Capillary electrophoresis

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  m

Electrophoretic migration Z i ： charge # of component i e 0 ： elemental charge [ 1.602× 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)

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)

 =  r    / 

+ - n EOF -+ + n - Net 毛細管電泳的向量圖

Effective Mobility and Apparent Mobility

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

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

(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

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

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

Reversal of EOF Using a Cationic Surfactant

(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

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)

(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)

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

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)

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 )

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 )

Chiral Capillary Electrophoresis (CCE)

Separation Mode: Capillary Gel Electrophoresis (CGE)

CGE: Protein Size Separation Using SDS Linear Polymer Solutions

Separation Mode: Capillary Isoelectric Focusing (CIEF)

Detection: Direct UV Detection

Detection: Indirect UV Detection

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

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

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) ?

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

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

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

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

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?

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

溶膠凝膠塗佈合成步驟示意圖 (a) 合成步驟。 (a)

溶膠凝膠塗佈毛細管內塗佈結果示意圖

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

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

CEC separation Mechanism

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

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

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

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

Compound Structure molecular weight Testosterone Methyltestosterone Progesterone Testosterone propionate Steroids structures used for C 18 packed CEC separation.

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.

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

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; Methyltestosterone; 3. Progesterone; 4:Testosterone propionat. Chromatographic separation of 4 steroids.

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

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

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

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..

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.

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

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.

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.

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

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.

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 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.

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

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

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

Structure of Marcrocyclic Glycopeptides

Teicoplanin Structure

Alprenolol Atenolol Fenoterol Metoprolol Pindolol Propranolol Sotalol

Reversed-phase mode

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%.

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.

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

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 ℃.

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

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

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

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

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.

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)

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)

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 ℃.

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 Alprenolol , , Pindolol , , Atenolol , , Fenoterol ,500 51, Metopropol , , Propranolol , , Sotalol , MeOH/TEAA (pH4) 1%, 85/15, v/v, 15kV, 15 ℃

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 ,00099, Pindolol ,00065, Atenolol ,90012, Fenoterol ,10025, Metopropol ,90038, Propranolol ,550042, Sotalol ,00069, MeOH/TEAA (pH4) 1%, 85/15, v/v, 15kV, 15 ℃

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

Lab-on-a-chip or CE on chip

Photograph of the microdevice with attached transfer capillary

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

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