Capillary electrophoresis

Electrophoretic migration

Zi： charge # of component ie0： elemental charge [ 1.602×10-19 C ]E=V/L ( V： applied voltage； L： length )

ri ： stokes radius of i componentη ： viscosity of the medium [Pa · S]Vi

0 ： migration velocity of i component

EeZqEF ie 0 (1)

00 6 iiid VrVkF (2)

i

ii r

EeZV6

00

i

iii r

eZE

V

6

00

0

μi0： electrophoretic mobility

μi0 和 (q, η, ri) 有關

Electroosmotic flow (EOF)

(3)

(4)

EV eoeo μeo： electroosmotic mobility (cm2/Vs)

(5)

'4 eo (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

eo

D

i

Deonetii t

LtLVVV )(

VLV

EV Tii

i

VtLL

i

TDi if no EOF

VtLL

EV

eo

TDeoeo

(7)

(8)

(9)

(10)

Efficiency

(11)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

對某一 analyte , μ 和 D 是 constant

VN V 固定 D 愈小 N 愈大 對大分子分離有利

Resolution

VVNRs

NxNxNxNx

HNx

HxNand

xH

VVxR

vx

VVxxor

VV

xx

xR

s

s

4

44

4

4

222

2

(19)

(20)

(21)

(22)

：平均距離 (cm) ：平均速度

eos

eo

eos

s

eo

eo

DVR

DVR

VVxR

DVN

VV

VV

21

21

21

21

177.0

241

4

2

代入 (24)

EOF 存在(23)

(24)

(25)

(26)

EOF 和 electrophoretic 反方向則 μeo↑ Rs↑Best Resolution 分離時間很長 eo

12

21

12

2

ttWWttRs

(22) 式 Rs=column eff. × selectivity factor selectivity →separation factorαin chromatography

Buffer compositionComplex formation

1. Borate complexation 2. ion pairing3. inclusion complex 4. metal complexation

Organic modifier1. After the polarity & viscosity of the mobile phase EOF & electrophoretic mobility changed2. 增加 solubility of analyte3. reduce zeta potential

1

2

01

02'1

'2

tt

tttt

kk

Reversal of EOF Using a Cationic Surfactant

Micellar Electrokinetic Chromatography

aq

mc

nnk '

mcaq

aq

nnn

k

'11

mcaq

mc

nnn

kk

'1'

mceo Vk

kVk '1

''1

1

mcmc

eo

Rs

tlV

tlV

tlV

0

fraction of analyte in aqueous phase

fraction of analyte in micelle

migration velocity (Vs)：

tR, t0, tmc： migration time of analyte, aqueous phase, and micelle

k’： capacity factornmc： no. of analyte molecules incorporated into the micellenaq ： no. of analyte molecules in aqueous phase

(1)

(2)

(3)

(6)

(5)

(7)

(4)

(5)(6)(7) 代入 (4)

mceo

mcmceo

eo

mceoR

ttk

kt

VVk

ktVkVktV

VkVkl

Vslt

0

000

'1

'1

'1

'1'

'1''1

mcR tk

t

'11

00 '1 tktttt Rmcmc (micelle 停在 capillary) <<

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

(8)

(10)

(9)

Resolution

'1'1

4

'1

1

'1'1

4 0

0

2

2

kkNR

ktt

tt

kkNR

s

mc

mcs

tmc→∞ 兩式相等 tmc→∞ 固定相

(11)

Effect of the capacity factor

'1

1

'1''

0

0

ktt

tt

kkkf

mc

mc

t0/tmc=0 tmc=∞ conventional chromatography

large capacity better resolutionlead to

in MEKC large k’ unfavor (11) 式最後一項→ 0

optimum k’ 和 t0/tmc 有關 將 (12) 式微分0

'ttk mc

opt

(12)

(13)

capacity factoraq

mc

VVkk '

k ： distribution coefficientVmc,Vaq ： volumes of micelle and aqueous phases

CMCCV sfmc

： specific volume of micelle

CMCCkk

CMCC

CMCCCMCC

VV

sf

sf

sf

sf

aq

mc

'

1

when << 1

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

(15)

(16)

(14)

(17)

(Vmc<<Vaq)

Effect of EOF on the resolution

10

1

)(

)(0

)(

eo

mceo

eo

mceo

eo

epeo

eo

mc

mc

eo

mc

mcepeoepeomc

EE

VV

VLVL

tt

EVVV

mcmc tt

tt 00 < 1

> >

愈小 Rs 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 LinearPolymer Solutions

Separation Mode: Capillary Isoelectric Focusing (CIEF)

Detection: Direct UV Detection

Detection: Indirect UV Detection

Lab-on-a-chip or CE on chip

Photograph of the microdevice with attached transfer capillary

-TAS concept:Miniture-Total Chemical Analysis system.