1. Capillary electrophoresis

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

4. Electrophoretic migration Z i ： charge # of component i e 0 ： elemental charge [ 1.602×10 -19 C ] E=V/L ( V ： applied voltage ； L ： length ) r i ： stokes radius of i component η ： viscosity of the elution [Pa · S] V i 0 ： migration velocity of i component (1) (2)

5. μ i 0 ： electrophoretic mobility μ i 0 和 (q, η, r i ) 有關 Electroosmotic flow (EOF) (3) (4) μ eo ： electroosmotic mobility (cm 2 /Vs) (5) (6) ξ ： zeta potential (V) 和 pH 值, ionic strength 有關 η ： viscosity of double layer ~ η (bulk)

8.  =  r    / 

10. + - n EOF -+ + n - Net 毛細管電泳的向量圖

11. Effective Mobility and Apparent Mobility

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

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

14. (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

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

16. 代入 (24) EOF 存在 (23) (24) (25) (26) EOF 和 electrophoretic 反方向則 μ eo ↑ R s ↑ Best Resolution 分離時間很長

17. Reversal of EOF Using a Cationic Surfactant

18. (22) 式 R s =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 1.After the polarity & viscosity of the mobile phase EOF & electrophoretic mobility changed 2. 增加 solubility of analyte 3. reduce zeta potential

23. Micellar Electrokinetic Chromatography fraction of analyte in aqueous phase fraction of analyte in micelle migration velocity (V s ) ： t R, t 0, t mc ： migration time of analyte, aqueous phase, and micelle k’ ： capacity factor n mc ： no. of analyte molecules incorporated into the micelle n aq ： no. of analyte molecules in aqueous phase (1) (2) (3) (6) (5) (7) (4)

24. (5)(6)(7) 代入 (4) (micelle 停在 capillary) << t 0 =∞ 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 (8) (10) (9)

25. Resolution t mc →∞ 兩式相等 t mc →∞ 固定相 (11)

26. Effect of the capacity factor t 0 /t mc =0 t mc =∞ conventional chromatography large capacitybetter resolution lead to in MEKC large k’ unfavor (11) 式最後一項 →0 optimum k’ 和 t 0 /t mc 有關 將 (12) 式微分 (12) (13)

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

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

32. Chiral Capillary Electrophoresis (CCE)

36. Separation Mode: Capillary Gel Electrophoresis (CGE)

37. CGE: Protein Size Separation Using SDS Linear Polymer Solutions

38. Separation Mode: Capillary Isoelectric Focusing (CIEF)

39. Detection: Direct UV Detection

42. Detection: Indirect UV Detection

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

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

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

46. EOFV EOF =  E  =  r    /  E:electrical field strength  :zeta potential Pressure-driven flow = d p :particle diameter Driving force of CEC and HPLC

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

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

50. Advantages and disadvantages of 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

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

52. 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 Preparation of PTMAFS Sol-Gel

53. 溶膠凝膠塗佈合成步驟示意圖 (a) 合成步驟。 (a)

54. 溶膠凝膠塗佈毛細管內塗佈結果示意圖

55. Electron micrographs of a PTMAFS coating inside a fused-silica capillary A:10 s coating, flushing with water 30 s, aging in water B:10 s coating, flushing with water 4 min, aging in water

56. Anion-exchange behavior of PTMAFS coated capillary Migration time (min) Absorbance Unit. Running buffer 20 mM phosphate pH 3.0; Analyte: terephthalic acid 60 mM co-anion a:citrate b:sulfate c: nitrate d:chloride

57. CEC separation Mechanism

58. a.a. b c d 燒結儀器裝置. a. 矽酸鹽溶液 b. 蠕動幫浦 c. 燒結器 d. 電源供應器

59. a. Ni-Cr wire Closed view of a Ni-Cr wire heating head

60. Experimental conditions: capillary, 34.5 cm in length,75um i.d., 375um o.d..Suspension of ODS-2 particles in 75/25 IPA/MeOH at concentration of 0.2g/mL, packing pressure with 5000psi. Preparation a packed CEC column

61. 75 μm Picture of a frit by optical microscope with digital camera

62. Compound Structure molecular weight Testosterone 288.4 17-Methyltestosterone 302.4 Progesterone 344.5 Testosterone propionate 314.5 Steroids structures used for C 18 packed CEC separation.

63. Effect of MeCN concentration on EOF Mobile phase: 10 mM Tris at pH 8.0. Column: 35 x 75  m I.D. Bed length: 27.5 cm. Injection: 5kV with 20s. Applied voltage: 20 kV. Detection: 254 nm.

64. Volume fraction of MeCN effect on the migration of cholesterols.  : 17-methyltestosterone, : testosterone propionate.

65. Column: 35cm×75um I.d. Bed length: 27.5cm. Mobile phase: MeCN / 10mM Tris pH 8.0. Sample: 15 mg/L. Applied voltage: 20kV. Injection: 5kV. Detection: 254nm. 1. Testosterone; 2. 17-Methyltestosterone; 3. Progesterone; 4:Testosterone propionat. Chromatographic separation of 4 steroids.

66. Online pre-concentration in CEC Solid phase micro extraction (SPME) concept for the stacking of analyte in CEC column during sample injection.

67. BGS + - SB + - BB SPME Procedure CEC Separation BGS + - SB SSM S1 S2 S3    SPME sample injection

68. Compound Structure molecular weight 17-Methyltestosterone 302.4 Testosterone 288.4 Progesterone 344.5 Steroids structures used for pre-concentration of SPME-CEC.

69. Effect of MeCN conc. in the sample solution on the electrochromatograms Samples: 1, 17-Methyltestosterone; 2, Testosterone; 3, Propionate. (A), V MeCN : V H2O =80/20; (B), V MeCN : V H2O =20/80..

70. Effect of injection time on peak width at half-height (  ) and peak height (  ) of 17-Methyltestosterone. Experiment conditions: sample solution, V MeCN : V H2O =40/60; other conditions were the same as shown in Fig. 8.3.

71. Electrochromatogram of neutral compounds with SPME-CEC technique. Experimental conditions: mobile phase, V MeCN : V H2O =80/20, 10mM Tris, pH 8.0. sample solution,V MeCN : V H2O =20/80; injections (A) 5kV, 1-s, (B) 5kV, 600-s; Sample concentration: (A) 10mg/L; (B) 0.3mg/L. DL: ~15ppb

72. Naphthalene Acenaphthylene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benz(a)anthracene Chrysene Benzo(b) fluoranthene Benzo(k)fluoranthene Benzo(a)pyrene Indeno(1,2,3-cd)pyrene Dibenz(a,h)anthracene Benzo(ghi)perylene PAHs structure used for pre-concentration of SPME-CEC.

73. Effect of MeCN conc. in the sample solution on the electrochromatograms of PAHs Samples: 1, Naphthalene; 2, Acenaphthene; 3, Pyrene. Sample solution (A), V MeCN : V H2O =100/0; (B), V MeCN : V H2O =40/60.

74. Effect of injection time on the peak width at half-height (  ) and peak height (  ) of pyrene. sample solution, V MeCN : V H2O =40/60.

75. Electrochromatogram of PAHs with SPME-CEC technique. Mobile phase, V MeCN : V H2O =80/20, 10mM Tris, pH 8.0. Samples: 16 PAHs mixture; sample solution, (A) V MeCN : V H2O =100/0, (B) V MeCN : V H2O =40/60; injections (A) 5kV, 6-s, (B) 5kV, 600-s.

76. D.L. enhancement of 16 PAHs mixture by SPME-CEC Analytes original concentration D.L. ---------------------------------------------------------------------------------- Naphthalene 5.0  g/mL 9.8 ng/mL a Acenaphthylene 5.0  g/mL 13.4 ng/mL a Acenaphthene 10.0  g/mL 45.9 ng/mL b Fluorene 1.0  g/mL 6.7 ng/mL b Phenanthrene 0.4  g/mL 1.0 ng/mL a Anthracene 0.2  g/mL 1.2 ng/mL a Fluoranthene 0.5  g/mL 5.1 ng/mL a Pyrene 1.0  g/mL 8.4 ng/mL a Benzo(a)anthracene 0.5  g/mL 5.3 ng/mL a Chrysene 0.5  g/mL 3.9 ng/mL a Benzo(b)fluorine 0.2  g/mL 7.1 ng/mL a Benzo(k)fluorine 0.2  g/mL 9.9 ng/mL a Benzo(a)pyrene 0.5  g/mL 20.7 ng/mL a Dibenzo(a,h)anthracene 2.0  g/mL 284.1 ng/mL a Benzo(g,h,i)perylene 0.8  g/mL 68.5 ng/mL a Indeno(1,2,3-cd)pyrene 0.5  g/mL 28.9 ng/mL a -------------------------------------------------------------------------------------------------- a.Column: Vydac 201 TP-51 packed CEC, injection 5kV 600-s. b.b. Column: ODS-2 packed CEC, injection 5kV 500-s.

77. Enantiomeric resolution of  -blockers by CEC using macro cyclic antibiotic stationary phases

78. 對掌性異構物 (enantiomers): 具有相同的物性及化性。 （乳酸）

80. 1.Dalgiesh 1952 three-point interaction 2. transient diastereomeric complex

81. Structure of Marcrocyclic Glycopeptides

82. Teicoplanin Structure

83. Alprenolol Atenolol Fenoterol Metoprolol Pindolol Propranolol Sotalol

84. Reversed-phase mode

85. Buffer types effect on the enantiomeric resolution of Alprenolol with Teicoplanin CSP. Mobile phase: MeCN/buffer (pH4), 85 : 15, v/v, 15kV, 15 ℃, 335mm  75  m i.d. x 25cm, 2-s injection at 10kV, 10 bar, and detection at 200nm. Buffers: ammonium nitrate,10mM; ammonium acetate, 20mM; TEAA 1%.

86. Buffer types affect on the electrochromatograms of enantiomeric separation of alprenolol on the Teicoplanin CSP. (A)10mM ammonium nitrate (B) 20mMammonium acetate (C) 1%TEAA.

87. pH affect on the enantiomeric resolution of alprenolol with Teicoplanin CSP. Mobile phase : MeCN/1% TEAA, 85 : 15, v/v, 15kV, 15 ℃

88. Buffer concentration affect on the enantiomeric resolution (  ) and theoretical plates(  ) of alprenolol with Teicoplanin CSP. Mobile phase : MeCN/TEAA (pH4), 85 : 15, v/v, 15kV, 15 ℃.

89. MeCN content in the aqueous TEAA buffer on the resolution (  ) and efficiency ()of Alprenolol.

90. Relationship of linear velocity with field strength (  ) and Its effect on plate height (  ).

91. Simultaneous enantiomeric separation of  -blockers drugs with eicoplanin CSP in reversed-phase CEC. Samples: 1, alprenolol; 2, Propranolol; 3, Pindolol; 4, Atenolol.

92. Polar organic mode General composition of eluent: MeOH / MeCN / TEA / HOAc X / 100-X / base / acid 1: MeOH / MeCN Ratio 2: TEA / HOAc Ratio 3. Total TEA / HOAc concentration

93. Effect pf acid-base content in the non-aqueous media on the resolution ()and efficiency (  )of Alprenolol with Vancomycin CSP in polar organic chiral CEC. Conditions: MeOH/MeCN, (25/75, v/v), 15kV, 15 ℃, 335mm  75  m i.d. (L d 250mm), 2-s injection at 10kV, 10 bar, and detection at 200nm.

94. MeCN content in the polar organic mobile effect on the migration behavior of alprenolol first-eluted enantiomer. Conditions: TEA/HOAc, (0.1/0.1, v/v)

95. MeCN content in polar organic phase effect on the resolution (  ) and theoretical plates () of first-eluted alprenolol enantiomer. Conditions: TEA/HOAc, (0.1/0.1, v/v)

96. Comparison CEC enantiomeric separation of alprenolol by reversed-phase mode and polar organic mode in Teicoplanin CSP. (A). MeCN/TEAA (pH4) 1%, 85 : 15, v/v, 15kV, 15 ℃ (B). MeOH/MeCN/TEA/HOAc(75/25/0.15/0.3, v/v/v/v), 15kV, 15 ℃.

97. Reversed-phase mode CEC chiral separations of  -Blocker enantiomers with Teicoplanin CSP Racematet R 1 mint R 2 minN 1 plates/mN 2 plates/m R S Alprenolol14.98 15.16444,000 431,000 2.60 Pindolol18.49 19.04 339,000 334,400 2.33 Atenolol 56.72 58.68 213,000 201,000 1.92 Fenoterol 42.84 45.15 63,500 51,000 1.56 Metopropol 22.18 24.12 154,000 123,0001.82 Propranolol17.50 18.13 372,000 381,000 2.72 Sotalol 32.83 34.07 70,200 55.200 1.08 MeOH/TEAA (pH4) 1%, 85/15, v/v, 15kV, 15 ℃

98. Reversed-phase mode CEC chiral separations of  -blocker enantiomers with Vancomycin CSP  Racematet R 1 mint R 2 minN 1 plates/mN 2 plates/mR S Alprenolol 12.3 11.6137,00099,4001.42 Pindolol14.314.7101,00065,0001.96 Atenolol12.713.020,90012,2000.68 Fenoterol 33.033.153,10025,3000.56 Metopropol17.017.759,90038,6001.07 Propranolol 23.825.668,550042,4000.89 Sotalol13.213.7102,00069,9001.11 MeOH/TEAA (pH4) 1%, 85/15, v/v, 15kV, 15 ℃

99. DOPAMethylDOPA 3-O-MethylDOPA ( L-dopa—Parkinson Disease)( L-methyldopa— High blood pressure ) ( DOPA’s metabolite)

104. Lab-on-a-chip or CE on chip

105. Photograph of the microdevice with attached transfer capillary

106.  -TAS concept: Miniture-Total Chemical Analysis system.

107. Thank you for your time