2013-04-08
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New Developments in Capillary
Electrophoresis with focus on
Bioanalysis
Lecture 5
Christian Nilsson
Lecture today
• 2D-CE
• CE-MS coupling
• More CEC
Complex Samples
• Proteomics
• Diagnostics
– Body Fluids
• Biomarker discovery
• Large difference in concentration
2D-CE
• General Setup
2D-LC/CE
• Reversed Phase LC and CE
• CE is a fast technique suitable as the second
dimension
• The two techniques are orthogonal in
separation mechanism
– Hydrophobicity
– Mass-to-charge ratio
2D-LC/CE - Examples
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2D-LC/CE - Examples 2D-LC/CE - Examples
Tryptic digest of ovalbumin
Fluorescentlabeled
2D-CE
• An alternative to 2D electrophoresis by slab
gels for proteomic studies
2D-CE - Examples
2D-CE - Examples
• Protein Separation
• On Column Tryptic Digestion
• Peptide Separation
• Mass Spectrometry Detection
• Data Analysis
2D-CE
• Peptides from one protein introduced into the
MS within a short time window
• Peptides not spread as if the proteins were
digested prior to separation
• Fewer peptides are detected simultaneously
as if digested just prior to introduction into
MS.
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2D-CE - Examples
• Use of magnetic nanoparticles
• Trypsin immobilized on nanoparticles
• Magnets used to hold the nanoparticles in the
capillary
• Nanoparticles are replaced before each
experiment
• Large surface-to-volume ratio
2D-CE - Examples
• Sufficient long time in microreactor
• Avoid band broadening
• Limited life time of certain microreactors, for
example, monoliths
• Separation of peptides after digestion
2D-CE - Examples
• Short microreactor to avoid band broadening
• The peptides are introduced to the second
capillary prior to MS detection
• At the same time fresh protein is introduced
into the microreactor and is digested during
the peptide separation
2D-CE – Examples
• Capillary Interface
– Cross that was machined into a plexiglas plate
2D-CE - Examples 2D-CE - Examples
First dimension – One capillary
Second dimension – An array of
capillaries
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2D-CE - Examples
2D separation on PDMS microchip
Isoelectric Focusing
Analysis of Biofluids for Diagnostics
• Combination of techniques
– Preconcentration
– A combination of techniques to:
• Separate
• Identify
• Quantify
• Large variation in abundance
Analysis of small proteins (below 20
kDa) and peptides in urine
• CZE coupled to ESI-MS
• High throughput
• Excellent resolution
• Biomarker often limited to the highly
abundant substances
CE-MS
• 2 Review articles uploaded among study
material
• Interface of CE with
– Electrospray
– MALDI
– ICP
CE-MS
• Compatibility of CE electrolyte with MS
detection have to be considered
• Avoid ion supression
• Use of low ionic strength
• Use of acetic or formic acid
• Use of organic solvents
Coupling of CE with ESI-MS
• Sheath Liquid
• Sheathless
• Liquid Junction Interface
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CE-ESI-MS Interface
• Electrical connection for adjusting spraying
potential and CE outlet potential
• Transfer of the analytes to the spray
• Continuous delivery of spray or sheath liquid
CE-ESI-MS – Sheath liquid
• The voltage is applied via a sheath liquid
• The sheath liquid is flowing outside the
separation capillary and is mixing with the
analytes at the spray tip
CE-ESI-MS – Sheathless interface
• Electrical contact directly via the fused silica
capillary
• Lower detection limits
CE-ESI-MS – Liquid Junction
Less dilution of the analytes
compared to the sheath flow
techniques
CE-ESI-MS – Liquid Junction
• Narrow gap between separation and spray
capillary
• The spray potential is applied to a spray liquid
which is surrounding the junction
• Separation voltage is determined by the field
at the inlet of the separation capillary and at
the spray liquid
CE-MALDI-MS
• Off-line coupling the most common
• Separation and detection can be performed
independently
• Reliable fraction collection
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Applications of CE-MS
• Proteomics and Glycomics most common
– Analysis of intact proteins
– Analysis of digested proteins
• Useful for biology/cell studies
• High throughput, High sensitivity and
resolution
Applications of CE-MS
• Intact proteins
– Characterization of protein isoforms in the
biopharmaceutical industry
– Impurities
– Protein modification
Applications of CE-MS
• Peptides
• Biomarkers
– Large differences in concentrations
• Tryptic digests of proteins
Nanoparticle-based CEC
Packed column CEC Monolithic column CEC
Open tubular CEC
Pseudostationary phase
CEC
Pretoriuset al. J. Chromatogr. 1974
Jorgenson et al. J. Chromatogr. 1981
Tsuda et al.
J. Chromatogr. 1982
Wallingford and Ewing Adv. Chromatogr. 1989
Nilsson et al. Electrophoresis2006
Hjertén et al.
J. Chromatogr. 1989
Capillary
Electrochromatography
CEC
PSP
Conventional Liquid Chromatography (LC)
• Complicated packing procedures
• Need for retaining frits
• Sample matrix can modify column
– Fouling
– Reduced reproducibility
Pressure
Adsorbed
material
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• Small particles (nanoparticles) ~5-100 nm
Pseudostationary phase –
Capillary electrochromatography
Large
surface area
Enhanced
mass transfer
Increased
efficiency
50
μm
20 kV+ •
Liquid Chromatography
– Pressure-driven flow
– 1-5 mm column diameter
– 2-5 μm particle diameter
• Capillary Electrochromatography
– Electro-driven flow
– ~50 μm column diameter
– 10-100 nm particle diameter
Nanoparticle-based PSP-CEC
• No packing or retaining frits
• One-time use of stationary phase
– No carry over
– Minimal column regeneration
– Low consumption of nanoparticles
– Can start a new separation before the previous one
finished
• Small particles (sub-micron)
– High surface-to-volume ratio
– High efficiency Detection
???
Continuous Full Filling
Detection
= Nanoparticles
= Sample
Detection
• Orthogonal ESI-MS
• Laser Induced Fluorescence (LIF)
• UV
• Other…
Separation of Small Molecules
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• Hydrophobic core
• Hydrophilic surface
TEM
Average Diameter 600 nm
Dextran-coated Polymer Nanoparticles Polymerisation
O
OH
O
O O
O
O
O
O
O O
O
O
O
O
O
O
O
O
O
O
O
O O
O
O O
O
O
O
O
O
O
O
O
O
AIBN
MAA
TRIM
Surface modification Orthogonal electrospray interface
+ -
-
-
--+
+ ++
+
+-
+
+
++-
-
-
-
-
-
-
-
++
+
+
+
+
+
+
+++++++++++++
+
+
+
+
+
+
+
+
+
+
+
+
+
+
++ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Analytes
Nanoparticles
Ortogonal electrospray interface Conclusions RP-CEC
• Nanoparticles could be used for separation
with an electrolyte with low acetonitrile
concentration
• It was possible to use high amount of
nanoparticles in continuous full filling without
contamination of the mass spectrometer
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Continuous full filling RP-CEC
Electrolyte: Acetonitrile and 10 mM
ammonium acetate, pH 5,6 (30:70
and 40:60 v/v)
Nanoparticle slurry: 0; 0,5 1,0; 2,0,
5,0 and 10,0 mg/ml.
30 kV
Continuous full filling RP-CEC
R2
= 0,7912
R2
= 0,7734
R2
= 0,2096
0,99
1,00
1,01
1,02
1,03
1,04
1,05
1,06
1,07
1,08
1,09
1,10
0 2 4 6 8 10
Slurry concentration [mg/ml]
Normalisedretentiontime
Dimethyl phthalate
Diethyl phthalate
Dipropyl phthalate
Dibutyl phthalate
0
2
4
6
8
10
12
14
0 2 4 6 8 10
Slurry concentration [mg/ml]
Peakintensity
Dimethyl phthalate
Diethyl phthalate
Dipropyl phthalate
Dibutyl phthalate
Separation of Small Molecules
Peak 2: >700 000 plates /m
Reversed Phase
Conclusions RP-CEC
• Separation was performed with high peak
numbers.
– High concentration of acetonitrile.
– High concentration of nanoparticles.
• Low consumption of nanoparticles
– 10 μl nanoparticle slurry per effective hour of
separation
Future improvements
• Further optimisation of acetonitrile
concentration, nanoparticle concentration and
pH.
• Use of other nanoparticles.
• Use of combinations of nanoparticles.
• Use of smaller nanoparticles.
• Start a new separation before last one finished
Separation of Proteins
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24 cm long
Capillary column
5-30 kV
Detection:
UV or LIF
50 μm
ProteinNanoparticle
Electron microscopy
of nanoparticles
(diameter 70 nm)
Protein CE/CEC
• Capillary Wall Adsorption
• Solutions to the problem
–Buffer pH (high or low)
–Salt or Zwitterionic Additives
–Static or Dynamic Coatings
–Nanoparticles?
...in bare silica capillary
...tricine, zwitterionic, low µA
...at neutral pH
Lipid Nanoparticles
~70 nm diameter
Lipid-based liquid crystalline
nanoparticles
• Average diameter 70 nm
• Bicontinuous Cubic Phase
• Porous (100 Å)
• Protein compatible
– Membrane proteins
– Drug delivery
• Easy to prepare
– One-step procedure
230
212
230
212
26 kDa
238 amino acids
pI=5.7
Green fluorescent protein (GFP)
Mutants
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Protein Separation
PSP- CEC cationic and anionic porous
lipid based nanoparticles
Cationic
Cationic
+ Anionic
Protein Separation
High resolution
Single-amino-acid-
substitution
High efficiency
Protein Separation
Separation of Proteins
GFP
(+)GFP
(-)GFP
Transfer to chip format
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GFP variants In Topas® Capillary
With nanoparticles
Separation on polymer chip
0 20 40 60 80 100 120 140 160 180
0
50
100
150
200
250
300
time / s
Fluorescencesignal/arbitraryunits
GFP with impurities
0 %
10 %
2 %
GFP sample
Weight %
Nanoparticles
Future of nanoparticle CEC?
–Separation with UV detection
–Separation of membrane proteins
–Analysis of biological nanoparticles