微分离系统中整体/混合毛细管色谱柱的制备与性能评价方法研究
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摘要
液相色谱-质谱联用技术(Liquid chromatography-mass spectrometry,LC-MS)是结合了色谱的分离及质谱的定性分析性能于一体的一种分析方法。LC-MS技术具有很高的选择性、灵敏度、分离度和分析速度。近年来,随着LC-MS新技术的不断发展和完善,该技术已经成为现代分析手段中不可缺少的组成部分,是小分子药物、复杂生物样本中多肽、蛋白质定性、定量分析的有力工具。
本论文首先研究了毛细管整体柱的制备方法。采用甲基丙烯酸月桂酯作为功能单体,乙二醇二甲基丙烯酸酯为交联剂,十二醇、1,4-丁二醇及二甲亚砜混合物为致孔剂,在内径为75μm、不同长度的石英毛细管内制备了具有良好机械性能和化学稳定性的反相毛细管整体柱,并对所合成的毛细管整体柱进行了电镜表征、流速测定、柱长与柱压的变化关系分析,表明毛细管整体柱的通透性良好,柱压低。因此,可通过延长柱长的方法进一步提高其分离度。将所制备的毛细管整体柱装于高效液相色谱仪上进行牛血清白蛋白和血浆样本的胰酶酶解液的分离,表明所制备的毛细管整体柱可以满足一般多肽混合物的分离分析。
在以上研究的基础上,制备了一种应用于液-质联用系统中的带尖头的新型混合型色谱柱。在毛细管喷头部位制备长度约为1 cm的反相毛细管整体填料作为筛板,然后在该毛细管色谱柱的其余部分,采用匀浆装柱法填充颗粒度为5μm的C18硅胶填料(Octadecylsilyl,ODS),再将其装于LC-MS系统中,以标准蛋白、酵母蛋白为分析对象,考察其在LC-MS中离子化效率、色谱柱性能和寿命,并与商品化的色谱柱进行色谱性能比较,未见显著性差异。在实际应用中分离分析鼠肝细胞全蛋白酶切液,将强阳离子交换的28个馏分分别进行分离分析,通过数据处理证实获得了较好的分离效果,在假阳性率为1%的条件下鉴定到1262个肽段,513个蛋白簇。实验结果表明该混合型毛细管色谱柱没有偏性,可以在液-质联用系统中获得应用。
Liquid chromatography coupled with mass spectrometry (LC/MS)is one of the analytical techniques widely used in analytical areas, which combines separating property of chromatography and identification power of mass spectrometry. The use of LC-MS enabled rapid and efficient separation and identification of samples.In recent years,LC-MS has emerged as an important tool in qualitative and quantitative assay of medicines and complicated bio-specimens.
Capillary monolithic columns (75μm i.d and different length) are prepared in this research by copolymerization of lauryl methacrylate as the basic monomer,ethylene dimethacrylate as the cross-linking agent and 1-dodecyl alcohol,1,4-butanediol and dimethyl sulfoxide as a porogenic mixture. The synthetic stationary phases have better mechanical property and chemical stability. A series of characterization and evaluation were performed on the capillary monolithic column including SEM image, the influence of polymer property of the capillary monolithic columns on the separation of peptide mixtures by changing the proportion of the porogen solution and cross-linking agent. The test of relationship between column length and back pressure shows that the capillary monolithic columns prepared have superior permeability, so longer capillary monolithic columns can be prepared to improve the resolving power. The prepared capillary monolithic columns were fitted on a nano-scale high performance liquid chromatography for the separation of tryptic digests of bovine serum albumin(BSA) and human plasma samples, and good results have been obtained.
On the basis of above research, we prepared the monolithic material in the emitter of capillary columns with an emiter as a cribellum by using lauryl methacrylate-ethylene dimethacrylate (LMA-EDMA),and then the other empty parts of capillary columns with an emiter were packed with octadecylsilica particles(ODS).The property of monolithic parts of the capillary columns is similar to that of reversed-phase packing materials used in the capillary columns which benefits the improvement on separation efficiency and avoid the clogging problem of the capillary columns in the tapered tips.Finally the parameters of the mixed columns such as peak capacity, ionization efficiency,signal intensity and the coverage of identified peptides or proteins were examinated. We also evaluated and compared the data with those obtained using both a mixed capillary column and a traditional packed capillary column to separate the tryptically digested proteins extracted from hepatic cells of mousse and appreciated 1262 peptides and 513 protein groups.The results show that the mix-capillary column had no bias,and this type of the column will play an important role in LC/MS system in the future.
本论文首先研究了毛细管整体柱的制备方法。采用甲基丙烯酸月桂酯作为功能单体,乙二醇二甲基丙烯酸酯为交联剂,十二醇、1,4-丁二醇及二甲亚砜混合物为致孔剂,在内径为75μm、不同长度的石英毛细管内制备了具有良好机械性能和化学稳定性的反相毛细管整体柱,并对所合成的毛细管整体柱进行了电镜表征、流速测定、柱长与柱压的变化关系分析,表明毛细管整体柱的通透性良好,柱压低。因此,可通过延长柱长的方法进一步提高其分离度。将所制备的毛细管整体柱装于高效液相色谱仪上进行牛血清白蛋白和血浆样本的胰酶酶解液的分离,表明所制备的毛细管整体柱可以满足一般多肽混合物的分离分析。
在以上研究的基础上,制备了一种应用于液-质联用系统中的带尖头的新型混合型色谱柱。在毛细管喷头部位制备长度约为1 cm的反相毛细管整体填料作为筛板,然后在该毛细管色谱柱的其余部分,采用匀浆装柱法填充颗粒度为5μm的C18硅胶填料(Octadecylsilyl,ODS),再将其装于LC-MS系统中,以标准蛋白、酵母蛋白为分析对象,考察其在LC-MS中离子化效率、色谱柱性能和寿命,并与商品化的色谱柱进行色谱性能比较,未见显著性差异。在实际应用中分离分析鼠肝细胞全蛋白酶切液,将强阳离子交换的28个馏分分别进行分离分析,通过数据处理证实获得了较好的分离效果,在假阳性率为1%的条件下鉴定到1262个肽段,513个蛋白簇。实验结果表明该混合型毛细管色谱柱没有偏性,可以在液-质联用系统中获得应用。
Liquid chromatography coupled with mass spectrometry (LC/MS)is one of the analytical techniques widely used in analytical areas, which combines separating property of chromatography and identification power of mass spectrometry. The use of LC-MS enabled rapid and efficient separation and identification of samples.In recent years,LC-MS has emerged as an important tool in qualitative and quantitative assay of medicines and complicated bio-specimens.
Capillary monolithic columns (75μm i.d and different length) are prepared in this research by copolymerization of lauryl methacrylate as the basic monomer,ethylene dimethacrylate as the cross-linking agent and 1-dodecyl alcohol,1,4-butanediol and dimethyl sulfoxide as a porogenic mixture. The synthetic stationary phases have better mechanical property and chemical stability. A series of characterization and evaluation were performed on the capillary monolithic column including SEM image, the influence of polymer property of the capillary monolithic columns on the separation of peptide mixtures by changing the proportion of the porogen solution and cross-linking agent. The test of relationship between column length and back pressure shows that the capillary monolithic columns prepared have superior permeability, so longer capillary monolithic columns can be prepared to improve the resolving power. The prepared capillary monolithic columns were fitted on a nano-scale high performance liquid chromatography for the separation of tryptic digests of bovine serum albumin(BSA) and human plasma samples, and good results have been obtained.
On the basis of above research, we prepared the monolithic material in the emitter of capillary columns with an emiter as a cribellum by using lauryl methacrylate-ethylene dimethacrylate (LMA-EDMA),and then the other empty parts of capillary columns with an emiter were packed with octadecylsilica particles(ODS).The property of monolithic parts of the capillary columns is similar to that of reversed-phase packing materials used in the capillary columns which benefits the improvement on separation efficiency and avoid the clogging problem of the capillary columns in the tapered tips.Finally the parameters of the mixed columns such as peak capacity, ionization efficiency,signal intensity and the coverage of identified peptides or proteins were examinated. We also evaluated and compared the data with those obtained using both a mixed capillary column and a traditional packed capillary column to separate the tryptically digested proteins extracted from hepatic cells of mousse and appreciated 1262 peptides and 513 protein groups.The results show that the mix-capillary column had no bias,and this type of the column will play an important role in LC/MS system in the future.
引文
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