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研究生:林盈瑩
研究生(外文):Ying-Ying Lin
論文名稱:毛細管電泳技術應用於奈米級顆粒的粒徑與形狀分析
論文名稱(外文):Size and shape separation of nanometer-sized particles by capillary electrophoresis
指導教授:吳劍侯
指導教授(外文):Chien-Hou Wu
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:2004
畢業學年度:92
語文別:英文
論文頁數:126
中文關鍵詞:毛細管電泳技術銀奈米粒子大小形狀PEOSDS
外文關鍵詞:Capillary electrophoresisgold nanoparticlessize separationpoly(ethylene oxide)SDSsilver nanoparticles
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本研究的主要目的為利用毛細管電泳技術建立分離方法並對快速鑑定奈米等級粒子大小與形狀之可行性進行評估。論文由兩部分組成,第一部分為以毛細管電泳技術建立金奈米粒子粒徑大小的分析方法。探討其分離機制,並應用於以微波加熱方法製備的真實樣品。第二部分為以毛細管電泳技術建立不同形狀之銀奈米粒子分離技術,配合線上濃縮概念,建立動態酸鹼梯度掃集毛細管微胞濃縮技術以分離不同形狀之銀奈米粒子。
添加陰離子界面性劑SDS與線性聚合物PEO於緩衝液中,利用毛細管電泳技術能成功的分離與鑑定不同大小的金奈米粒子。在粒徑5到40奈米範圍,遷移時間和粒徑大小有很好的線性關係存在,且遷移時間的再現性佳(CV<4.1%),可用以推估出真實樣品粒徑大小。添加SDS於緩衝液中對於分離效果有明顯的提升,應是基於其吸附於粒子表面藉以改變電荷體積比之故;添加PEO也有助於分離,應是由於其與粒子間的作用力而使得分離效果提升。分析真實樣品,比對所建立之分析方法得到結果與以掃描式電子顯微鏡觀測統計後得到粒徑大小之分佈結果,兩者有相當好的一致性。
第二部分為建立動態酸鹼梯度掃集毛細管微胞濃縮技術並應用於分離不同形狀之銀奈米粒子。研究所用之銀奈米粒子經掃描式電子顯微鏡觀測統計後,證實其為棒狀與球狀奈米粒子以27比73之混合溶液。由於所使用之奈米粒子差異不大,在本研究中,利用酸鹼值與導電度梯度以及SDS濃度差,使奈米粒子能有效的被集中並達到分離。於此,探討線上濃縮的分離機制並推估銀奈米粒子與SDS之作用機制應不同於金奈米粒子。本實驗建立之分析方法的遷移再現性良好(CV<1.8 %),最後利用分段收集法收集被分離之奈米粒子,並以穿透式電子顯微鏡確認其形狀與大小,證實本方法確實可行。
The feasibility of separation of nanoscale metal particles either by size or shape with capillary electrophoresis (CE) is demonstrated. This dissertation is composed of two main parts (i) size separation of gold nanoparticles and (ii) shape separation of silver nanoparticles. In the first part, a separation method by adding anionic surfactant and polymer was established. The separation mechanism of surfactant and polymer was probed. In the second part, a dynamic pH gradient sweeping on-line concentration was build up and separation of different shaped silver nanoparticles can be achieved. The separation can be confirmed with fraction collection to take TEM images.
CE with anionic surfactant, sodium dodecyl sulfate (SDS), and linear polymer, poly (ethylene oxide) (PEO), can successfully separate gold nanoparticles with different sizes. This work demonstrates the feasibility of employing CE to separate gold particles in nanoscale regimes. After addition of SDS and PEO to the buffer, particles with different sizes were separated simultaneously. Parameters including buffer concentration, SDS concentration, percentage of PEO, pH value, and applied voltage are investigated to obtain the optimized separation resolution. The separation mechanism of PEO and the effect of SDS are also discussed here. Most important of all, a linear relationship between the migration time and particle size is obtained in the particle diameters range of 5 – 40 nm. The coefficient of variation of migration time for 5 nm and 20nm gold nanoparticles are 3.5 and 4.1 %, respectively. Real samples made by microwave heating method have been analyzed and the analytical results of CE show a good fit with the statistical size distribution from SEM images. This study provides an alternative method for rapid separation and characterization of nanoscale gold with different particle sizes.
This study also demonstrated the feasibility of CE use to separate silver nanoparticles with different shapes. Herein, a dynamic pH gradient-sweeping on-line concentration method had been established and employed to separate silver nanoparticles. The concentration of SDS and pH difference between leading and terminal buffer were optimized. More than 12 runs showed a very similar(CV<1.8 % in migration time)on-line concentration effect. Besides, the mechanism of the dynamic pH gradient-sweeping is proposed in this study. To confirm the effect of this separation method, a fraction collection is used to collect the separated silver nanoparticles and observe by TEM. From TEM images, spherical nanoparticles are successfully separated by CE and the particles of 2nd peak in electropherogram were collected.
Abstract(Chinese)…………………………………… i
Abstract(English)…………………………………… ii
Acknowledgement……………………………………… iv
Content Index ………………………………………….. vi
Figures Index………………………………………….... xi
Table Index……………………………………………... xv
Abbreviation and Symbol……………………………… xvi


Chapter 1 General Introduction………………………… 1
1.1 The history of capillary electrophoresis Technique......... 2
1.2 The separation theory of capillary electrophoresis…….. 4
1.2.1 Electrophoresis………………………………………. 4
1.2.2 Electroosmotic flow and zeta potential……………… 5
1.3 The separation mode of capillary electrophoresis……... 8
1.3.1 Capillary zone electrophoresis………………………. 8
1.3.2 Micellar electrokinetic chromatography…………….. 9
1.3.3 Capillary gel electrophoresis………………………… 10
1.4 Selectivity and the use of additives……………………. 13
1.4.1 Buffer selection……………………………………… 14
1.4.2 Buffer pH…………………………………………….. 15
1.4.3 Surfactants…………………………………………… 15
1.4.4 Chiral selectors………………………………………. 16
1.4.5 Temperature………………………………………….. 17
1.4.6 Sample injection width………………………………. 18
1.5 Reference……………………………………………… 20
Chapter 2 Experimental Section………………………… 22
2.1 Apparatus……………………………………………… 23
2.2 Capillary Rinsing Protocols……………………………. 24
2.3 Procedure………………………………………………. 25
2.3.1 Buffer preparation…………………………………… 25
2.3.2 Sample……………………………………………….. 25
2.4 Calculation…………………………………………….. 25
2.4.1 Resolution……………………………………………. 25
2.4.2 Mobility and migration time…………………………. 26
2.4.3 Injection volume……………………………………... 27
2.5 Sample Injection Method……………………………… 29
2.6 Buffer Replenishment and Set…………………………. 30
2.7 Reference………………………………………………. 31
Chapter 3 Size Separation of Gold Nanoparticles by Capillary Electrophoresis................................ 33
3.1 Introduction…………………………………….……… 34
3.2 Experimental section…………………………………... 38
3.2.1 Apparatus………………….......................................... 38
3.2.2 Chemicals and reagents……………………………… 40
3.2.3 Nanoparticles samples……………………………….. 40
3.2.4 Preparation of PEO solution……………..................... 41
3.2.5 Buffers……………………………………………….. 42
3.3 Results and discussion………………………………… 42
3.3.1 Optimization of this separation method……………... 42
3.3.1.1 Co-additive finding………………………………… 42
3.3.1.2 Optimization of pH value………………………….. 57
3.3.1.3 Separation optimization……………………………. 60
3.3.1.4 Optimization of working condition……………….. 61
3.3.1.5 Summary of separation method optimization……... 67
3.3.2 Real sample analysis………………………………… 71
3.4 Conclusion……………………………………………... 75
3.5 Reference………………………………………………. 76
Chapter 4 Shape Separation of Silver Nanoparticle by Capillary Electrophoresis…................... 80
4.1 Introduction……………………………………………. 81
4.2 Experimental section…………………………………… 84
4.2.1 Apparatus…………………………………………….. 84
4.2.2 Reagents……………………………………………... 84
4.2.3 Silver nanoparticles…………………………………. 84
4.2.3.1 Preparation…………………………………………. 84
4.2.3.2 Sample description ………………………………... 85
4.2.4 Buffers……………………………………………… 86
4.2.5 Fraction collection………………………………….. 86
4.3 Results and discussion…………………………………. 87
4.3.1 Co-additive optimization……………………………. 87
4.3.2 Separation parameters optimization…......................... 90
4.3.2.1 Concentration of SDS……………………………… 90
4.3.2.2 Effect of buffer type and pH value………………… 93
4.3.2.3 Concentration of CAPS…………………………… 95
4.3.3 On- line concentration……………………………… 96
4.3.3.1 On-line concentration by pH gradient…………… 98
4.3.3.2 On-line concentration by sweeping………………. 103
4.3.3.3 Effect of dynamic pH gradient-sweeping………... 106
4.3.4 Proposed mechanism of stacking…………………... 107
4.3.4.1 Mechanism of dynamic pH-gradient……………... 107
4.3.4.2 Mechanism of sweeping………………………….. 109
4.3.4.3 Mechanism of dynamic pH gradient-sweeping…... 110
4.3.5 Separation Results………………………………….. 110
4.4 Reproducibility……………………………………….. 112
4.5 Conclusion….…………………………………............ 112
4.6 Reference……………………………………………... 114
Chapter 5 Conclusion.……………………………………... 119
5.1 Conclusion.………………………………………….. 120
5.2 Suggestions and Future Perspectives………………… 122
5.3 Reference…………………………………………….. 124
The Author…………………………………………….. 125








Figure Index
Figure 2-1 Hardware overview of the Lsuerlabs’ Butler replenishment system.…………………………………………... 24
Figure 3-1 UV spectrum of five standards in running buffer.………….. 39
Figure 3-2 The relationship between concentration of SDS and migration time.…………………………………………….. 45
Figure 3-3 Effect of buffer concentration on the migration time of gold particles………………………………………….. 46
Figure 3-4 Electropherograms of 5nm and 20nm mixture separated with different percentage of PEO 8,000,000……….................. 51
Figure 3-5 The effect of different pH and comparison of different percentage of PEO………………………………….. 54
Figure 3-6 Electropherograms of 5nm and 20nm mixture separated with different molecular weights of PEO.……………………. 55
Figure 3-7 Relationship between the migration time and particle diameter.. 56
Figure 3-8 Effect of pH on (a) Rs and (b) migration time on Au nanoparticles............................................................. 59
Figure 3-9 Comparison of Rs with six different buffer compositions..…... 60
Figure 3-10 Influences of temperature on Rs and current and comparison of their electropherograms.………………………….. 63
Figure 3-11 Effect of voltage on (i) Rs and (ii) maximum current.………. 64
Figure 3-12 Electropherograms of using segmental filling method to separate the mixture of 5nm and 20nm standards.………… 67
Figure 3-13 Standard electropherograms of 5 standards........................ 69
Figure 3-14 The SEM images of a mixture of 20nm and 40nm gold nanoparticles……………………………………... 69
Figure 3-15 Electropherograms of 30nm and 40nm gold nanoparticles.…. 70
Figure 3-16 Standard curve.…………………………................ 70
Figure 3-17 SEM images and statistic size distribution graphs of real samples.…………………………………………. 72
Figure 3-18 Electropherograms of four real samples under optimized separation condition.……………………………... 73
Figure 3-19 The correlation of CE and SEM determined particle size.…… 74
Figure 4-1 UV/VIS absorption spectrum of silver nanoparticle sample used thoughout this study. ………………………….......... 85
Figure 4-2 SEM image of the silver nanoparticle sample used in this study. 86
Figure 4-3 Effect of adding Brij35 as co-additive to the running buffer….. 88
Figure 4-4 Effect of adding PEG as co-additive to the running buffer…… 89
Figure 4-5 Effect of adding PEO as co-additive to the running buffer…… 89
Figure 4-6 Electropherogram obtained for silver nanoparticle separation at different concentration of SDS.………………………... 91
Figure 4-7 UV spectrum of 0.01mM silver nanoparticle at pH 10 using different SDS concentration…………………………. 92
Figure 4-8 Electropherograms of 5nm gold nanoparticle at different concentration of SDS.………………………….......... 92
Figure 4-9 Electropherograms of silver nanoparticle with different buffer composition.……………………………………… 93
Figure 4-10 Electropherograms of silver nanoparticle at different pH value..................................................................... 94
Figure 4-11 Electropherograms of different concentration of CAPS.......... 95
Figure 4-12 Schematic illustration of capillary buffer system…………. 99
Figure 4-13 Effect of different pH at inlet and outlet buffer.………....... 100
Figure 4-14 Comparison of different pH gradient method...................... 102
Figure 4-15 Effect of different injection volume……………………. 102
Figure 4-16 Comparison of the electropherograms with different pH gradient.………………………………………… 103
Figure 4-17 Effect of SDS gradients.…………………………….. 105
Figure 4-18 Comparison of different SDS concentration of outlet and flush vial.…………………………………………….. 106
Figure 4-19 Schematic overview of proposed mechanism of dynamic pH-gradient sweeping of silver nanoparticles.…………… 108
Figure 4-20 TEM images of separated silver nanoparticles collect by fraction collection method.………………………….. 111
Figure 4-21 Injection volume test……………………………….. 113
Figure 4-22 Effect of stacking or not…………………………….. 113




















Table Index
Table 3-1 Altered parameters and effects of adding amines to improve the separation efficiency.……………………………….. 43
Table3-2 Conditions tested to increase the separation efficiency with segmental filling…………………………………… 66
Table 3-3 Size distribution calculated from electropherogram with standard curve……………………………………... 73
Table 3-4 Comparison between mean of two methods and random error with 95% confidence level…………………………… 75
Table 4-1 Conditions tested to study pH-gradient effect.……………... 99
Table 5-1 The optimum conditions and results of this thesis…….......... 120
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