary zone electrophoresis with a buffer solution of pH3.2,360mmol/L tartaric acid. Cation-selective exhaustive injection, as an on-column preconcentration method, was also investigated for the determination of the alkaloids of cigarette samples.With the proposed method, both the resolution and detection limit of the alkaloids were improved comparing with those in phosphate buffer.
(3)Using chiral purity hydroxy acid salt as buffer system, through their hydrogen bonds stereo selective, the separation of amino acid chiral isomers were accomplished with the help of stereo selective hydrogen bonds interaction. The method peoposed a new strategy for the separation of chiral isomers.
(4) Because hydroxy acid salt can bond to DNA, which is rich in lonely pair electronics, the longer the DNA molecular chain, the more the hydrogen bond between hydroxy acid salt and DNA forms, so more resistance DNA receives, more slowly the DNA move towards to detect window, then DNA fragments separate out one by one according to the length of DNA. The experiments demonstrated the interation. separation of DNA Marker was completed successfully using citric acid salt as buffer system.
(5) Basing on DNA transient entanglement coupling and improvement of mechanisms of sieve theory in the non-cross-linked polymer solution under high electric field intensity, especially the intra-molecular hydrogen-bonding interaction between DNA and buffer solution, fully utilizing the principle of the separation of DNA:when DNA molecules migrating, DNA fragments tangling or colliding with buffer solution molecules, the larger the DNA,the more the amount of buffer solution molecules encountered and tangled, leading the lower mobility, so separated on the size of DNA fragment. Citrate buffer solution, as a buffer and screening media, was applied to separate single-strand DNA by high performance capillary zone electrophoresis, the analytical method was high precision and good reproducibility.
(1)在集中关注缓冲体系与被分离物之间的氢键作用力存在的同时,发现了一种在烟草生物碱麦斯明阳离子与柠檬酸盐阴离子子之间的类似络合物螯合作用方式,其作用保持了麦斯明电泳分离过程中环亚胺结构的稳定性,成功解决了烟草中麦斯明的毛细管区带电泳分离检测问题。
(2)以pH3.2、360mmol/L的酒石盐作缓冲溶液,利用阳离子选择性耗尽在柱富集技术,实现了烟草生物碱的毛细管区带电泳基线分离。与磷酸盐缓冲溶液进行比较,卷烟样品生物碱测定的数量多、检测限低。
(3)从手性异构体分子结构上的差异,依靠同样具有同分异构体的醇酸盐缓冲体系,通过相互间氢键的立体选择性作用,实现了对氨基酸手性异构体的分离,为今后手性异构体的拆分提供了新思路。
(4)设想醇酸盐与富含氢键作用源(碱基)的DNA片段作用,将导致DNA片段在醇酸盐溶液中定向运动时,由于分子链越长,形成的氢键越多,所受阻力越大,迁移速率越小,进而可以据此将不同长度的DNA片段一一分开。通过实验研究,证实以醇酸(柠檬酸)盐作缓冲体系的毛细管区带电泳可以成功地分离DN A Marker。
(5)借鉴DNA在较高电场强度下的无交联高分子溶液筛分理论之瞬态缠结偶合(Transient entanglement coupling,TEC)及其改进机理,特别是DNA和缓冲溶液分子之间的氢键特殊相互作用,充分利用当DNA分子在缓冲溶液分子中进行迁移时,DNA片段与缓冲溶液分子相互缠结或碰撞,且较大的DNA分子具有更大的可能性与更多的缓冲溶液分子相遇并缠结,导致淌度下降更多,从而可以按照大小对DNA进行分离的原理。创新地将柠檬酸盐既作缓冲溶液,又作筛分介质,应用于单链DNA的高效毛细管区带电泳分离；分析方法的精密度高,重现性好。
Selecting hydroxy acid (lactic acid, Malic acid, tartaric acid, citric acid) salt as a buffer system of high performance capillary zone electrophoresis, according to the possible different forces from the formation of all hydrogen bonds betweet objects such as nicotine nornicotine, myosmine, anabasine, DNA, amino acids, amino acid isomers and alcholic acid, a series separation studies were completed. The separation conditions such as buffer pH, alkyd concentration, electrophoresis run voltage, and sample injection lengh andelectrophoresis run temperature were investigated. The optimal conditions were applied in different appications successfully. The main results and innovation points are as follows:
(1) While concerning whether the hydrogen bond between the buffer and the separated object exist or not, an important similar complex formed by hydrogen bond between tobacco biological alkaloid myosmine cation and citric anion are found,which mantaining stable separation behavior of myosmine in CZE. Basing on the principle, myosmine can be analyzed successfully.
(2) Tobacco alkaloids were separated on baseline by capillary zone electrophoresis with a buffer solution of pH3.2,360mmol/L tartaric acid. Cation-selective exhaustive injection, as an on-column preconcentration method, was also investigated for the determination of the alkaloids of cigarette samples.With the proposed method, both the resolution and detection limit of the alkaloids were improved comparing with those in phosphate buffer.
(3)Using chiral purity hydroxy acid salt as buffer system, through their hydrogen bonds stereo selective, the separation of amino acid chiral isomers were accomplished with the help of stereo selective hydrogen bonds interaction. The method peoposed a new strategy for the separation of chiral isomers.
(4) Because hydroxy acid salt can bond to DNA, which is rich in lonely pair electronics, the longer the DNA molecular chain, the more the hydrogen bond between hydroxy acid salt and DNA forms, so more resistance DNA receives, more slowly the DNA move towards to detect window, then DNA fragments separate out one by one according to the length of DNA. The experiments demonstrated the interation. separation of DNA Marker was completed successfully using citric acid salt as buffer system.
(5) Basing on DNA transient entanglement coupling and improvement of mechanisms of sieve theory in the non-cross-linked polymer solution under high electric field intensity, especially the intra-molecular hydrogen-bonding interaction between DNA and buffer solution, fully utilizing the principle of the separation of DNA:when DNA molecules migrating, DNA fragments tangling or colliding with buffer solution molecules, the larger the DNA,the more the amount of buffer solution molecules encountered and tangled, leading the lower mobility, so separated on the size of DNA fragment. Citrate buffer solution, as a buffer and screening media, was applied to separate single-strand DNA by high performance capillary zone electrophoresis, the analytical method was high precision and good reproducibility.
引文
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