Bài giảng Phương pháp điện di (Electrophoresis) – Nguyễn Đức Tuấn

Phương Pháp Điện Di (Electrophoresis) PGS.TS. Nguyễn Đức Tuấn Bộ môn Phân Tích – Kiểm Nghiệm Khoa Dược – Đại học Y Dược TPHCM Nguyễn Đức Tuấn Đại học Y Dược TPHCM Phương Pháp Điện Di Nguyễn Đức Tuấn Đại học Y Dược TPHCM Mục tiêu - Hiểu được nguyên tắc hoạt động của điện di mao quản Dàn bài - Lịch sử - Định nghĩa - Phân loại - Điện di mao quản Lịch sử Nguyễn Đức Tuấn Đại học Y Dược TPHCM 1791 Faraday Laws of Electrolysis 1877 Helmholtz Charged Solvent Layer Closed to Surface of a Wall 1897 Nernst Properties of Small Ions 1897 Kohlrausch Kohlrausch Function describing the Order of Migration of Ions and their Concentration 1923 Kendall, Crittenden Rare Earth Metal Separation by "Ion Migraion Method" 1930 Tiselius Thesis: Moving Boundary Method for Electrophoresis of Proteins (Nobel Price 1948) 1939 Svenson Development of Zone and Displacement Electrophoresis 1950 Haglund, Tiselius Electrophoresis Tube filled with Glass Beads and Glass Powder 1955 Smithies Gel Electrophoresis 1958 Hjertén Electrophoresis in Free Solution 1967 Martin, Everaerts Displacement Electrophoresis in Glass Tube with Hydroxyethylcellulose 1967 Hjertén Elimination of Electroosmosis by Coating of Glass Tubes 1969 Giddings Non-Diffusional Model of Concentration Distribution in Free Zone Electrophoresis 1969 Virtanen Glass Capillaries 0.2 - 0.5 mm I.D. 1970 Everaerts, Capillary Isotachophoresis 1970 Arlinger, Routs UV-Detection 1972 Verheggen Conductivity Detection 1979 Mikkers Use of High Voltage and TEFLON Capillaries 1981 Jorgenson Use of 75 µm I.D. Open Tubular Glass Capillaries: "High Performance Capillary Electrophoresis – HPCE" 1984 Terabe Combination of electrophoretic and chromatographic Separation: "Micellar Electrokinetic Capillary Chromatography – MECC" 1991 Jandik, Jones Use of Surface Active Electrolyte Additives for Reversal of Electroosmotic Flow 1991 Knox "Capillary Electrochromatography – CEC" Anion hữu cơ và vô cơ Nguyễn Đức Tuấn Đại học Y Dược TPHCM 89 seconds 1.5min 3.1min 1 10 20 30 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 10 19 21 22 23 24 25 26 27 28 29 Standard 1...thiosulfate 16..phosphite 2...bromide 17..chlorite 3...chloride 18..galacturate 4...sulfate 19..carnonate 5...nitrite 20..acetate 6...nitrate 21..ethanesulfonate 7...molybdate 22..propionate 8...azide 23..propanesulfonate 9...tungstate 24..butyrate 10.fluorophosphate 25..butyrsulfonate 11..chlorate 26..valerate 12..citrate 27..benzoate 13..fluoride 28..glutamate 14..formate 29..pentanesulfonate 15..phospahate 30..D-gluconate W.R. Jones, P. Jandik J. Chromatogr. 546 (1991), 445 Định nghĩa Nguyễn Đức Tuấn Đại học Y Dược TPHCM  Quá trình tách các tiểu phân đã ion hóa và hòa tan hay phân tán trong dung dịch điện giải dưới tác dụng của điện trường  Độ dịch chuyển điện di (linh độ điện di: electrophoretic mobility,  EP ) phụ thuộc:  Bản chất tiểu phân: hình dạng, kích thước, điện tích  Dung dịch điện giải: bản chất, nồng độ, pH, độ nhớt,. Định nghĩa Nguyễn Đức Tuấn Đại học Y Dược TPHCM  ep(C)  ep (A) A B  ep(D)  ep (B) + + + + C D  Chiều chuyển dịch các ion phụ thuộc vào điện tích của nó Phân loại Nguyễn Đức Tuấn Đại học Y Dược TPHCM Điện di dung dịch tự do (Moving Boundary Electrophoresis)  Không dùng chất mang   EP phụ thuộc vào điện trường E, bản chất tiểu phân  Xác định các chất có PTL lớn và ít khuếch tán  Kiểm tra độ tinh khiết protein + - Moving Boundary Electrophoresis Phân loại Nguyễn Đức Tuấn Đại học Y Dược TPHCM Điện di vệt (điện di vùng: Zone Electrophoresis)  Sử dụng chất mang: giấy, cellulose acetat, gel agar, gel polyacrylamid   EP phụ thuộc vào E, bản chất tiểu phân, dòng bay hơi (nhiệt Joule), dung dịch điện giải  Tách các chất có PTL nhỏ và kích thước nhỏ, lượng mẫu ít Điện di trên gel Điện di mao quản (Capillary Electrophoresis) Nguyễn Đức Tuấn Đại học Y Dược TPHCM High Voltage Power Supply 0-±30 kV 5-150 A Injection hydrostatic, vacuum, electromigrative Data Processing Separation Electrolyte Salt solution (borate, phosphate); pH 1-12 organic solvent (0-100%) Pt Electrodes Capillary o.d. 200-400µm i.d. 5-100µm (2µm) Fused Silica, Teflon coated (RP, Ion exchange) or filled (RP, ...) Detector UV, Fluorescence (direct, indirect); electrochemical conductometric MS Điện di mao quản Nguyễn Đức Tuấn Đại học Y Dược TPHCM 50µm 12µm 363µm Fused Silica Capillary Hydrodynamic flow profile and chromatographic peak form a) pressure driven b) electroosmosis a) b) Ohm's Law: U=R.I U [kV] I [µA] 0 10 20 50 100 150 0 30 200 250 300 Liquid cooling 10m/s air cooling w/o cooling Dòng điện thẩm (Electroosmotic Flow) Nguyễn Đức Tuấn Đại học Y Dược TPHCM Origin of Electroosmotic Flow: a) Formation of negatively charged silica-surface b) Hydrated cations at surface c) Bulk flow of whole capillary contents towards cathode after application of electric field  i iiA czeN Tk ....1000 .. 22    = Thickness of Layer  = dielectricity constant k = Boltzmann-constant T = temperature NA = Avogadro-constant e = charge per unit surface area z = charge of ion c = molar concentration - + + + + + oriented and non- oriented water molecules outer Helmholtz-Layer (diffuse) inner Helmholtz-Layer (adsorbed, rigid) r Y1 Y2 C a p il la ry Electrolyte Solvated Cations Solvated Anions (van der Waals) + +    e4 21 Y Y Silanol groups in fused silica capillaries Si O O Si O O O O O H H H O Si O Si O O H H O Si O H O O H H O primary sekundary tertiary Hydrolysis: Si-O- Thickness of Layer    eof  = Zeta-Potential [V] Y1 = Stern-Potential [V] Y2 = Potential of bulk solution [V] µeof = Mobility of EOF [cm 2V-1sec-1]  = Dielectricity constant of electrolyte  = Viscosity of electrolyte Electroosmotic mobility Linh độ điện di (Electrophoretic Mobility) Nguyễn Đức Tuấn Đại học Y Dược TPHCM F qE E )( )( F E ep FForcefrictional FForceelectric  F r F ep 6  eprqE  6  ep qE r 6   ep ep E q r 6 Mobility in Infinitely Diluted Solutions C+ N A- 0 1 2 3 4 5 C+: Cations Trimethylphenylammonium bromide Histamine 4-Aminopyridine N: Neutral Molecules Benzylalcohol Phenol A-: Anions Syringaldehyde 2-(p-Hydroxyphenyl)acetic acid Benzoic acid Vanillic acid 4-Hydroxybenzoic acid Overlay of Migration of Charged Ions and Molecules with EOF a) Cations to Cathode (Detection before EOF). b) Neutral Moleculese (Detection together EOF). c) Anions to Anode (Detection for |µAnion| < |µeof| after EOF; no Detection for |µAnion| > |µeof |) Linh độ điện di (Electrophoretic Mobility) Nguyễn Đức Tuấn Đại học Y Dược TPHCM Dissociation of Weak Electrolytes % i o n h ĩ a Nguyên tắc của điện di mao quản vùng (Capillary Zone Electrophoresis, CZE) obs(A + ) = EOF + EP(A + ) obs(C - ) = EOF - EP(C - ) obs(N) = EOF 0 1 2 3 4 5 6 (t) A+ B+ C- D- EOF N - + - µ - ep + µ + ep electrophoretic mobility cations from the electrolyte µeof very important parameter! electroosmotic flow Separation principle of MEKC capillary wall µEOF µep+eof (A+) = µep+eof (B-) µep (A + ) µep (B-) µmicelle SDS A+ C µep(A+solub) µobs (A + ) µobs (B-) µobs (C) µobs (M) 0 1 2 3 4 C A+ EOF B- M 5 6 (t) B- Thông số thực nghiệm trong CE Nguyễn Đức Tuấn Đại học Y Dược TPHCM Analyte geometry molecular weight, structure pKA ionic strength effective charge capillary wall capillary length high voltage V field strength E=V/L  wall    eof r q ep   6 tot = eof + ep ep = µep.E eof = µeof.E viscosity  permittivity  adsorption pH solvation Electrolyte Instrument Carbohydrat Nguyễn Đức Tuấn Đại học Y Dược TPHCM 1...mannuronic acid (n.a.) 2...glucuronic acid (3.20) 3...galacturonic acid (3.48) 4...gluconic acid (3.76) 5...N-acetylneuraminic acid (2.60) 6...fructose (12.03) 7...rhamnose (n.a.) 8...glucose (12.35) 9...galactose (12.35) 10...2-deoxy-D-ribose (12.65) 11...sucrose (12.51) A. Zemann, D.T. Nguyen, G. Bonn Electrophoresis 18 (1997) 1142 1 4 EOF 7 2 5 8 9 6 3 10 11 1.5 2.0 2.5 3.0 3.5 min Conditions: Capillary: uncoated fused silica, i.d. 50M; L=32cm, l=24.5cm; Electrolyte: 6mM sorbate, 0.001% HDB, pH12.1; Injection: 3sec hydrostatic (10cm); Detection: indirect UV @ 254nm; Instrumentation: WATERS Quanta 4000; U=-10kV, I=29,2A, T=amb. Carbohydrat Nguyễn Đức Tuấn Đại học Y Dược TPHCM 45 60 75 90 sec Orange Juice Apple Juice Coca Cola 1 1 1 2 2 2 3 3 3 EOF EOF EOF indirect UV detection analysis of soft drinks 1..... fructose 2..... glucose 3..... sucrose Conditions: Capillary: uncoated fused silica, i.d. 50M; L=32cm, l=24.5cm; Electrolyte: 6mM sorbate, 0.001% HDB, pH11.9; Injection: 3sec hydrostatic (10cm); Detection: indirect UV@254nm; Instrumentation: WATERS Quanta 4000; U=-22kV, I=34,4A, T=amb. A. Zemann, D.T. Nguyen, G. Bonn Electrophoresis 18 (1997) 1142 Acid amin Nguyễn Đức Tuấn Đại học Y Dược TPHCM 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min 5.0 200 220 240 260 nA 1 2 3 4 5 6 Conditions: Capillary: 20 µm I.D.; 375 µm O.D.; l=60 cm; U=30 kV; I=1.3 µA BGE: 3 mM Tetraborate, pH 10.5 Injection: 20 kPa, 7 sec Detection: amperometric; Coated-Wire-Cu-Electrode; U=+1125 mV Sample Concentration: 100 ppm 1...Arginine 2...Lysine 3...Proline 4...Histidine 5...Threonine 6...Tyrosine counter-electroosmotic CE Hợp chất carbonyl Nguyễn Đức Tuấn Đại học Y Dược TPHCM -113,50 -113,00 -112,50 -112,00 -111,50 -111,00 -110,50 -110,00 -109,44 2.0 2.5 3.0 3.5 4.0 4.5 min 5.0 1 2 3 4 5 6 7 8 9 10 11 12 Capillary, I.D. 50 µm, L=60 cm, leff=52.5 cm Electrolyte, 20 mM borate, 0.001% HDB, pH=11.5, 45% acetonitrile, Detection, UV @ 254 nm Peak assignments: 1... formaldehyde 2... acetaldehyde 3... benzaldehyde 4... crotonaldehyde 5... m-tolualdehyde 6... acetaldehyde 7... propioaldehyde 8... butyraldehyde 9... valeraldehyde 10... hexaldehyde 11... acetone 12... butanone Anion hữu cơ và vô cơ Nguyễn Đức Tuấn Đại học Y Dược TPHCM 89 seconds 1.5min 3.1min 1 10 20 30 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 10 19 21 22 23 24 25 26 27 28 29 Standard 1...thiosulfate 16..phosphite 2...bromide 17..chlorite 3...chloride 18..galacturate 4...sulfate 19..carnonate 5...nitrite 20..acetate 6...nitrate 21..ethanesulfonate 7...molybdate 22..propionate 8...azide 23..propanesulfonate 9...tungstate 24..butyrate 10.fluorophosphate 25..butyrsulfonate 11..chlorate 26..valerate 12..citrate 27..benzoate 13..fluoride 28..glutamate 14..formate 29..pentanesulfonate 15..phospahate 30..D-gluconate W.R. Jones, P. Jandik J. Chromatogr. 546 (1991), 445 Các thuốc kháng HIV Nguyễn Đức Tuấn Đại học Y Dược TPHCM Capillary, L=48.5 cm, leff=40 cm, 50 µm; electrolyte, 16 mM phosphate, 0.001% HDB, pH 2.5 Injection, 20 sec @ 10 mbar; standard concentration, 5 ppm; Separation, -30 kV Detection, UV @ 195 ± 5 nm (bubble cell 200 µm). AMP...amprenavir; RTV...ritonavir; SQV...saquinavir; NFV...nelfinavir; IDV...indinavir 0 1 2 3 4 1 2 3 4 5 min 6 AMP RTV SQV NFV IDV EOF mAU (195nm) Nguyen D.T., A. Zemann J. Chromatogr. A, 922 (2001) 313 – 320 protease inhibitors Các thuốc kháng HIV Nguyễn Đức Tuấn Đại học Y Dược TPHCM 0 4 8 1 2 3 4 5 6 7 8 min 285 nm 240 nm 195 nm mAU AMP RTV SQV NFV DLV ABC NVP 3TC DDC IDV EOF 12 16 20 Capillary, L=42.5 cm, leff=34 cm, i.d.=50 µm Electrolyte, 16 mM H3PO4, 0.001% HDB, pH 2.2 Injection, 20 sec @ 10 mbar; standard concentration, 5 ppm; Separation, U=-30 kV Detection, UV (bubble cell 200 µm). AMP...amprenavir; RTV...ritonavir; SQV...saquinavir; NFV...nelfinavir ; IDV...indinavir NVP...nevirapine; DLV...delavirdine; ABC...abacavir; 3TC...lamivudine; DDC...zalcitabine D.T. Nguyen, A. Zemann Journal of Chromatography A, 982 (2002) 153 – 161. protease and reverse transcriptase inhibitors Group 1: Chemical structure of CDs • ACE inhibitors Captopril (CAP) Enalapril (ENA) Lisinopril (LI) • Diuretics Hydrochlorothiazide (HCT) Furosemide (FURO) NHS COOH CH 3 O H N N O COOH CH 3 OOH 3 C H N N COOH H 2 N O HOOC N H S NH O O H2N S O O Cl H 2 N S O O COOH N H O Cl Group 1: ACE inhibitors and diuretics • Optimized electrophoretic conditions Electrophoretic conditions: 60 mM orate buffer at pH 8.6; fused-silica capillary (57 cm x 50 m i.d., 48.5 cm); injection: 5s at 50 mbar; 18 kV; 25oC; detection wavelength: 214 nm nm 240 260 280 300 320 340 mAU 0 2 4 6 8 10 12 HCT LI ENA FURO CAP Separation principle of MEKC capillary wall µEOF µep+eof (A+) = µep+eof (B-) µep (A + ) µep (B-) µmicelle SDS A+ C µep(A+solub) µobs (A + ) µobs (B-) µobs (C) µobs (M) 0 1 2 3 4 C A+ EOF B- M 5 6 (t) B- MECC – Các NSAID Nguyễn Đức Tuấn Đại học Y Dược TPHCM min5 10 15 20 mAU -10 0 10 20 30 40 50 60 DAD1 C, Sig=210,20 Ref=off (CELE\CELE0047.D) c e l e c o x i b m e l o x i c a m n i m e s u l i d e p i r o x i c a m + t e n o x i c a m r o f e c o x i b Hệ đệm: dung dịch dinatri tetraborat 25 mM pH 9,3; 50 mM SDC Cột mao quản: silica nung chảy 72/80,5 cm x 50 µm Nhiệt độ cột: 25 o C, điện thế: 30 kV Lượng mẫu tiêm: 50 mbar x 3 s; Bước sóng phát hiện: 210 nm Application – Multi-components Conditions: Background electrolyte 10 mM borate, 10 mM phosphate, pH 9.2, 5% ACN, 50 mM SDS. Capillary 39.5/48 cm, 50 µm I.D. Temperature 25oC. Detection 210 nm. Applied voltage 20 kV. Injection 50 mbar x 20 sec 1 2 3 4 1. Paracetamol 2. Phenylpropanolamine hydrochloride 3. Pseudoephedrine hydrochloride (IS) 4. Chlorpheniramine maleate Group 2: Chemical structure of CDs • Beta blockers H2N O O H3C O H N CH3 CH3 OH R Atenolol (ATE) Metoprolol (METO) Propranolol (PRO) • Calcium channel antagonists H N CH 3 H 3 C O CH 3 O H 3 C O NO 2 O 5 H N O NH 2 O CH 3 O H3C O H 3 C Cl O Amlodipine (AM) Nifedipine (NI) Group 2: -blockers and Ca channel antagonists • Optimized electrophoretic conditions Electrophoretic conditions: 10% methanol in 100 mM tris buffer at pH 12.0 containing 100 mM SDC; fused-silica capillary (57 cm x 50 m i.d., 48.5 cm); injection: 5s at 50 mbar; 25 kV; 25oC; detection wavelength: 225 nm nm 240 260 280 300 320 340 mAU 0 2.5 5 7.5 10 12.5 15 17.5 20 (2) ATE METO NI PRO AM Group 3: Chemical structure of CDs • Statin derivatives Lovastatin (LOV) Simvastatin (SIM) Atorvastatin (ATOR) N O OOHOH F . Ca 2+ , 3H 2 O 2 N H O CH 3 H 3 C H O O CH 3 H O O H 3 C OH CH 3 H H 3 C O OH H O CH 3 H 3 C CH 3 O O H H 3 C H CH 3 Group 3: Statin derivatives • Optimized electrophoretic conditions Electrophoretic conditions: 15% methanol in 15 mM borate buffer at pH 8.0 containing 50 mM SDC; fused-silica capillary (57 cm x 50 m i.d., 48.5 cm); injection: 5s at 50 mbar; 30 kV; 30oC; detection wavelength: 237 nm nm 240 260 280 300 320 340 mAU 0 2 4 6 8 10 ATOR LOV SIM Application – Natural products Analyte Additives Ref. Flavonoid (rutin, isoquercitrin, quercitrin, kaempferol, quercetin, etc) 50 mM SDS P.G. Pietta, et al; J. Chromatogr. (549) 367 Opium alkaloids (6 mixture) 12 mM SDS + 25 mM Tween 20 I. Bjornsdottir, et al; J. Pharm. Biomed. Anal. (13) 687 Amphetamines and related substances 25 mM CTAB + 11% DMSO + 1% ethanol V.C. Trenerry, et al; J. Chromatogr. A (708) 169 Cocaine and related substances 50 mM CTAB + 7.5% ACN V.C. Trenerry, et al; Electrophoresis (15) 103 Codeine and its by products 40 mM SDS M. Korman, et al; J. Chromatogr. (645) 366 Application – Optical purity testing of drugs • Use area percentage method for purity testing of drugs as in HPLC • Normalize peak areas with migration times • Identify impurities above apparent levels of 0.1% Application – Dexchlorpheniramine maleate min. 2.5 5 7.5 10 12.5 15 17.5 mAU 0 2 4 6 8 10 12 14 1 8 ,3 9 3 1 4 ,4 6 9 1 7 ,9 9 4 1 2 3 Optical purity testing of dexchlorpheniramine maleate by CE with -CD Conditions: Capillary: 76.5 cm (68 cm effective length) x 50 m I.D.; Background electrolyte: 0.05 M Tris buffer pH 3.5 + 5 mM -CD; Detection: 214 nm; Applied voltage: 20 kV; Injection: 50 mbar x 10 sec.; Temperature: 25oC. 1. Pseudoephedrine HCl (IS) 2. Levochlorpheniramine maleate 3. Dexchlorpheniramine maleate Chemical structure of drug substances Nefopam NCH 3 O * 1 5 O N CH 3 CH 3 1 2 3 * OH Propranolol * Brompheniramine Ketoconazole N ON CH 3 O O O N N Cl Cl * * 1 2 3 4 5 Miconazole Cl Cl O Cl Cl N N * 1 2 2'' 2' 4''4' N O NH 2 COOCH 2 CH 3 Cl CH 3 CH 3 OOC 1 3 4 5 6 2 3 4 6 1 * 5 2 Amlodipine Ofloxacin N O N N CH 3 CH 3 OH F O O 1 2 * 3 4 5 6 7 8 10 9 Promethazine S N N CH 3 CH 3 CH 3 *1 10 2 Effect of the CD types and their concentrations on Rs Electrophoretic conditions: 50 – 100 mM tris-phosphate buffer pH 2.5 – 3.0, 20% methanol (for propranolol) or 25% acetonitrile (for nefopam); capillary (63.5 cm x 50 m i.d., 54 cm);  = max of each compound; 25oC; 20 kV; injection: 5s at 50 mbar. Rs Electropherograms for the chiral separation of enantiomers Optimized electrophoretic conditions: 50 mM tris-phosphate buffer pH 2.5; capillary (63.5 cm x 50 m i.d., 54 cm);  = 228 nm, 25oC; 20 kV; injection: 5s at 50 mbar. Miconazole 5 10 15 0 1 2 3 4 5 6 min. mAU 2 mM -CD min. 5 10 15 0 1 2 3 4 5 6 mAU 7 2 mM HP- -CD 5 10 15 0 1 2 3 4 5 6 min. mAU 2 mM HB- -CD Electropherograms for the chiral separation of enantiomers Optimized electrophoretic conditions: 50 mM tris-phosphate buffer pH 3.5; capillary (63.5 cm x 50 m i.d., 54 cm);  = 230 nm, 25oC; 20 kV; injection: 5s at 50 mbar. Brompheniramine 10 mM - CD 25 mM HP- -CD 15 mM HB- -CD Electropherograms for the chiral separation of enantiomers Optimized electrophoretic conditions: 50 mM tris-phosphate buffer pH 2.5, 25% MeCN; capillary (63.5 cm x 50 m i.d., 54 cm);  = 275 nm, 25oC; 20 kV; injection: 5s at 50 mbar. Nefopam 30 mM -CD 30 mM HP--CD 20 mM HB--CD Electropherograms for the chiral separation of enantiomers Optimized electrophoretic conditions: 50 mM tris-phosphate buffer pH 2.5; capillary (63.5 cm x 50 m i.d., 54 cm);  = 360 nm, 25oC; 20 kV; injection: 5s at 50 mbar. Amlodipine min . 5 10 15 20 25 mAU 7 1 3 5 0 2 4 6 8 10 mM HP-- CD 5 mM HB-- CD min. 5 10 15 20 25 mAU 0 2 4 6 8 Ofloxacin Optimized electrophoretic conditions: 50 mM tris-phosphate buffer pH 2.5; capillary (63.5 cm x 50 m i.d., 54 cm);  = 294 nm, 25oC; 20 kV; injection: 5s at 50 mbar. 5 10 15 20 25 min. 0 2 4 6 8 10 mAU 30 mM HP-- CD 5 10 15 20 mAU 0 2 4 6 8 min. 30 mM HB--CD Electropherograms for the chiral separation of enantiomers Optimized electrophoretic conditions: 50 mM tris-phosphate buffer pH 2.5, 20% MeOH; capillary (63.5 cm x 50 m i.d., 54 cm);  = 288 nm, 25oC; 20 kV; injection: 5s at 50 mbar. Propranolol Ketoconazole Promethazine Optimized electrophoretic conditions: Tris-phosphate; 63.5 cm x 50 m i.d., 54 cm; 25oC; 20 kV; injection: 5s at 50 mbar. KET: 50 mM BGE pH 3.0;  = 227 nm. PRM: 100 mM BGE pH 2.5, 30% MeOH;  = 254 nm 10 20 30 40 0 2 4 6 8 mAU min . 30 mM HP-- CD min. 20 30 40 0 2 4 6 8 mAU 10 20 mM HB--CD 5 10 15 20 min. mAU 0 2 4 8 6 10 15 mM HB--CD 5 15 25 35 min. 0 2 4 6 8 mAU 30 mM HB--CD Application 10 5 15 20 25 min. mAU 0 2 4 6 8 R -a m lo d ip in e S -a m lo d ip in e Electrophoretic conditions: 50 mM tris-phosphate buffer pH 2.5, 10 mM HP--CD; capillary (63.5 cm x 50 m i.d., 54 cm);  = 360 nm, 25oC; 20 kV; injection: 5s at 50 mbar. min . 5 15 25 mAU 0 10 20 30 S -a m lo d ip in e R -a m lo d ip in e min. 5 10 15 20 mAU -5 0 5 10 S -a m lo d ip in e Amlodipine racemic spiked R-amlodipine S-amlodipine drug substance S-amlodipine tablet Application Electrophoretic conditions: 50 mM tris-phosphate buffer pH 2.5, 30 mM HB--CD; capillary (63.5 cm x 50 m i.d., 54 cm);  = 294 nm, 25oC; 20 kV; injection: 5s at 50 mbar. Ofloxacin racemic spiked levofloxacin Levofloxacin tablet min. 5 10 15 20 mAU 0 4 8 12 16 L e v o fl o x a c in O fl o x a c in min. 5 10 15 20 mAU 0 4 8 12 16 L e v o fl o x a c in Conclusion CZE and MEKC can be used for drug analysis as a complementary or alternative method to HPLC Conclusion Advantage • One-run separation of every kind of drug, including cationic, neutral and anionic is possible within a relatively short time • MEKC is especially powerful for the separation of complex mixtures because of its high resolution • Direct enantiomer separation also can be successful using chiral selectors Disadvantage • For much wider use it is still desirable for the precision in quantitative analysis to be improved to be comparable to those in HPLC