capillaryelectrophoresis

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Capillary Electrophoresis - Fundamentals of zone electrophoresis (CZE) - Various other modes ... CGE, IEF, MEKC, CD-based ...- MS detection - examples

Food Toxicants Analysis: Techniques, Strategies and Developmentsvon 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)

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Historical

Volta, Napoleon, Reuss & Helmholtz

Electrokinetics: Reuss early in 1808 … water movement in clay suspensions …-> electrophoretic movement of environmental colloids

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

Reuss, F F. 1809. Sur un nouvel effect de l'électricitéglavanique. Mémoires de la Societé Impériale des

Naturalistes de Moscou , 2, 327- 337

Allesandro Volta ( 1745 – 1827)

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



Historical

Electrophoresis U-tube assembled with electrode containers from Tiselius 1948;Electrophoresis of normal human plasma (from Tiselius ´s Nobel lecture 1948)

A R N E W . K . TI S E L I U SElectrophoresis and adsorption analysis as aids ininvestigations of large molecular weight substancesand their breakdown productsNobel Lecture, December 13, 1948

1960ies

2000ies

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Capillary Electrophoresis

Capillary Electrophoresis - System setup

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Capillary length: 20 cm to 100 cm

Capillary i.d.: 50 µm to 75 µm

Hydrodynamic injection

Applied voltage: -30 to 30 kV

pH range: 2 to 12

Aqueous, non aqueous

coated uncoated capillary

Instrumental setup

Detection:

- Optical: direct, indirect UV-Vis, laser inducedfluorescence (LIF), Chemoluminescence

- Electrochemical: potentiometric, conductivity,amperometric,

- Mass selective with APCI, ESI, CI

- Radioactivity (β) [email protected]



Capillary Electrophoresis Further miniaturization = Chip

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50 µm i.d.

50 cm L

Total volume980 nl

20 cm/min6.5 nl/sec

6.5 nl sample load(3.3 mm)

CE

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50 µm i.d.

50 cm L

1 cm i.d.

100 m

Total volume980 nl 80 l

20 cm/min6.5 nl/sec

20 cm/min

0.5 l/min

6.5 nl sample load(3.3 mm)

0.5 l sample load(66 cm)

CE

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(with ΔP the difference in pressure across the capillary in Pascals, d isthe capillary inner diameter, t the time of pressure application, η the

viscosity and L the capillary length)

34

10128

⋅⋅⋅

⋅⋅⋅Δ=L

t d PV injη

π

Hydrodynamic injection

What makes CE different from Chromatography?

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Electroosmotic flow


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The driving force: the electroosmotic flow

κ x0 eψψ

−=

kT

enz

ε

π κ

228=

thickness of the diffuse doublelayer or Debye length

The potential ψ drops to ψ 0/e at adistance of x = κ–1, which is calledthe thickness of the diffuse double

layer or also Debye length .

(A) Helmholtz’s capacitor model (B) Gouy and his diffuse layer model(C) Stern and his model as coupled diffuse layer charge distribution withcorresponding drops of potential (D) Overall model of the double-layer showing

presence of solvent molecules Schematic representation of the fused silica capillarysurface at high (E) and low pH (F) values and consequences for the electroosmoticflow when a difference of potential is applied across the capillary.

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

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The driving force: the electroosmotic flow

Concequence -> low dispersions= sharp peaks

high theoretical plates >100000

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Capillary Electrophoresis - Capillary zone electrophoresis of small molecules

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Capillary Zone Electrophoresis

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Separation in free Solution

CZE

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V t

LLm

t d

⋅

⋅

== E

v

µ

emes

eof mesef μμf μ −=

eof m

meof tdeff

ttV

)t- (tLL µ

⋅⋅

⋅⋅=


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

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30 kV22 kV

Time [min] Time [min]

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

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Qualitativ effects …quantitative effects

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Separation in free Solution

CZE

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Capillary Electrophoresis - Capillary zone electrophoresis Structure dependancy

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Basically the mobility of an analyte in free solution is defined as the ratioof its electric charge Z (Z = q.e, with e the charge if an electron and q the valance)to its electrophoretic friction coefficient f.

f

eq ⋅=μ

Charge

Size

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(A) Schematic representation of a charged particle and its different charged layers. (B) Relative distortions of thelocal field by particle and double layer; A shematic representation of the dependence of electrophoretic mobility onκR for different ζ potential values. The stars represent the decrease in mobility of a particle of a given size due to a decreaseof 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 extremeand relaxation effects thus can be neglected for approximations. sc

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The electrostatic potential at the shear surface ( ζ potential) controls to a large extentthe 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:

)(3

2Rf ⋅⋅

⋅

⋅⋅= κ

η ζ ε

μ

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.

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κ is the reciprocal of the Debye length (electric double layer thickness), which is afunction 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 between3.8 nm (for I = 6 mM) and 17.6 nm (I = 0.3 mM) .

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 [email protected]




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 polarizationso 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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Structure versus Mobility

schm

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schm

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schm

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C ill El h i




schm

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C ill El t h i




Other Capillary Electrophoresis methods

schm

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CD-Capillary Zone Electrophoresis

schm

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Cyclodextrines as chiral selectors



Cyclodextrines as chiral selectors

www.science-forum.de

Interaction: 3 points

Other use: controlled synthesis (orientation)controlled release (flavors, biocides, pesticides)...

schm

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Capillary electrophoresis



EOF

(neutral)

: anions

: neutral cyclodextrin

Capillary electrophoresis

F

M D

2,4-D

herbicidalactive

- Concentration and buffer solution dependence- Migration time function of binding strength

(pH dependence, substitution dependence)

Miniaturization: CE on chipused on Mars Mission (NASA)

Derivatization and laser induced fluorescence (LIF)detection to detect D/L amino acids!

Enantiomeric ratio, enantiomeric excess,enantiomeric fraction can be defined from CE, LC, GC

(no mixture problems as with polarimetry)

schm

itt-kopplin@

gsf.de



Micellar Capillary Electrophoresis

Partial filling MEKC for coupling to MS

schm

itt-kopplin@

gsf.de

Mi ll C ill El h i




schm

itt-kopplin@

gsf.de

Mi ll C ill El t h i




Capacity factor -> LogP

schm

itt-kopplin@

gsf.de





Strong binding to micelleneutral, cationic

schm

itt-kopplin@

gsf.de

CD Micellar Capillary Electrophoresis



CD-Micellar Capillary Electrophoresis

schmitt-kopplin@

gsf.de



Environmental chemicals:- POPs (PCBs, HCH)- agrochemicals- antibiotics/pharmaceuticals(fluoroquinolones, ibuprofen)

7%26%

1725628524100

201701420growth prom.

6012854415fungicides

107523306990insecticides

570250011275herbicidesenantiopureracematetotal1996

- racemates in production / field application

- impurity within the formulation

- metabolization into racemic compounds

Ex: - phenoxy acids R(+) killing weeds

General accepted rule: changes in ER due to biotic effects

- 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

schmitt-kopplin@

gsf.de

Affinity Capillary Electrophoresis



y p y p

-0,007

-0,006

-0,005

-0,004

-0,003

-0,002

-0,001

0

-0,016-0,015-0,014-0,013-0,012-0,011-0,01-0,009

L-leu L-leu (n=5)

L-leu D-leu (n=8)

D-leu L-leu (n=9)

D-leu D-leu (n=5)

mobility [cm²/Vs]

AU 254 n

(reconstructed electropherogram)

schmitt-kopplin@

gsf.de




-0.02

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.0007 0.0008

Concentration in humic acid [kg/l]

capacity factor k´

hydroxyatrazine

atraton

ametryn

ameline

Kp = 357

Kp = 269

Kp = 198

Kp = 151

HCMC30 mg/l

schmitt-kopplin@

gsf.de




Fluoroquinolones

-0.016

-0.014

-0.012

-0.01

-0.008

-0.006

-0.004

-0.002

00 200 400 600 800 1000 1200

µeff [cm²/V.s]

HA [mg/L]

enroflaxacin

cyproflaxacin

n° 5

n° 7

n° 8

enroflaxacin

decarboxylated

n° 8decarboxylated

metabolite A n° 8

metabolite B n° 8

0

0.5

1

1.5

2

2.5

3

3.5

0 0.5 1 1.5 2

LogP

LogK

Structure dependant BindingPartial charge dependant

When ACE compared to MEKC-> specific binding sch

mitt-kopplin@

gsf.de



Capillary Isoelectric Focussing



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gsf.de




Measurement of isoelectric points

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Capillary Electrochromatography



schmitt-kopplin@

gsf.de

Coupling to mass spectrometry



CE-ESI/MS

schmitt-kopplin@

gsf.de

Interfacing nanoLC and CE to ESI/ion trap MS



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

adapted to nanoLC

schmitt-kopplin@

gsf.de

Interfacing CE to ESI/MS



Schmitt-Kopplin & Englmann, Electrophoresis , 2005, Survey on CE-MS schmitt-kopplin@

gsf.de

Interfacing CE to ESI/MS



Schmitt-Kopplin & Englmann, Electrophoresis , 2005, Survey on CE-MS schmitt-kopplin@

gsf.de




Schmitt-Kopplin & Frommberger, Electrophoresis , 2003, review on CE-MS, 24, 3837-3867 [email protected]




structure information

from separation(CE, CEC, LC, GC)

exact structure formula

from FTICR Palmblad et al. UpsalaSweden




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. [email protected]




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Interfacing CE to ESI/ion trap MS



-> Adduct formation (NH 4+)

Negative modus[M-H] -

Positive modus[M+NH 4]+

-> Thermal degradation

No benzoic acid adduct

CE-ESI/MSAmonium carbonate pH 9

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Applications

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Paraquat ist eine quartäre Ammoniumverbindung , die alsKontakt herbizid 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 demHandelsnamen Gramoxone® auf den Markt.

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dithiocarbamates



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dithiocarbamates

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Pressure flush with 0.1 mol/L NaOH for 15 min followed by milli Qwater for 15 min then with air for 5 min.

Storing of capillary(more then one week)

Pressure flush with 0.1 mol/L NaOH for 30 min followed by BGEfor 15 min.

Capillary preconditioning(new capillary only)

Replenishment of both separation vials and injection vial ispreferred after each injection.

Replenishment

~50 μA.Typical current

20 kV .Separation voltage

Hydrodynamic injection of sample 50 mbar for 10 s(injected volume 58 nl).

Injection

2 min pressure with 0.1 mol/L NaOH, then 2 min pressure withBGE.

Capillary preconditioning

Cartridge set to 30 oC.Temperature

196 nmDetector cell size:100*800 μm.

Detection

Uncoated fused-silica, inner diameter: 75 μm,effective length: 50 cm, total length: 57 cm.Capillary

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