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研究生:詹富閔
研究生(外文):Jan,Fu-Min
論文名稱:利用形體篩分管柱配合線上同位素稀釋感應耦合電漿質譜法及二維形體篩分/逆相層析法進行人體血清中含金屬元素生物分子之定量及含砷生物分子之結構研究
論文名稱(外文):Characterization of Essential Elements and As-containing Biomolecules in Human Serum by Size-Exclusion coupled with On-line Isotope Dilution Method and 2-D SEC/RPLC Chromatographic Techniques
指導教授:楊末雄楊末雄引用關係
指導教授(外文):Yang, Mo-Hsiung
學位類別:碩士
校院名稱:國立清華大學
系所名稱:原子科學系
學門:工程學門
學類:核子工程學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:144
中文關鍵詞:形體篩分管柱逆相層析管柱感應耦合電漿質譜儀生物分子
外文關鍵詞:size exclusion columnreverse phase columnICP-MSbiomolecule
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摘要
含微量元素之生物分子在生物體內扮演許多重要之生化功能。相關的物種分析工作,近年來已成為分析化學的一個重要研究主題。此項研究有助於了解微量元素在生物體內的可利用性(bioavalibility)、循環(cycling)、轉化(transformation)及生物累積(bioaccumulation)等生化作用之機制。但由於此類含微量元素生物分子在體內之濃度甚低,且樣品基質十分複雜,因此在方法的發展上仍有諸多困難有待解決。
本研究利用連線系統的概念,建立二維形體篩分/逆相層析管柱配合感應耦合電漿質譜儀,進行此類生物金屬(biometals)之分析工作。首先探討SEC-(UV)-ICP-MS連線系統的最佳化條件,繼再以其應用於人體血清中生物金屬(Cu、Zn、Cd、Pb、Co及As-containing biomolecule)的分離及測定。實驗結果顯示,人體血清中有一分子量6,500 Da之生物分子與Cu、Zn、Cd、Pb及Co等元素鍵結;另有一分子量1,500 Da之生物分子則與As元素鍵結。由於As是一致癌性元素,為進一步鑑定此含As分子之化學結構,後續利用二維形體篩分/逆相層析管柱法對於此含As之生物分子進行多次收集,並利用冷凍乾燥法進行濃縮,再進行電灑質譜之分析。初步的結果顯示,電灑質譜圖中有一m/z為1,500 Da之質量蜂,恰與SEC-(UV)-ICP-MS所得者相近。據此,推測人體血清中十分可能有此一分子量為1,500 Da之含As生物分子存在,此一發現迄未見於文獻的報導中。至於此一分子化學結構的解析,則仍有待後續作進一步ESI-CID-MS/MS的探討。
研究中另對於體內之必需元素Cu,開發線上同位素稀釋配合感應耦合電漿質譜儀之連線系統(on/line-ID-ICP-MS),在不需標準品的情況下即可進行精確定量之一種分析方法。所建立之分析系統應用於標準血清樣品(NIST SRM 1598及SeronormTM Trace Elements Serum 311089)中Cu的測定。所測得的結果與確認值頗為相符,顯示方法的可行性。接著,以此連線系統連接SEC管柱(SEC-on/line-ID-ICP-MS)進行人體血清中含Cu之生物分子之線上分離及定量。所得的結果顯示,此含Cu生物分子之分子量約為60,000 Da,推測此含Cu分子可能即為Cu-albumin。接著以同位素稀釋法求得此分子的濃度,估計其約佔血清中總Cu濃度的90%,此結果與文獻上的報導頗相一致。
Abstract
The speciation of trace element, in clinical and biological samples has become a focusing discipline, which provides significant information for biochemist to explore the mechanism of availability, cycling, transformation, bioaccumulation and final fate of these “biometals”. In view of the important role of bio-molecules played in the biological systems, development of reliable analytical method aiming at elucidating the structural and quantitative information about these materials is highly demanded.
For the purpose of characterizing biometals which bind with various bio-molecules, a hyphenation technique coupling size exclusion chromatographic separation and ICP-MS detection was explored. Various parameters of SEC, such as mobile phase flow rate and sample volume on the effect of separation efficiency were investigated. Following the optimized separation condition, trace amount of Cu, Zn, Cd, Pb and Co in human serum were found bound with the biomolecule of M.W.~6,500 Da and a broad As-containing peak corresponding to M.W.~1,500 Da was observed on SEC-ICPMS chromatograph. The identification of the species was prelimernarily investigated with a molecular specific ESI-MS technique.
In this study an on-line SEC-ID-ICPMS system for the quantitative determination of binding and non-binding species of Cu in human serum has also been developed. The accuracy of this hyphenation system for the determination of total concentration of Cu in human serum was verified with the use of NIST SRM 1598 and 311089. With this SEC-ID-ICP-MS system, the presence of the Cu-biomolecule of molecular weight of about 60,000 Da, was found to be about 90% of the total Cu concentration, which is in good agreement with that reported in the literature.
目錄
中文摘要…………………………………………………………….….………..….I
英文摘要…………………………………………………………….….………..…II
謝誌……………………………………………………….…..……………….…...III
目錄……………………………………………………...…………………….…...IV
表目錄………………………………………………...………………….….…...VIII
圖目錄………………………………………………………...……….…………..IX
第一章 前言…………………………….……………………………...………….....…….…...1
1.1生物體內微量元素的重要性……………………………...……….…….….….1
1.2生物體內元素物種分析的重要性………………………...…….……..…....….1
1.2.1元素物種分析定義之討論及其應用………………………..….…….…..…..…...….2
1.2.2生物體內之元素物種分析的難題…………………………..………….……..…..….4
1.2.3元素物種分析技術的需求……………………………………………...……..…..….6
1.3金屬與類金屬(metalloid)之生理意義……………………..……….....……..…8
1.3.1銅(copper)……………………………………………………...………………...…....8
1.3.2鋅(zinc)…………………………………………………………..……………....……8
1.3.3鎘(cadmium)……………………………………………………..…………...….…....9
1.3.4鉛(lead).….……………………………………………………….………...…….….10
1.3.5鈷(cobalt)…..………………………………………………………..……….……....10
1.3.6砷(arsenic)…...……………………………………………………….....……………11
1.4文獻回顧……………………………………………………..……..….…....….12
1.5研究目的……………………………………………….……………………….14
第二章 分析儀器原理……………………………………………………….…….15
2.1形體篩分層析法(size exclusion chromatography,SEC)….…………..…..…15
2.1.1 SEC的分離原理與影響SEC分離能力之因素……………………….……....…...16
2.1.2 SEC的填充材料……………………………….…………………….………....…...19
2.1.3分子量校正法……………………………….…………………………………....…21
2.1.4非形體篩分效應(nonsize exclusion effect)………………………….……….…....21
2.1.5 SEC的優缺點……………………………………………………………….…..…..21
2.2高效能逆相層析管柱原理(reverse phase chromatography, RPC)…….….…22
2.2.1逆相層析管柱的填充材料…………………………………………….……...…...23
2.2.2胺基酸的基本性質…………………………………………….……………...…...23
2.2.3 RPC的特殊分離模式…………………………………………….…...……...…....25
2.2.4 RPC的優缺點…………………………………………….…...……………...…....26
2.3感應耦合電漿質譜儀分析法(ICP-MS)….…………………………....….….26
2.3.1感應耦合電漿離子源(the ICP as an ion source)………………..…….…….…......27
2.3.2離子萃取(ion extraction)………………..……………………………………….....29
2.3.3離子聚焦(ion focusing)………………..……………………………...……………29
2.3.4四極柱質量分析器(quadrupole mass analyzer).…………………...……….….….29
2.3.5離子偵側(ion detection)………………..……………………………..……...…….30
2.3.6感應耦合電漿質譜儀的分析特性與限制……………………………..…..………31
2.4電灑串聯質譜儀分析法(ESI-MS/MS)………………………………….…...…33
2.4.1電灑過程的參數.……………………………………………………...……..…….37
2.4.1.1電灑針頭位置………………………………………………….....………...37
2.4.1.2屏幕氣體流速…………………………………………………...……….....37
2.4.1.3電灑電壓………………………………………………………...……….....38
2.4.1.4加速氣體流速與溫度…………………………………………...……….....38
2.4.1.5霧化氣體流速………………………………………………………………38
2.4.1.6溶劑組成……………………………………………………………………39
2.4.1.7樣品內離子濃度……………………………………………………………39
2.4.2串級質譜的偵測模式………………………….………………………...………...39
2.4.3電灑質譜之解析原則………………………………………………...…...…….....41
2.5同位素稀釋質譜法(isotope dilution mass spectrometry,IDMS)….....….....42
2.5.1同位素稀釋法配合質譜儀測定元素的原理…………………………………...…42
2.5.2線上同位素稀釋配合管柱層析進行物種分析之方法…………………………...44
2.5.3影響同位素稀釋法準確度之因素與其解決方法…………………………..…….45
第三章 實驗部分…………………………………………..………..……….…..49
3.1儀器裝置………………………………………………………………….......49
3.2藥品及用水………………………………………………………….……......49
3.3容器清洗…….…………………………………………...…………...……...50
3.4實驗室空氣…….………………………………………...…….……....…….51
3.5層析流洗液之配製…….………………………….…..…….…………...…..51
3.6分離管柱之使用及保存…….……………………………………...….….....51
3.7感應耦合電漿質譜儀的操作條件……...……………………………..…….52
3.8連線系統的建立…….………………………….…..…………..……..…......52
3.9實驗流程…….……………………………….………..……………..….…...53
第四章 結果與討論………………………………………...…………..…..…...56
4.1建立SEC-(UV)-ICP-MS連線分析系統…………………….…….…….….56
4.1.1 SEC參數之最佳化…………………………………….….………………….…..56
動相之選擇………………………………………………………..……...…….....56
pH值…………………………………………………………………….……..….58
SEC管柱的選擇……………………………………………….….…..………..…58
動相流速……………………………………………….…………………….…....58
樣品注入量…………………………………………………………….…...….….59
4.1.2 SEC-ICP-MS操作參數的最佳化…………………………………..………..…..59
RF power………………………………………………………….………....….....60
霧化氣體流速………………………………………………….….………………60
4.1.3人體血清中含金屬元素的生物分子………………………..…….…….....…….60
4.2血清中含As生物分子之探討………………………………..………….....63
4.2.1 RPC-UV-ICP-MS系統之建立…………………………..………………..……...64
4.2.1.1 RPC分離參數之探討………………………………………………………..64
RPC管柱的選擇……………………………………………………………....…..64
動相種類之選擇…………………………………………………………….....….64
梯度流洗程式之探討………………………………………………………….….65
4.2.1.2 RPC-(UV)-ICP-MS系統探討…………………………………...……….….65
RF power………………………………………………………………………..…65
霧化氣體流速……………………………………………….………………….....65
有機溶劑在電漿中造成的影響……………………………………………….….66
通氧之影響…………………………………………………...……………….…..67
4.2.1.3利用SEC-RPC-(UV)-ICP-MS系統分析含As之物種………………..…...68
4.2.2血清中含砷分子之結構探討…………………………………………….…....…68
4.2.2.1小分子砷物種存在的可能性探討…………………………….……....…….68
4.2.2.2砷與穀胱酸生成錯和物(GSH-As)之研究…………...…….……….….....71
4.2.3人體血清中含砷元素生物分子之探討…………...………….……...…….…….74
4.3利用SEC-ID-ICP-MS進行人體血清中含Cu生物分子之定量……...…..75
4.3.1 SEC-ID-ICP-MS系統的建立………………...……………………….….….......76
4.3.2質量歧視效應以及基質干擾效應…………………………………….…….......76
4.3.3方法準確度與精密度………………………..……………………….……….....77
4.3.4 SRM與真實樣品之總濃度與物種濃度的測定…………………….….….........78
第五章 結論…………...……………………………………………....…….….81
第六章 參考文獻………..…………………………………………...……........82
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