Capillary electrophoresis

(KV)(Capillary Electrophoresis-CE) I.D. 25-100 mm

Electrophoretic migrationZicharge # of component ie0elemental charge [ 1.60210-19 C ]E=V/L ( Vapplied voltageLlength ) ri stokes radius of i componentviscosity of the elution [Pa S]Vi0migration velocity of i component(1)(2)

i0electrophoretic mobility i0(q, , ri)Electroosmotic flow (EOF)(3)(4)eoelectroosmotic mobility (cm2/Vs)(5)(6)zeta potential (V)pH, ionic strengthviscosity of double layer ~ (bulk)

m = er e0x / h

+-nEOF-++n-Net

Effective Mobility and Apparent Mobility

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

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

(16)(17)(18)analyte , D constantV D N

Resolution(19)(20)(21)(22) (cm)

(24)EOF(23)(24)(25)(26)EOFelectrophoreticeo RsBest Resolution

Reversal of EOF Using a Cationic Surfactant

(22)Rs=column eff. selectivity factor selectivity separation factorin chromatographyBuffer compositionComplex formation1. Borate complexation 2. ion pairing3. inclusion complex 4. metal complexationOrganic modifierAfter the polarity & viscosity of the mobile phase EOF & electrophoretic mobility changed2. solubility of analyte3. reduce zeta potential

Micellar Electrokinetic Chromatographyfraction of analyte in aqueous phasefraction of analyte in micellemigration velocity (Vs)tR, t0, tmcmigration time of analyte, aqueous phase, and micelle kcapacity factornmcno. of analyte molecules incorporated into the micellenaq no. of analyte molecules in aqueous phase(1)(2)(3)(6)(5)(7)(4)

(5)(6)(7)(4)t0= EOF is completely suppressed, in MEKC EOF is not essential.(+)k(8)(10)(9)

Resolutiontmc tmc(11)

Effect of the capacity factort0/tmc=0 tmc= conventional chromatographyin MEKC large k unfavor (11)0optimum kt0/tmc (12)(12)(13)

kdistribution coefficientVmc,Vaqvolumes of micelle and aqueous phaseswhen

Effect of EOF on the resolution< 1>> Rsseparation factor micelle (stationary phase in RP-HPLC) aqueous (mobile phase in RP-HPLC)

type of surfactants(from 11)

Chiral Capillary Electrophoresis (CCE)

Chiral Capillary Electrophoresis (CCE)

Separation Mode: Capillary Gel Electrophoresis (CGE)

CGE: Protein Size Separation Using SDS LinearPolymer 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 chromatographyWhat is Capillary electrochromatography (CEC) ?

EOFVEOF = mE m = er e0x / h E:electrical field strengthx:zeta potential

Pressure-driven flow= dp:particle diameter

Driving force of CEC and HPLC

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

CapillaryFunctional group coated surfaceSide view of open-tubular CEC column

Advantages and disadvantages of open-tubular columnAdvantages: 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 SiO2 in the form of glass-like material1.Hydrolysis:2.Condensation:3.Polymerization:What is Sol-Gel?

DI.water180 l 0.1M HCl 15 l +500l TMOS305TMSPTMA 340 l 30PTMAFS sol-gelPreparation of PTMAFS Sol-Gel

(a)

Electron micrographs of a PTMAFS coating inside a fused-silica capillaryA:10 s coating, flushing withwater 30 s, aging in waterB:10 s coating, flushing withwater 4 min, aging in water

Anion-exchange behavior of PTMAFS coated capillaryMigration time (min)Absorbance Unit .Running buffer 20 mM phosphate pH 3.0; Analyte: terephthalic acid60 mM co-aniona:citrateb:sulfatec: nitrated:chloride

CEC separation Mechanism

a.bcd . a. b. c. d.

a. Ni-Cr wireClosed 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 mPicture of a frit by optical microscope with digital camera

Compound Structure molecular weight Testosterone 288.4

17-Methyltestosterone 302.4

Progesterone 344.5

Testosterone propionate 314.5 Steroids structures used for C18 packed CEC separation.

Effect of MeCN concentration on EOFMobile phase: 10 mM Tris at pH 8.0. Column: 35 x 75 mm 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: 35cm75um 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.

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

BGS+-SBBGS+-BBSPME ProcedureCEC SeparationBGS+-SBSSMS1 S2 S3SPME sample injection

Compound Structure molecular weight 17-Methyltestosterone 302.4

Testosterone 288.4

Progesterone 344.5

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), VMeCN: VH2O =80/20; (B), VMeCN: VH2O =20/80..

Effect of injection time on peak width at half-height () and peak height () of 17-Methyltestosterone. Experiment conditions: sample solution, VMeCN: VH2O =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, VMeCN: VH2O =80/20, 10mM Tris, pH 8.0. sample solution,VMeCN: VH2O =20/80; injections (A) 5kV, 1-s, (B) 5kV, 600-s; Sample concentration: (A) 10mg/L; (B) 0.3mg/L.DL: ~15ppb

Naphthalene AcenaphthyleneAcenaphtheneFluorene PhenanthreneAnthracene FluoranthenePyrene Benz(a)anthraceneChrysene Benzo(b) fluoranthene

Benzo(k)fluorantheneBenzo(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), VMeCN: VH2O =100/0; (B), VMeCN: VH2O =40/60.

Effect of injection time on the peak width at half-height () and peak height () of pyrene.sample solution, VMeCN: VH2O =40/60.

Electrochromatogram of PAHs with SPME-CEC technique.Mobile phase, VMeCN: VH2O =80/20, 10mM Tris, pH 8.0. Samples: 16 PAHs mixture; sample solution, (A) VMeCN: VH2O =100/0, (B) VMeCN: VH2O =40/60; injections (A) 5kV, 6-s, (B) 5kV, 600-s.

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

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

(enantiomers):

1.Dalgiesh 1952 three-point interaction2. 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, 335mm75m 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 enantiomericseparation 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 () andIts effect on plate height ().

Simultaneous enantiomeric separation of -blockers drugs witheicoplanin 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 Ratio2: TEA / HOAc Ratio3. 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, 335mm75m i.d. (Ld 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 b-Blocker enantiomers with Teicoplanin CSP RacematetR1 mintR2 minN1 plates/mN2 plates/m RS

Alprenolol14.98 15.16444,000 431,000 2.60Pindolol18.49 19.04 339,000 334,400 2.33Atenolol 56.72 58.68 213,000 201,000 1.92Fenoterol 42.84 45.15 63,500 51,000 1.56Metopropol 22.18 24.12 154,000 123,0001.82Propranolol17.50 18.13 372,000 381,000 2.72Sotalol 32.83 34.07 70,200 55.200 1.08

MeOH/TEAA (pH4) 1%, 85/15, v/v, 15kV, 15

Reversed-phase mode CEC chiral separations of b-blocker enantiomers with Vancomycin CSP RacematetR1 mintR2 minN1 plates/mN2 plates/mRS

Alprenolol 12.3 11.6137,00099,4001.42Pindolol14.314.7101,00065,0001.96Atenolol12.713.020,90012,2000.68Fenoterol 33.033.153,10025,3000.56Metopropol17.017.759,90038,6001.07Propranolol 23.825.668,550042,4000.89Sotalol13.213.7102,00069,9001.11

MeOH/TEAA (pH4) 1%, 85/15, v/v, 15kV, 15

DOPAMethylDOPA3-O-MethylDOPA( L-dopaParkinson Disease)( L-methyldopaHigh blood pressure )( DOPAs metabolite)

Effect of different types of organic modifier

Sample : 3-O-methyldopa Sample injection : 10 kV, 2 sec.Apply voltage : 15 kV Detector : UV at 200 nmBuffer : organic solvent / pH 5, 50 mM ammonium acetate = 60 / 40Temperature : 15

reverse phasebufferbuffer,ex:phosphate,ammonium nitrate,sodium nitrateammonium acetate

A comparison of CEC with HPLC(optimized conditions)

3-O-methyldopa

dopa

methyldopa

A comparison of CEC with HPLC( at the same linear flow rate)*

methyldopa

dopa

3-O-methyldopa

Separation performances of CEC and HPLC

N: CEC 3.3~5.3 HPLC at optimized conditionsRs: CEC 1.7~2.8 HPLC

N: CEC 2.7~3.3 HPLC at the similar linear velocity,Rs: CEC 1.1~3.3 HPLC

Sheet1

CECHPLC

NRsNRs

dopa1095274.89dopa248271.71

methyldopa1350541.6methyldopa253751.58

3-mo-dopa1473163.563-mo-dopa447302.05

NRs

dopa248271.71

methyldopa253751.86

3-mo-dopa447302.05

NRs

dopa336141.56

methyldopa494691.44

3-mo-dopa439721.01

Sheet2

Sheet3

Sheet1

CECHPLC

NRsNRs

dopa1095274.89dopa336141.56

methyldopa1350541.59methyldopa494691.44

3-mo-dopa1473163.563-mo-dopa439721.01

NRs

dopa248271.71

methyldopa253751.86

3-mo-dopa447302.05

NRs

dopa336141.56

methyldopa494691.44

3-mo-dopa439721.01

Sheet2

Sheet3

Lab-on-a-chip or CE on chip

Photograph of the microdevice with attached transfer capillary

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

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