1 / 105

Capillary electrophoresis

Capillary electrophoresis. 資料處理 系統. 毛細管. 毛細管 I.D. 25-100 m m. 偵測器. 注入端. 偵檢端. 白金電極. 電解質緩衝溶液. 電解質緩衝溶液. 高電壓 (KV). 毛細管電泳 (Capillary Electrophoresis-CE) 儀器結構簡圖. Electrophoretic migration. (1). Z i ： charge # of component i e 0 ： elemental charge [ 1.602 ×10 -19 C ]

quiana
Download Presentation

Capillary electrophoresis

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Capillary electrophoresis

  2. 資料處理 系統 毛細管 毛細管 I.D. 25-100 mm 偵測器 注入端 偵檢端 白金電極 電解質緩衝溶液 電解質緩衝溶液 高電壓(KV) 毛細管電泳(Capillary Electrophoresis-CE)儀器結構簡圖

  3. Electrophoretic migration (1) Zi：charge # of component i e0：elemental charge [ 1.602×10-19 C ] E=V/L ( V：applied voltage；L：length ) (2) ri ：stokes radius of i component η：viscosity of the elution [Pa · S] Vi0：migration velocity of i component

  4. (3) (4) μi0：electrophoretic mobility μi0和(q, η, ri)有關 Electroosmotic flow (EOF) (5) μeo：electroosmotic mobility (cm2/Vs) (6) ξ：zeta potential (V) 和pH值, ionic strength有關 η：viscosity of double layer ~ η (bulk)

  5. m = er e0x / h

  6. + + n - - EOF + n Net - 毛細管電泳的向量圖

  7. Effective Mobility and Apparent Mobility

  8. (7) (8) (9) if no EOF (10)

  9. Efficiency (11) (12) (13) (14) (15)

  10. (16) (17) (18) 對某一analyte , μ和D 是constant V固定 D愈小 N愈大 對大分子分離有利

  11. Resolution (19) (20) ：平均速度 ：平均距離 (cm) (21) (22)

  12. (23) EOF存在 (24) 代入(24) (25) (26) EOF和electrophoretic反方向則μeo↑ Rs↑ Best Resolution 分離時間很長

  13. Reversal of EOF Using a Cationic Surfactant

  14. (22)式Rs=column eff. × selectivity factor selectivity →separation factorαin chromatography • Buffer composition • Complex formation • 1. Borate complexation 2. ion pairing • 3. inclusion complex 4. metal complexation • Organic modifier • After the polarity & viscosity of the mobile phase EOF & • electrophoretic mobility changed • 2. 增加solubility of analyte • 3. reduce zeta potential

  15. Micellar Electrokinetic Chromatography k’：capacity factor nmc：no. of analyte molecules incorporated into the micelle naq ：no. of analyte molecules in aqueous phase (1) fraction of analyte in aqueous phase (2) fraction of analyte in micelle (3) (4) migration velocity (Vs)： (5) tR, t0, tmc：migration time of analyte, aqueous phase, and micelle (6) (7)

  16. (micelle停在capillary) << (5)(6)(7)代入(4) (8) (9) t0=∞ EOF is completely suppressed, in MEKC EOF is not essential. 這時分析物向正(+)移動，k’值愈大移動速率愈快 (10)

  17. Resolution (11) tmc→∞ 兩式相等tmc→∞固定相

  18. large capacity better resolution lead to Effect of the capacity factor (12) t0/tmc=0 tmc=∞ conventional chromatography in MEKC large k’ unfavor (11)式最後一項→0 optimum k’和t0/tmc有關 將(12)式微分 (13)

  19. capacity factor ：specific volume of micelle (14) k：distribution coefficient Vmc,Vaq：volumes of micelle and aqueous phases (15) (16) when (Vmc<<Vaq) << 1 (17) k’ vs conc. of surfactant Csf linear if k is constant 可調整Csf來調整k‘ RP-HPLC調整phase ratio對k‘改變小

  20. hydrophilic (ionic group) hydrophobic Effect of EOF on the resolution > > 愈小 Rs愈大 (from 11式) < 1 separation factor ← micelle (stationary phase in RP-HPLC) aqueous (mobile phase in RP-HPLC) type of surfactants：

  21. Separation Mode: Capillary Gel Electrophoresis (CGE)

  22. CGE: Protein Size Separation Using SDS LinearPolymer Solutions

  23. Separation Mode: Capillary Isoelectric Focusing (CIEF)

  24. Detection: Direct UV Detection

  25. Detection: Indirect UV Detection

  26. Peak profile with long injection time (A) No stacking; (B) stacking.

  27. Injection time:200, 500, 700, 900 sec with SRMM stacking

  28. What is Capillary electrochromatography (CEC) ? CEC = CE + HPLC Electroosmotic flow(EOF) + HPLC stationary CE separation efficiency + HPLC selectivity P.S. CE: Capillary electrophoresis HPLC: High performance liquid chromatography

  29. Driving force of CEC and HPLC EOF VEOF = mE m = er e0x / h E:electrical field strength x:zeta potential Pressure-driven flow <V>= dp:particle diameter

  30. Cross-section view of different types of CEC column a.LC stationary phase- packed column b.Open-tubular column c. Countinuous-bed or monolithic column

  31. Side view of open-tubular CEC column Capillary Functional group coated surface

  32. Advantages and disadvantagesof open-tubular column Advantages: No bubble formation， easy to operate Disadvantages: 1.Low phase ratio 2. On-column UV detection is difficult – optical path-length is short and difficult to align 3. The synthesis of matrix with homogeneous is not easy

  33. What is Sol-Gel? Sol-Gelthat is made from solution-gelation process: Hydrolysis of TMOS under acidic or basic condition yield SiO2 in the form of glass-like material 1.Hydrolysis: 2.Condensation: 3.Polymerization:

  34. Preparation of PTMAFS Sol-Gel DI.water180 μl ＋0.1M HCl 15 μl +500μl TMOS 冰震30分鐘 冰埋5小時 加入TMSPTMA 340 μl 震盪30秒 PTMAFS sol-gel

More Related