(aciii ephoresis biolchem 2013 [kompatibilitätsmodus])

8/13/2019 (ACIII Ephoresis BiolChem 2013 [Kompatibilittsmodus])

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(+) anode (-) cathode

-

+

++

n

Electrophoresis Separation is based on differences in

migration of charged analyte ions in anelectric field in solution


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Electrophoresis

Separation can be carried out in free (buffered)

solutionCAPILLARY ELECTROPHORESIS (CE)

or in (buffered) solution immobilized in a gel oron a similar support


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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)


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Capillary zone electrophoresis

Instrumentation

carrierelectrolytevial

carrierelectrolytevial

samplevials

0+-30 kV

High-voltage

power supply

fused-silica capillary(filled with carrier electrolyte) detector


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fused silica360 m outer diameter

polyimidecoating

15 m thick

inner diameter25-100 m


Fused silica capillary:

30-100 cm length


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Acceleration:

Kac = z e E z charge numbere 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


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Steady-state: Kac = Kfr

vep/E = z e / 6 r

vep/E electrophoretic mobility ep

ep = / F !! (F Faraday constant

ionic equivalence conductance)


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- - - - - - - - - - - - -

- - - - - - - - - - - - -

+ + + + + + + + + + + + +

+ + + + + + + + + + + + +

(+)anode

(-)cathode

flow (EOF)

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


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electroosmotic flow profile hydrodynamic flow profile

laminar


electroosmotic flow:


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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


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Dependence of electroosmotic mobility on pH

pH

Electro

osmoticm

obility


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ep / eo/ apphigh- and medium-molecular-mass analyte ions

eo

ep

!"-

!#-

!3+

app!3

+

!"-

!#-

low-molecular-mass analyte ionseo

ep

!"-

!#-

!3+

app !3+

!"-

!#-

!3+$%&'marker$!"

-$!#-

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

-+


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Ld = length of capillary from injector to detectort = migration time

Lt = total length of capillaryV = voltage applied to capillary

VtLL

LVtL

EV td

t

dappapp ===

vapp


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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


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Effects of EOF-modifiers

CATHODEANODEelectroosmotic flow eo


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Sample injection

very small sample sizes - 10-9 liters

hydrodynamic injection, uses pressure toforce sample onto column

electrokinetic injection based on ep - thusinjected sample has different relativecomposition of analytes


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Sample injection techniques


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direct and indirect UV detection:


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Capillary zone electrophoresisSummarizing the mechanisms:

Capillary filled with (buffered) carrier electrolyte

Separation according to electrophoretic mobilities

Optimization of separation can be done by changing thepH (changes in degree of dissoziation of the analytes,changes of charge) or by addition of complex-formingagents (changes in charge and/or size) to the carrierelectrolyte


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Capillary zone electrophoresisUse 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 analytesand the chiral selectors

-, - or -cyclodextrinmethyl derivatives of -CD2-hydroxypropyl--CDcarboxymethyl--CDsulfated -CD

sulphobutylether--CD2-hydroxypropyltrimethyl-

ammonium--CD.


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Capillary zone electrophoresisChiral separations by CZE

Fenoprofen

Ketoprofen

Ibuprofen

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

25 mM TM--CD20 mM TEA/phosphoric acid

pH 5


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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 pseudostationaryphase

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)


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eo

ephoretic (Micelle) apparent (Micelle)


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(EOF)

(pseudostationary phase)

(analyte)

Analytes are separated according to their interaction with micelles as a

pseudostationary phase (and their charge / size ratio, unless they are

neutral)


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Micellar electrokinetic chromatography! good start for %*

Capillary: 50 m inner diameter, 60 cm length

20 mM borate buffer pH 9, containing 50100 mMsodium dodecylsulfate (SDS)

Voltage: + 20 kV


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,etention factor for %*

t t0

t0 (1 - t/tmc)k =

t0 migration time of a neutral markerit!out interaction it! t!e micelle"

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


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Microemulsion electrokinetic

chromatography (MEEKC)

*arrier electrolyte contains oil droplets sta.ili/ed .y a

surfactat (eg 121) and a co-surfactant (.utanol)

2roplets (if 121 is used) act as a negatively chargedpseudo-stationary phase

1eparation according to partition .etween the aueousphase and the pseudo-stationary phase (and theircharge4si/e ratio in case of charged analytes)

1uited for the separation of neutral analytes (hydropho.icanalytes prefer the oil and need a longer time to reach thedetector at the cathodic side)


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www.ceandcec.com


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Migration time


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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


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Capillary electrochromatography


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!dvantages

- *hromatographic selectivity can .e easilymanipulated .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


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Capillary gel electrophoresis

Application of CGE:

* DNA molecules

*SDS CGE of proteinsSDS is used to form negatively charged complexes withproteins. Thereby, a constant ratio of charge/mass isachieved for all proteins, allowing a separation in the gel due

to differences in size.


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Capillary gel electrophoresis

Application of CGE:

* DNA molecules


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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 separatedaccording to their pI values. Analytes migrate in the pHgradient untill they reach the pH zone corresponding totheir pI value and become neutral.


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Isotachophoresis (ITP)

1eparation of either cations or anions

*apillary filled with leading electrolyte6 followed.y terminating electrolyte

%7ample

1eparation of anions !"-6 !#-6 !3-

8eading electrolyte ,+8-

9erminating electrolyte ,+9-

ep(8-

) $ ep(!"-

6!#-

6!3-

) $ep (9-

)


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Isotachophoresis

:efore in;ection

,+8-,+9-

!pplication of voltage results in migration of 9- and 8-


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ELECTROPHORESIS WITH


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ELECTROPHORESIS WITH

(ANTICONVECTIVE) SUPPORT

?olyacrylamide gels

!garose gels

*ellulose acetate

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


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Cellulose acetate


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Application: Serum protein electrophoresis


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electroblotting

soaked in transfer buffer

soaked in transfer buffer

Electrophoresis with


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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)


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Coupling of analyticalseparation techniques

with mass spectrometry


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GC with MS detection

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

Mobile phase (He) + Analyte


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GC with MS detection

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

Scan ModeAquisition of full spectra (eachspectrum is recorded within avery short time)

SIM Modus

(selected ion monitoring)Mass analyzer is adjusted formeasurement of only a fewm/z corresponding to theanalytes

(target analysis)

Mobile phase (He) + Analyte


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GC -MS

Chromatogram(Totalion chromatogram)

Mass spectrum ofpeak at retention timeof 63.34 min


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HPLC with MS detection


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APCI (atmospheric pressure chemical ionization)

coronadischarge

needle



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APPI (atmospheric pressure photoionization)

MS

highvacuum


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CE with MS detection


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ESI

MShighvacuum

CE with MS detection


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APPI

MShighvacuum

(aciii ephoresis biolchem 2013 [kompatibilitätsmodus])

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