(ACIII Ephoresis BiolChem 2013 [Kompatibilitätsmodus])
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Transcript of (ACIII Ephoresis BiolChem 2013 [Kompatibilitätsmodus])
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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
Capillary zone electrophoresis
Fused silica capillary:
30-100 cm length
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Capillary zone electrophoresis
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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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)
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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
Capillary zone electrophoresis
electroosmotic flow:
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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
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Capillary zone electrophoresis
Dependence of electroosmotic mobility on pH
pH
Electro
osmoticm
obility
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Capillary zone electrophoresis
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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Capillary zone electrophoresis
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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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
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Capillary zone electrophoresis
Effects of EOF-modifiers
CATHODEANODEelectroosmotic flow eo
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Capillary zone electrophoresis
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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Capillary zone electrophoresis
Sample injection techniques
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Capillary zone electrophoresis
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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Micellar electrokinetic chromatography
eo
ephoretic (Micelle) apparent (Micelle)
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Micellar electrokinetic chromatography
(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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Micellar electrokinetic chromatography
,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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Micellar electrokinetic chromatography
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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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Microemulsion electrokinetic
chromatography (MEEKC)
www.ceandcec.com
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Microemulsion electrokinetic
chromatography (MEEKC)
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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Capillary electrochromatography
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Capillary electrochromatography
!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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HPLC with MS detection
APCI (atmospheric pressure chemical ionization)
coronadischarge
needle
HPLC with MS detection
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HPLC with MS detection
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