Capillary Electrophoresis

- Fundamentals of zone electrophoresis (CZE)

- Various other modes ... CGE, IEF, MEKC, CD-based ...
- MS detection
- examples

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Food Toxicants Analysis: Techniques, Strategies and Developments
von Yolanda Pico von Elsevier (Gebundene Ausgabe - Februar 2007)

Capillary Electrophoresis. Methods and Protocols (Methods in Molecular Biology)

von Philippe Schmitt-Kopplin von Springer, Berlin (Gebundene Ausgabe - September 2007)
 Historical

Volta, Napoleon, Reuss & Helmholtz

Allesandro Volta (1745 – 1827)

Voltaic Pile (1800) 1801; Napoleon, made Volta a "Count" for his discovery

Electrokinetics: Reuss early in 1808 … water movement in clay suspensions …

-> electrophoretic movement of environmental colloids

Reuss, F F. 1809. Sur un nouvel effect de l'électricité

glavanique. Mémoires de la Societé Impériale des
Naturalistes de Moscou, 2, 327- 337

Later on theoretical aspects Helmholtz, Smouluchowski, Gouy, Debye and Huckel

 Historical

Electrophoresis U-tube assembled with electrode containers from Tiselius 1948;

Electrophoresis of normal human plasma (from Tiselius´s Nobel lecture 1948)

1960ies

2000ies

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A R N E W . K . TI S E L I U S
Electrophoresis and adsorption analysis as aids in
investigations of large molecular weight substances
and their breakdown products
Nobel Lecture, December 13, 1948
 Capillary Electrophoresis

Capillary Electrophoresis
- System setup

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 Capillary Electrophoresis
Instrumental setup

Capillary length: 20 cm to 100 cm

coated uncoated capillary
Capillary i.d.: 50 µm to 75 µm
Hydrodynamic injection
Applied voltage: -30 to 30 kV
pH range: 2 to 12
Aqueous, non aqueous

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Detection:
- Optical: direct, indirect UV-Vis, laser induced
fluorescence (LIF), Chemoluminescence
- Electrochemical: potentiometric, conductivity,
amperometric,
- Mass selective with APCI, ESI, CI
- Radioactivity (β)
 Capillary Electrophoresis
Further miniaturization = Chip

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 Capillary Electrophoresis

CE
50 µm i.d.

6.5 nl sample load

(3.3 mm)

20 cm/min
50 cm L 6.5 nl/sec

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Total volume
980 nl
 Capillary Electrophoresis

CE 1 cm i.d. 0.5 l sample load

(66 cm)
50 µm i.d.

6.5 nl sample load

(3.3 mm)
20 cm/min
0.5 l/min
20 cm/min 100 m
50 cm L 6.5 nl/sec

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Total volume
980 nl 80 l
 Capillary Electrophoresis

What makes CE different from Chromatography?

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Hydrodynamic injection
Capillary Electrophoresis Hydrodynamic injection
What makes CE different from Chromatography?

ΔP ⋅ d 4 ⋅ π ⋅ t
Vinj = ⋅10 3
128 ⋅η ⋅ L
(with ΔP the difference in pressure across the capillary in Pascals, d is
the capillary inner diameter, t the time of pressure application, η the
viscosity and L the capillary length)

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 Capillary Electrophoresis

What makes CE different from Chromatography?

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chmitt kopplin@gsf de
Electroosmotic flow
 Capillary Electrophoresis
The driving force: the electroosmotic flow
thickness of the diffuse double
layer or Debye length
− κx
ψ = ψ0e 8πnz 2 e 2
κ=
εkT
The potential ψ drops to ψ0/e at a
distance of x = κ–1, which is called
the thickness of the diffuse double
layer or also Debye length.

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The Debye length (κ–1) can be estimated with
κ–1 = 3.288.√I for a monovalent electrolyte at 25 °C

(A) Helmholtz’s capacitor model (B) Gouy and his diffuse layer model
(C) Stern and his model as coupled diffuse layer charge distribution with
corresponding drops of potential (D) Overall model of the double-layer showing
presence of solvent molecules Schematic representation of the fused silica capillary
surface at high (E) and low pH (F) values and consequences for the electroosmotic
flow when a difference of potential is applied across the capillary.
 Capillary Electrophoresis
The driving force: the electroosmotic flow

κ-1

The electroosmotic flow (µeo) is

also dependant on the chemistry,
the viscosity η and the dielectric
κ-1
constant ε of the separation buffer:

εζ
μeo =
4πηr

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ξ is the zeta potential measured at the plane of shear close to the liquid-solid interface
(slipping plane). An increase in the concentration of the electrolyte decreases the EOF,
Strongly adsorbed ions will have the same effect.
 Capillary Electrophoresis
What makes CE different from Chromatography?

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 Capillary Electrophoresis
The driving force: the electroosmotic flow

Concequence -> low dispersions

= sharp peaks

high theoretical plates >100000

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 Capillary Electrophoresis

Capillary Electrophoresis
- Capillary zone electrophoresis of small molecules

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Capillary Zone Electrophoresis

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 Capillary Zone Electrophoresis

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chmitt kopplin@gsf de
 Capillary Electrophoresis

Separation in free Solution

CZE

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 Capillary Electrophoresis
What makes CE different from Chromatography?

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 Capillary Electrophoresis
What makes CE different from Chromatography?

ve Ld ⋅ Lt
µmes = =
E tm ⋅ V
Ld ⋅ Lt ⋅ (teof - tm)
µeff =
V ⋅ tm ⋅ teof
μeff = μmes − μeof

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pH independant normalisation:
Field strength reduced velocity (cm/min)/(V/cm)
Capillary Electrophoresis

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Capillary Electrophoresis

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 22 kV 30 kV

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Time [min] Time [min]

Effective mobility [cm²/Vmin] Effective mobility [cm²/Vmin]

 Capillary Electrophoresis

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Qualitativ effects …
quantitative effects
 Capillary Electrophoresis
What makes CE different from Chromatography?

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 Capillary Electrophoresis
What makes CE different from Chromatography?

schmitt-kopplin@gsf.de
Capillary Electrophoresis

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 Capillary Electrophoresis

Separation in free Solution

CZE

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 Capillary Electrophoresis

Capillary Electrophoresis
- Capillary zone electrophoresis Structure dependancy

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 Capillary Electrophoresis

Basically the mobility of an analyte in free solution is defined as the ratio

of its electric charge Z (Z = q.e, with e the charge if an electron and q the valance)
to its electrophoretic friction coefficient f.

Charge

q⋅e

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μ=
f
Size
 Capillary Electrophoresis

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(A) Schematic representation of a charged particle and its different charged layers. (B) Relative distortions of the
local field by particle and double layer; A shematic representation of the dependence of electrophoretic mobility on
κR for different ζ potential values. The stars represent the decrease in mobility of a particle of a given size due to a decrease
of the electric double layer by changes in ionic strength. Humic substances were reported to have ζ potentials ranging from
–39 mV to –69 mV for Suwannee River fulvic acid at pH 4.5 and pH 11 respectively. These values are not extreme
and relaxation effects thus can be neglected for approximations.
 Capillary Electrophoresis

The electrostatic potential at the shear surface (ζ potential) controls to a large extent
the mobility of the particle. An estimation of electrophoretic mobility is given
in the case of small potentials, assuming a rigid and non conducting spherical particle
of Radius R, moving in a medium of viscosity η and permitivity ε, such as:

2 ⋅ε ⋅ζ
μ= ⋅ f (κ ⋅ R )
3 ⋅η

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The function f(κR), called Henry function, ranges
from 1 at κR << 1 (Hückel limit)
to 1.5 at κR >> 1 (Smoluchowski limit) as a function of the particle shape.
 Capillary Electrophoresis

κ is the reciprocal of the Debye length (electric double layer thickness), which is a
function of the ionic strength of the solution.
The Debye length (κ–1) can be estimated with κ–1 = 3.288.√I for a monovalent
electrolyte at 25 °C (nm-1; ionic strength I(M)) i.e. can vary between
3.8 nm (for I = 6 mM) and 17.6 nm (I = 0.3 mM).

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It is obvious that the entire diameter of the particle is composed from the diameter
of the rigid core and the contribution of the double layer.
Therefore the thickness of the double layer will also determine the electrophoretic
mobility of the particles
 Capillary Electrophoresis

The counterions and the charged species it contains may create retardation effects

Furthermore the distortion of the diffuse layer in the applied field leads to a polarization
so that the particle and the counterions tend to be drawn back together.
This is especially true for high ζ potentials and is known as a relaxation effect

The last effect is due to the distortion of the local electrical field by the particle.
When the particle size is small in relation to the double layer (κR << 1),
this effect is negligible (Hückel´s theory)

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Capillary Electrophoresis

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Structure versus Mobility

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Structure versus Mobility

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Structure versus Mobility

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Structure versus Mobility

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Structure versus Mobility

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Capillary Electrophoresis

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 Capillary Electrophoresis

Other Capillary Electrophoresis methods

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CD-Capillary Zone Electrophoresis

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Cyclodextrines as chiral selectors

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Interaction: 3 points

Other use: controlled synthesis (orientation)

controlled release (flavors, biocides, pesticides)
...

www.science-forum.de
 Capillary electrophoresis

M D
EOF

(neutral)

F 2,4-D

: anions

: neutral cyclodextrin

Enantiomeric ratio, enantiomeric excess,

enantiomeric fraction can be defined from CE, LC, GC
(no mixture problems as with polarimetry)

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- Concentration and buffer solution dependence
- Migration time function of binding strength
(pH dependence, substitution dependence)
Miniaturization: CE on chip
used on Mars Missionherbicidal
(NASA)
active
Derivatization and laser induced fluorescence (LIF)
detection to detect D/L amino acids!
 Biotic effects

-+ + + + +
-
-

Garison et al (1996)

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Different soils: different R/S

amendment: homogenization

environment change -> affect ER

Romero et al (1996) mainly biotic effects (µorganisms)

abiotic effects ?
Example: behavior of R/S-mecoprop and dichlorprop after field application
Micellar Capillary Electrophoresis

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Partial filling MEKC
for coupling to MS
Micellar Capillary Electrophoresis

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 Micellar Capillary Electrophoresis

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Capacity factor Å-> LogP
Micellar Capillary Electrophoresis

Strong binding to micelle

neutral, cationic

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CD-Micellar Capillary Electrophoresis

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 1996 total racemate enantiopure
Environmental chemicals:
-herbicides
racemates in production
11275 /2500
field application
570
- POPs (PCBs, HCH)
insecticides 6990 2330 1075
- agrochemicals -fungicides
impurity within4415
the formulation
1285 60
- antibiotics/pharmaceuticals
growth prom. 1420 170 20
(fluoroquinolones, ibuprofen) - metabolization into racemic compounds
24100 6285 1725
Ex: - phenoxy acids R(+) killing weeds 26% 7%
- metolachlor 2R and 2S forms (S-forms are 10 times more active)
- (R)-enantiomer of o,p´-DDT has more estrogenic activity than the (S)-enaniomer

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General accepted rule: changes in ER due to biotic effects
Affinity Capillary Electrophoresis

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(reconstructed electropherogram)
mobility [cm²/Vs]
-0,009 -0,01 -0,011 -0,012 -0,013 -0,014 -0,015 -0,016
0

-0,001

-0,002

-0,003
AU 254 nm

-0,004
D-leu D-leu (n=5)
-0,005
L-leu L-leu (n=5)
-0,006
D-leu L-leu (n=9)
-0,007
L-leu D-leu (n=8)
Affinity Capillary Electrophoresis

0.16

hydroxyatrazine
0.14 Kp = 357

0.12

capacity factor k´
0.1
atraton
Kp = 269
0.08

ametryn
0.06 Kp = 198
HCMC
ameline
30 mg/l Kp = 151
0.04

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0.02

0
0 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.0007 0.0008

-0.02 Concentration in humic acid [kg/l]

 Affinity Capillary Electrophoresis

HA [mg/L]

0 200 400 600 800 1000 1200

0

-0.002

n° 8
decarboxylated
-0.004

enroflaxacin
decarboxylated
µeff [cm²/V.s]

-0.006

metabolite A n° 8 Fluoroquinolones
-0.008

metabolite B n° 8
-0.01
enroflaxacin

n° 7
-0.012

cyproflaxacin
-0.014 n° 8

n° 5
-0.016

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LogK
3.5
3
Structure dependant Binding
2.5 Partial charge dependant
2
1.5

When ACE compared to MEKC

1
0.5

-> specific binding

0
0 0.5 1 1.5 2
LogP
Capillary Gel Electrophoresis

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Capillary Isoelectric Focussing

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Capillary Isoelectric Focussing

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Capillary Isoelectric Focussing

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Measurement of isoelectric points
Capillary Electrochromatography

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Coupling to mass spectrometry

CE-ESI/MS

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 Interfacing nanoLC and CE to ESI/ion trap MS

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adapted to nanoLC

Schmitt-Kopplin & Frommberger, Electrophoresis, 2003, review on CE-MS, 24, 3837-3867

Interfacing CE to ESI/MS

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Schmitt-Kopplin & Englmann, Electrophoresis, 2005, Survey on CE-MS
Interfacing CE to ESI/MS

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Schmitt-Kopplin & Englmann, Electrophoresis, 2005, Survey on CE-MS
Interfacing nanoLC and CE to ESI/ion trap MS

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Schmitt-Kopplin & Frommberger, Electrophoresis, 2003, review on CE-MS, 24, 3837-3867
 Interfacing nanoLC and CE to ESI/ion trap MS

Palmblad et al. Upsala Sweden

structure information exact structure formula
from separation from FTICR
(CE, CEC, LC, GC)
 Interfacing nanoLC and CE to ESI/ion trap MS

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Laboratory constructed microspray interface.
ST, polymer tee;
SY, syringe needle;
SN, stainless steel nut;
SL, sheath liquid channel;
SC, separation capillary;
PN, polymer nuts;
EF, elastomer ferrule.
Interfacing nanoLC and CE to ESI/ion trap MS

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Interfacing nanoLC and CE to ESI/ion trap MS

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 Interfacing CE to ESI/ion trap MS

CE-ESI/MS
Amonium carbonate pH 9

Negative modus
[M-H]-
-> Thermal degradation

No benzoic acid adduct

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Positive modus
[M+NH4]+
-> Adduct formation (NH4+)
 Applications

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schmitt-kopplin@gsf.de
Paraquat ist eine quartäre Ammoniumverbindung, die als
Kontaktherbizid aus der Familie der Bipyridinium-
Herbizide eingesetzt wird. Es wurde von der englischen
Firma ICI (Agrarsparte heute Teil der Schweizer Syngenta)
1955 entwickelt und kam 1962 erstmals unter dem
Handelsnamen Gramoxone® auf den Markt.

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 dithiocarbamates

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 dithiocarbamates

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 Uncoated fused-silica, inner diameter: 75 μm,
Capillary effective length: 50 cm, total length: 57 cm.
Detection 196 nm
Detector cell size:100*800 μm.
Temperature Cartridge set to 30 oC.
Capillary preconditioning 2 min pressure with 0.1 mol/L NaOH, then 2 min pressure with
BGE.
Injection Hydrodynamic injection of sample 50 mbar for 10 s
(injected volume 58 nl).
20 kV.

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Separation voltage
Typical current ~50 μA.
Replenishment Replenishment of both separation vials and injection vial is
preferred after each injection.
Capillary preconditioning Pressure flush with 0.1 mol/L NaOH for 30 min followed by BGE
(new capillary only) for 15 min.
Storing of capillary Pressure flush with 0.1 mol/L NaOH for 15 min followed by milli Q
(more then one week) water for 15 min then with air for 5 min.
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