Electrophoresis

Separation is based on differences in migration of charged analyte ions in an electric field in solution

(+) anode -

+ n ++

(-) cathode

Electrophoresis
Separation can be carried out in free (buffered) solution CAPILLARY ELECTROPHORESIS (CE) or in (buffered) solution immobilized in a gel or on a similar support

Modes of CE
Capillary zone electrophoresis (CZE) Micellar electrokinetic chromatography (MEKC) Microemulsion electrokinetic chromatography (MEEKC) Capillary gel electrophoresis (CGE) Isoelectric focussing (IEF) Isotachophoresis (ITP) Capillary electrochromatography (CEC)

Capillary zone electrophoresis Instrumentation

fused-silica capillary (filled with carrier electrolyte) detector

0+-30 kV High-voltage power supply carrier electrolyte vial carrier electrolyte vial

sample vials

Capillary zone electrophoresis Fused silica capillary:

polyimide coating 15 m thick fused silica 360 m outer diameter

inner diameter 25-100 m

30-100 cm length

Capillary zone electrophoresis Acceleration: Kac = z e E

z charge number e charge unit E electric field strength (E = voltage / length)

Friction (Stokes Law): Kfr = 6 r vep vep electrophoretic migration velocity

(cm / s) r radius of ion viscosity

Steady-state: Kac = Kfr

Capillary zone electrophoresis

Steady-state: Kac = Kfr vep/E = z e / 6 r vep/E electrophoretic mobility ep

ep = / F !!

(F Faraday constant ionic equivalence conductance)

Capillary zone electrophoresis

electroosmotic flow (EOF) fused silica has exposed silanol groups (Si-OH), which become deprotonated at pH 3 Causes a layer of cations from the carrier electrolyte to build up at the inner surface

+ + + + + (+) anode + + + + + - - - - - -

+ + + + + + + +
flow (EOF) +- +- +- + - + - + -+ + -

(-)
cathode

Capillary zone electrophoresis electroosmotic flow:

laminar electroosmotic flow profile hydrodynamic flow profile

Capillary zone electrophoresis electroosmotic flow velocity veo = eoE:

eo electroosmotic mobility; depends on the zeta potential (charge of the inner surface) eo = prop /

apparent mobility of an analyte ion: app = ep + eo apparent velocity of an analyte ion: vapp = vep + veo

Capillary zone electrophoresis Dependence of electroosmotic mobility on pH

Electroosmotic mobility

pH

Capillary zone electrophoresis ep / eo / app

high- and medium-molecular-mass analyte ions

ep

!# -

!" !3 + !"
-

eo

!3 + !3+$%&'marker$!"-$!#-

app

!# -

low-molecular-mass analyte ions !# !" !3 + !" !3 + !3+$%&'marker$!"eo

ep

app

!#

Capillary zone electrophoresis

app

Ld t Ld Lt = = = E V Lt Vt

V vapp app

Ld = length of capillary from injector to detector t = migration time Lt = total length of capillary V = voltage applied to capillary

Capillary zone electrophoresis Manipulation of EOF

Increase of ionic strength of carrier electrolyte decreases EOF Organic solvents in carrier electrolyte generally decrease EOF Addition of EOF-modifiers can reduce or reverse EOF

Example for EOF-modifier: Tetradecyltrimethylammonium

Capillary zone electrophoresis Effects of EOF-modifiers

ANODE electroosmotic flow eo

CATHODE

Capillary zone electrophoresis Sample injection

very small sample sizes - 10-9 liters hydrodynamic injection, uses pressure to force sample onto column electrokinetic injection based on ep - thus injected sample has different relative composition of analytes

Capillary zone electrophoresis Sample injection techniques

Capillary zone electrophoresis Detection:

UV absorbance (direct or indirect mode)
(on-capillary detection)

Fluorescence
(on-capillary detection)

Conductivity Detection Mass Spectrometry

Capillary zone electrophoresis direct and indirect UV detection:

Capillary zone electrophoresis

Summarizing the mechanisms:

Capillary filled with (buffered) carrier electrolyte Separation according to electrophoretic mobilities Optimization of separation can be done by changing the pH (changes in degree of dissoziation of the analytes, changes of charge) or by addition of complex-forming agents (changes in charge and/or size) to the carrier electrolyte

Capillary zone electrophoresis

Use of complex-forming reagent in the carrier electrolyte Chiral separations by CZE Addition of cyclodextrins (chiral selectors) to the carrier electrolyte. Formation of diastereomeric complexes between the enantiomeric analytes and the chiral selectors -, - or -cyclodextrin methyl derivatives of -CD 2-hydroxypropyl--CD carboxymethyl--CD sulfated -CD sulphobutylether--CD 2-hydroxypropyltrimethylammonium--CD .

Capillary zone electrophoresis

Chiral separations by CZE

Fenoprofen Ketoprofen

25 mM TM--CD 20 mM TEA/phosphoric acid pH 5

Ibuprofen

M.Blanco et al. J.Chromatogr.A 793 (1998) 165

Micellar electrokinetic chromatography (MEKC)

Same instrumentation as for capillary zone electrophoresis Carrier electrolyte contains micelle-forming additives (sodium dodecylsulfate (SDS), ...) SDS-micelles act as negatively-charged pseudostationary phase Separation according to partition between the aqueous phase and the pseudostationary phase Suited for separation of neutral analytes (hydrophobic analytes prefer the micelles and need a longer time to reach the detector at the cathodic side)

Micellar electrokinetic chromatography

ephoretic (Micelle)

apparent (Micelle)

eo

Micellar electrokinetic chromatography

Analytes are separated according to their interaction with micelles as a pseudostationary phase (and their charge / size ratio, unless they are neutral)

(pseudostationary phase)

(analyte) (EOF)

Micellar electrokinetic chromatography

! good start for (%)*+
Capillary: 50 m inner diameter, 60 cm length 20 mM borate buffer pH 9, containing 50100 mM sodium dodecylsulfate (SDS) Voltage: + 20 kV

Micellar electrokinetic chromatography

,etention factor for (%)*
0 ------------------

k=

tt

t0 (1 - t/tmc)

t0

migration time of a neutral marker it!out interaction it! t!e micelle"

tmc migration time of a neutral micelle marker (#er$ !$%ro&!o'ic)

Micellar electrokinetic chromatography

Microemulsion electrokinetic chromatography (MEEKC)

*arrier electrolyte contains oil droplets sta.ili/ed .y a surfactat (e0g0 121) and a co-surfactant (.utanol) 2roplets (if 121 is used) act as a negatively charged pseudo-stationary phase 1eparation according to partition .etween the a3ueous phase and the pseudo-stationary phase (and their charge4si/e ratio in case of charged analytes) 1uited for the separation of neutral analytes (hydropho.ic analytes prefer the oil and need a longer time to reach the detector at the cathodic side)

Microemulsion electrokinetic chromatography (MEEKC)

www.ceandcec.com

Microemulsion electrokinetic chromatography (MEEKC)

! good start for (%%)*+
Capillary: 50 m inner diameter, 60 cm length 0.81 g octane 6.61 g butan-1-ol 3.31 g sodium dodecylsulfate 89.27 g sodium tetraborate (sonication) Voltage: + 20 kV

www.ceandcec.com

Microemulsion electrokinetic chromatography (MEEKC)

Migration time

Capillary electrochromatography (CEC)

Fused-silica capillaries with a stationary phase Column entirely filled with the stationary phase: packed columns monolithic columns

Stationary phase only present on the capillary surface: open tubular (OT) CEC

EOF acts as pump for the mobile phase

Capillary electrochromatography

Capillary electrochromatography

Capillary electrochromatography

!dvantages+ *hromatographic selectivity can .e easily manipulated .y changing the stationary phase Very high efficiency 5nteresting selectivities due to com.ined mechanism (chromatography4electrophoresis)

2isadvantages+ ,eproduci.ility may .e a pro.lem ,o.ustness may .e a pro.lem 'lushing of the column difficult

Capillary gel electrophoresis (CGE)

Capillary filled with (buffered) carrier electrolyte in a gel (polyacrylamide gels, ...) Separation according to size cross-linked gels linear gels (capillaries can be flushed and refilled)

Acr$lami%e CH(=CH-CO-NH(

linear

cro""-linke%

Capillary gel electrophoresis

Application of CGE: * DNA molecules *SDS CGE of proteins SDS is used to form negatively charged complexes with proteins. Thereby, a constant ratio of charge/mass is achieved for all proteins, allowing a separation in the gel due to differences in size.

Capillary gel electrophoresis

Application of CGE: * DNA molecules

Isoelectric focussing
Capillary filled with mixture of ampholytes (aminocarboxylic acids of different pH) leading to a pH gradient after applying voltage. Ampholytic analytes (proteins, ...) are separated according to their pI values. Analytes migrate in the pH gradient untill they reach the pH zone corresponding to their pI value and become neutral.

Isotachophoresis (ITP)
1eparation of either cations or anions *apillary filled with leading electrolyte6 followed .y terminating electrolyte %7ample+ 1eparation of anions !"-6 !#-6 !38eading electrolyte ,+89erminating electrolyte ,+9ep(8 ) $ ep(!" 6!# 6!3 ) $ ep (9 )

Isotachophoresis
:efore in;ection

,+9-

,+8-

!pplication of voltage results in migration of 9- and 8- < 8- would migrate at higher speed6 which would result in a /one without anions< therefore6 the electric field strength will change in the leading and terminating electrolyte0 v" =
"

%"<

v# =

%#
"

v" = v# 1ame speed for all /ones >

= %# 4 %"

Isotachophoresis
Separation of !3 !# !"
!3 !# !" 98-

t isotachopherogram

/one length depends on analyte concentration in in;ected sample

ELECTROPHORESIS WITH (ANTICONVECTIVE) SUPPORT

?olyacrylamide gels !garose gels *ellulose acetate

Cro""-"ection %iagram" of gel a&&aratu" %e"ign"

agarose

Cellulose acetate

Application: Serum protein electrophoresis

Electrophoresis with (anticonvective) support

2etection of separated /ones+ 1taining techni3ues (sometimes preceeded .y transfer of the separated species from the gel onto a nitrocellulose or other mem.rane (.lotting))

electroblotting

soaked in transfer buffer

soaked in transfer buffer

Electrophoresis with (anticonvective) support

2-dimensional electrophoresis (2D-electrophoresis) can be used for efficient separation of proteins: Separation in first dimension: isoelectric focussing (separation according to pI) Separation in second dimension: SDS-PAGE (polyacrylamide gel electrophoresis) (separation according to size)

Coupling of analytical separation techniques with mass spectrometry

GC with MS detection

Electron Impact Ionization (EI) or Chemical Ionization (CI)

Mobile phase (He) + Analyte

GC with MS detection

Electron Impact Ionization (EI) or Chemical Ionization (CI)

Scan Mode Aquisition of full spectra (each spectrum is recorded within a very short time) SIM Modus (selected ion monitoring) Mass analyzer is adjusted for measurement of only a few m/z corresponding to the analytes (target analysis)

Mobile phase (He) + Analyte

GC -MS

Chromatogram
(Totalion chromatogram)

Mass spectrum of peak at retention time of 63.34 min

HPLC with MS detection ESI (electrospray ionization) APCI (atmospheric pressure chemical ionization) APPI (atmospheric pressure photoionization)
from HPLC column

ESI +

HPLC with MS detection

APCI (atmospheric pressure chemical ionization)

corona discharge needle

HPLC with MS detection

APPI (atmospheric pressure photoionization)

MS high vacuum

CE with MS detection ESI APPI

Detection window voltage voltage

Kapillare

Kapillare

Carrier electrolyte vials

Carrier electrolyte vial

UV-vis absorbance detection

MS detection

CE with MS detection ESI

MS high vacuum

CE with MS detection APPI

MS high vacuum