2013-04-15
1
New Developments in Capillary
Electrophoresis with focus on
Bioanalysis
Lecture 8
Christian Nilsson
Western Blotting using CE
• Drawback slab gel Western blotting
– Manually time consuming
– Gel preparation, separation, electro blotting,
incubation
– Sensitivity in ng range
– Difficult to transfer large proteins from gel
Anal. Chem. 2011, 83, 1350-1355
Western Blotting using CE
• Microscale western blotting system
• Based on Capillary Gel electrophoresis for
separation of SDS-protein complexes
• Deposition on blotting membrane
• Grounding through a sheath capillary
Anal. Chem. 2011, 83, 1350-1355
Western Blotting using CE
• Translation stage to move blotting membrane
past the outlet of the capillary
• Membrane moistured by methanol/buffer
• Polymer solution for sieving
Anal. Chem. 2011, 83, 1350-1355
Western Blotting Using CE
Anal. Chem. 2011, 83, 1350-1355
Western Blotting Using CE
Anal. Chem. 2011, 83, 1350-1355
Detection limit, 10 pg
2013-04-15
2
Western Blotting using CE
• Faster analysis
• No electro blotting
• Automation
• Lower consumtion of reagent and sample
• Lower detection limits, 10 pg
– Little optimization
Anal. Chem. 2011, 83, 1350-1355
Western Blotting using CE
Anal. Chem. 2011, 83, 1350-1355
Band broadening ~1.7 fold
Microelectrodialysis coupled to CE
• Pretreatment and analysis of inorganic cations
in biological matrices
• No adsorption of high molecular weight
compounds to the capillary wall.
• Cellulose acetate dialysis membrane with
molecular weight cut-off of 500 Da
– Can be used approximately 100 times before
replaced
Electrophoresis 2011, 32, 464-471
Microelectrodialysis coupled to CE
Electrophoresis 2011, 32, 464-471
• Less than 1 μl of sample is needed
Microelectrodialysis coupled to CE
Electrophoresis 2011, 32, 464-471
Microelectrodialysis coupled to CE
Electrophoresis 2011, 32, 464-471
2013-04-15
3
Use of additives to CE/CEC
• Since the Terabe introduced micelles as
additive for CE, a large amount of different
additives have been used
• Follows the development in material science
and nanotechnology
Use of additives in CE/CEC
• Requirements for nanoparticles to be used
analytes
Use of additives in CE/CEC Use of additives – Silica particles
• Early use of particles as pseudostationary
phase
• Use of reversed phase particles (1.5 μm)
• Surfactants used to coat the particles to form
stable suspensions
• A partial filling approach was used due to the
light scattering at the particles
• Nine phenol derivates were separated
J. Chromatogr. A 1994, 688, 283-292
Use of additives – Silica particles
UV detection
The particles were relatively big
which caused problem with
suspension stability and thereby
separation reproducibility.
Could be improved by sonicating
the particle suspension every
hour
The mobility of the particles was
larger and in the opposite
direction compared to the EOF
Use of additives – Silica particles
• Improvement of method
• Fluorescence detection was used circumvent
the complications with light scattering of the
particles when UV detection is used
• Smaller particles was used (500 nm in
diameter)
J. Chromatogr. A, 1997, 768, 320-324
2013-04-15
4
Use of additives – Silica Particles
• Addition of cyclodextran or urea to the mobile phase was
necessary to prevent ”too” strong binding of the analytes to
the particles, causing assymmetric peaks and band
broadening
Use of additives – Silica particles
• Particles with a diameter of 500 nm
• Covalent modification of the particles to
introduce carboxylic groups
• Fluorescence detection
• Migration window optimized by changing the
pH. The widest window was observed at pH
7.0
Use of additives – Silica Particles
• The main factors for reducing the plate
numbers were concluded to be:
– Mass transfer resistance at the particle surface
– Different velocity of the particles
Use of additives – Molecular micelles
• Molecular micelles are micelles that are
covalently linked
• They have a zero CMC
• They are stable in presence of organic solvent
Use of additives – Molecular micelles
• Amino acid-based molecular micelles have
been used for enantioseparation of eight β-
blockers
• Possible with UV as well as MS detection
Use of additives – Molecular micelles
2013-04-15
5
Additives in CE/CEC – DNA analysis
• Conventional DNA analysis in CE is based on
using a physical gel (i.e. polymer solution)
– Limited by the high viscosity of solution
– Replenishment cumbersome
• An alternative is to use additives in the gel:
– Nanoparticles
– Carbon nanotubes
Additives in CE/CEC – DNA analysis
• Separation of DNA is possible with lower
polymer concentration in presence of
nanoparticles
Additives in CE/CEC – DNA analysis
• Gold nanoparticles
– Relatively easy to prepare
– Need to be stabilized
• For example by polymers, e.g. poly (ethylene oxide)
– Have affinity for thiols (R-SH)
Gold nanoparticles
Additives in CE/CEC – DNA analysis
• Separation of DNA using PEO-coated gold
nanoparticles
• A continuous full filling approach is used
– The capillary is filled with nanoparticles
• Laser induced fluorescence as detection
• Intercalation of Ethidium bromide to detect
DNA
Additives in CE/CEC – DNA analysis
• Lower viscosity of gold nanoparticle
suspensions
• The capillary was dynamically coated with PVP
to suppress EOF and prevent interactions
between DNA and capillary wall
• A suggested separation mechanism involve
that DNA temporary interwines with the PEO
on the gold nanoparticles
Additives in CE/CEC – DNA analysis
2013-04-15
6
DNA analysis on chip using
nanoparticles
Additives in CE/CEC – Carbon nanostructures
• For example:
– Fullerenes
– Carbon nanotubes
– Carbon nanohorns
• Low solubility in aqueous buffers
• Can be solved by:
– Oxidization of surface
• Sonication in sulfuric and nitric acid
• Carboxylic group on surface
– Addition of surfactants (i.e. SDS)
Additives for CE/CEC - Fullerenes
• Discovered in 1985
• Commercially availible with different surface
chemistries
Additives for CE/CEC - Fullerenes
• SDS used to solubilize fullerenes in water
• Use of fullerene-SDS complexes for separation
polycyclic aromatic hydrocarbons (PAHs)
• Similar separation mechanism as MEKC
• Fullerenes enhance separation
J. Chromatogr,. 2000, 873, 257-267
Nonaqueous CE of Fullerenes
• Separation of different variants of fullerenes
– C60 and C70 fullerenes as well as C60 variants
Anal. Bioanal. Chem. 2012, 404, 307-313
Nonaqueous CE of Fullerenes
Anal. Bioanal. Chem. 2012, 404, 307-313
2013-04-15
7
Nonaqueous CE of Fullerenes
Anal. Bioanal. Chem. 2012, 404, 307-313
Additives in CE/CEC – Carbon nanotubes
• Diameter of a few nm up to a few tens of nm.
• Length up to several micrometers
Additives in CE/CEC – Carbon nanotubes
• Separation of homologues of caffeine and
theobromine
• Distinct changes in the separation occur at a
certain concentration
• The nanotubes formed sieving networks in the
capillary that acted as pseudostationary phase
• The nanotube network prevented the
diffusion of the analytes to the capillary wall
and thereby minimized the adsorption to the
capillary wall
Electrophoresis 2003, 24, 4181-4188
Additives in CE/CEC – Carbon nanotubes
Electrophoresis 2003, 24, 4181-4188
SEM
Additives in CE/CEC – Carbon nanotubes
Electrophoresis 2003, 24, 4181-4188
Additives in CE/CEC – Carbon nanohorns
• Diameter of approximately 2 nm
• Length 30-50 nm, which is substantially
shorter than the carbon nanotubes
• Conical ends
• Forms flower-like structures with a diameter
of 80-100 nm, with a large surface area.
2013-04-15
8
Additives in CE/CEC – Carbon nanohorns Example of other additives in CE/CEC
• Molecular imprinted polymer nanoparticles
• Dendrimers
Template Orientation
at o/w Interface Using a Functional Surfactant
O
OH
NH2+
Template
(S)-propranolol
O
O
O
O
Cross-linker
EDMA
NH
O
O
O Functional
surfactant
N-undecenoyl glcinate
NH
O
O
O
NH
O
O
O
NH O
O
O
N
H
O
O
O
N
H
O
O
O
N
H
O
O
O
N
H
O
O
O
N
H
O
O
O
NHO
O
O
N
H
O
O
O
-
-
-
-
-
-
-
-
-
-
O
OH
NH2
O
OH
NH2
O
OH
NH2
O
OH H2
N
O
OH
H2
N
O
OH
H2
N
O
OH
H2
N
O
OH
NH2
O
OH
NH2
O
OH
NH2
+
+
+
+
+
+
+
+
+
+
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
MIP Nanoparticle Synthesis
(Mini-emulsion Strategy)
CEC Separation of Racemic mixture of Propanolol
1,3 1,4 1,5 1,6 1,7 1,8
0,000
0,025
Absorbance(mAU)
Time (min)
R S
Nanoparticles; hydrodynamic injection 0.5psi for 10 s,
sample injection 3 s 6 kV, electrolyte 10 mM phosphate/20% acetonitrile,
16 kV, T, 30 ºC. L, 30 cm
Monoclonal Imprints!!!