Notion d echelle dans les methodes de separation VF English Y. FRANCOIS 01

16/04/2020
Outline
• Introduction.
– Interests and specificities of nanometric scale for separation methods.
• Last insights in nanometric scale for chromatography,
Nanoscale in Analytical Chemistry
– from micro to nano flow rate. Evolution of new stationary phases, i.e.
monoliths.
Miniatirization of separation methods
• Another alternative to chromatography: electrophoretic separation.
– From 2D Gel to capillary electrophoresis.
• Lab on chip, the future of miniaturization for separation methods.
- Microsystem production
- Microsystems for sample preparation
Contact : Yannis FRANCOIS, Lab. de Spectrométrie de Masse
des Interactions et des Systèmes, Institut Lebel, 4 rue Blaise
Pascal, 67000 Strasbourg
email: [email protected]
Key points of miniaturization history
60’:
What the need in separation chemistry?
Gaz chromatography (GC) using packed columns
Characteristic of the method
First generation of mass spectrometer (elemental analysis)
Speed, high throughput
Reduction of analysis time
70’:
Introduction of GC with capillary columns
Low cost
Decreased consumption of solvents and samples, releases
Introduction of HPLC
 « in situ » analysis
Introduction of capillary electrophoresis
Characteristic of samples
Miniaturiation of the device  Integrated systems
90’:
Mass spectrometry: API, MALDI, ICP,…
Separation methods coupled to MS: >50% of instrumental market
Growing role of biology in analytical developments
Small volume
Diminution of sample consumption
Complexity of the mixture
Highly resolutive methods
Low concentration level
2000 -…:
Miniaturization is the key word in every analytical domains.
coupling with sensible detector
Bioanalytical chemistry requests high throughput and rapid diagnostic
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HPLC column: diminution of diameter
Reduction of solvent consuption
Description
Dimensions
HPLC
4 mm  i.d.  5 mm
narrow-bore column
2 mm  i.d.  4 mm
Tipical flow rate
0,3 – 3 mL/min
micro-bore column
1 mm  i.d.  2 mm
50 – 1000 µL/min
Capillary column
100 µm  i.d.  1 mm
0,4 – 200 µL/min
Nano-column
25 µm  i.d.  100µm
25 – 4000 nL/min
1 – 5 mL/min
Inner
diameter(mm)
Section (mm2)
Flow rate
(µL/min)
Solvent
consuption
4,6
16,6
1200
100%
2
3,1
225
19%
1
0,8
56
5%
0,5
0,2
15
1,2%
0,25
0,05
3,5
0,3%
nanoLC
HPLC
Improvement of sensitivy
Sensitivity
Detection of concentration: Response = Peak area

A = Response.dt
h = Response max
Inner
diameter(mm)
Section (mm2)
Flow rate
(µL/min)
4,6
16,6
1200
1
2
3,1
225
5,3
1
0,8
56
21
Gain in
sensitivity *
0,5
0,2
15
80
0,25
0,05
3,5
335
* for identical optical paths
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Theoretical aspect
The plate theory
 The plate theory is probably the best theory for explaining
chromatographic separation phenomena.
 Gaussian peak
 Calculation of theoretical plate number
Theoretical aspect
The kinetic theory
 The kinetic theory considers the chromatographic peak as
representative of the statistical distribution of the retention times
of the molecules of a given substance on the column.
 Limitations :
 Absence
of
consideration
of
diffusion
phenomena
 Absence of kinetic consideration (speed of
exchanges between the two phases
 Impossibility of introducing the entire sample in
an infinitely small volume
 The kinetic theory considers the diffusion phenomena and mass
transfer
Theoretical aspect
The kinetic theory
Diffusion phenomena
Longitudinal molecular diffusion
Theoretical aspect
The kinetic theory
Mass transfert
a b
 t0, molecules a and b of the
same substance are on the same
line
 ti, a will stay in the grain pore of
the stationary phase and b in the
mobile phase
Turbulent diffusion
 tf, b will go faster than the
molecule a
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Theoretical aspect
The kinetic theory
Theoretical aspect
The kinetic theory
Solution to minimize diffusion phenomena
 Improvement of the homogeneity of the stationnary phase:
 Absence of heterogeneities
 Absence de bubbles
 Homogenize the flow rate of the mobile phase
 Réduction of the diameter of particules dp
Van Deemter equation
H = A + B/ū + C.ū
 Decrease the size of particule pores
ū = mean linear velocity of flow of the mobile phase in the column
Theoretical aspect
Increase in efficacity (N)
The kinetic theory
Van Deemter equation
Summary
 For particules
Hopt = f(dp)
 Reduction of the diameter of particules
uopt = f(1/dp)
 Small size
 Low porosity
dp1
dp2<dp1
Hopt
 Chromatography
 Rapid separation
 With miniaturized stationnary phases
uopt
 Experimental condition
Limitation: Increased pressure at the top or the column
 At low temperature
 By reducing dead volumes
Darcy’s low: P = Lu / k0dp2
Limited particule diamenter
between 1,5 - 3µm
permeability
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Non-porous particles of particles with a porous surface layer
Monoliths
Inorganic or organic
Improvement of mass
transfert phenomena
Continuous structure containing interconnected pores
Bimodal structure: macroporous and mesoporous network
High permeability
Faster separation
Improvement in mass transfert
HEPT (µm)
35
P (bars)
300
Column: Poroshell 300SB-C18 75 mm x 2.1mm i.d.
Mobile phase: (A) 0.1%TFA, (B) 0.07%TFA in ACN
30
colonne particulaire 3µm
250
Gradient: 5-100%B in 1 min
20
colonne particulaire 5µm
150
T=70°C
colonne particulaire 3µm
15
100
UV à 215nm
colonne particulaire 5µm
25
200
Flow rate: 3mL/min - P=260bar
10
50
colonne monolithique
5
colonne monolithique
0
0
0
2
4
6
8
10
0
2
4
6
8
10
débit (mL/min)
débit (mL/min)
Characteristics of the porous network
Inorganic monolith synthesis
Tetramethoxysilane (TMOS)
Bimodal distribution of macropores and
mesopores
+ Acetic acid (catalyst)
+ PEG : porogene
Independent control of both pore sizes
Modling
Freezing - Aging
Structure of the monolith
Si(OR)4 + H2O
Si-OH + Si-OH
Si-OH + Si-OR
x Si-O-Si
Si(OH)(OR)3 + ROH
Si-O-Si + H2O
Si-O-Si + ROH
(Si-O-Si)x
Dissolution-Reprecipitation
Generation of mesoporous
Macroporosity : generated by PEG
Mesoporosity: generated by NH4OH treatment
(or urea)
Hydrolysys
Condensation
Polycondensation
Calcination
Drying
PEG removal
Skeletal strenghening
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Interest of organic monolith
Preparation of organic monolith
acrylamide-based
Synthesys in aqueous medium: low solubility of "hydrophobic" monomers in
water, difficulty in controlling pore size
 Simple synthesis
Synthesys in organic medium
 Adaptable to microdevice
 Methacrylic ester-based
Major work on monolith
 Stability in a high range of pH : 1 -13
Simple synthesis
 Polystyrene-based (LC packing, Dionex)
Physical aspect of silicium monolith
Initiation
By heating: T=55-70°C, t20h
Easy to make
By UV irradiation: =365nm, t<1h
Fast reaction
Polymerization zone easy to delimit
Slow
Possibility of cracks
Low penetration depth
Difficulty to delimit polymerization
zone
More complex implementation
Monolith made from a mold: L=10cm,
d=1cm
Micro-column
Micro-system
Thesis work J. Chamieh
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Monoliths
Column 4,6 mm i.d.
Inorganic or organic
Continuous structure containing interconnected pores
Bimodal structure: macroporous and mesoporous network
High permeability
Faster separation
Improvement in mass transfert
HEPT (µm)
35
P (bars)
300
30
colonne particulaire 3µm
250
colonne particulaire 5µm
25
200
20
colonne particulaire 5µm
150
colonne particulaire 3µm
15
100
10
50
colonne monolithique
5
colonne monolithique
0
0
0
2
4
6
8
10
débit (mL/min)
0
2
4
6
8
10
7mL/min  91bars
débit (mL/min)
Why the nano-flow rate?
Column 100 µm i.d.
When the sample concentration is very low

need of miniaturization

in classical columns, pressure problem
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Issues concerning nano-flow rate
Technological constraints:
 Pumping process
 The ability to generate gradients
 Measurement and control of nano-flow rate
 Leak detection,…
Capillary electrophoresis:
Fundamental notions
Options:
The "classic" pumping system can be used with “split "
Electrokinetic pumping
Contact : Yannis FRANCOIS, Lab. de Dynamique et Structure
Moléculaire par Spectrométrie de Masse, institut de Chimie, 1
rue Blaise Pascal, 67000 Strasbourg
email: [email protected]
History
History
1937
1939
Separation of protein
By electrophoresis on paper
1954
1967
Separation of protein
S. Hjerten : 300 µm i.d. capillaries
In the Human serum
1981
From J.L. Veuthey, Univ. de Genève
J. Jorgenson : 75 µm i.d. capillaries
From J.L. Veuthey, Univ. de Genève
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Electrophoresis: a big familly
Outline
1. Migration phenomenon in CE
Electrophoresis
Isoelectric
focalisation
Isotachophoreris
1.1 Electrophoretic mobility
1.2 Electroosmotic mobility
2. The separation in CE
Zone
electrophoresis
2.1 Efficacity
2.2 Resolution
3. Improvement of selectivity
Paper
Gel
Capillary
4. Quantitative analysis
4.1 Injection
4.2 Detection
CZE
CGE
CEC
MEKC
5. Capillary isoelectric focalisation (CIEF)
Introduction capillary electrophoresis
CE as a separation method
HPLC
Principle
CE
analyte
E
soluté
Mobile
phase
Stationary
phase
No Stationary phase
 instrumentation: No pressure device, no injection device
+
-
+Capillary
Injection
Detector
MINIATURISATION
Low sample consumption
Inexpensive method
High EFFICACITY of separation
High voltage generator
 Low SENSIBILITY of detection
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Principle of separation under electric fields
Experimental device
Capillary
+
Detector
Important question to ask
-
o So.. How it works?
Electrodes
o How to see neutrals and anions ?
o How to calculate electroosmotic and electrophoretic mobilities ?
Electrolyte
vials
o How to detect all anions and cations during the separtation ?
High voltage generator
o What are the parameters to optimize the separation ?
Classical capillaries: bare fused silica
lenght : 20 - 100 cm
inner diameter : 20 - 100 µm
Voltage: 5 - 30 kV
Principle of separation under electric fields
Principle of separation under electric fields
Voltage
30 kV
Voltage
30 kV
Electric Field
Detector
Electric Field
Detector
Cathode
Background electrolyte = Salted solution
Anode
Cathode
Cathode
Background electrolyte = Salted solution
Anode
+
Conductivity
–
++
Conductivity
–
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Principle of separation under electric fields
Principle of separation under electric fields
Voltage
30 kV
Voltage
30 kV
Electric Field
Cathode
Cathode
++
Detector
Electric Force
Friction Force
+
Electric Field
Anode
Cathode
Cathode
–
++
Principle of separation under electric fields
+
+
FF = 6   r vep
: viscosity
r : hydrodynamic radius
vep : ion velocity
Detector
++
+
Electrophoretic Mobility
µep
–
ANODE
FF: friction force
+
FE: electric force
Cathode
Cathode
Anode
Electrophoretic Mobility
µep
Electrophoresis
Voltage
30 kV
Electric Field
Detector
Anode
–
-
CATHODE
qE
Vep =
6r
FE = q E
q: ion charge
E: electric field
q
µep =
6r
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+
+
++++
+
++
++
+
-
+
-
+
-
++
++
+
++++
+
-
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+
-
++ ++
+
+
++
-
++
+
Principle of separation under electric fields
Voltage
30 kV
Detector
+
++
++
+
+
Cathode
Cathode
ELECTROPHORESIS can separate:
 Molecules bearing DIFFERENT CHARGES,
 Molecules with the SAME CHARGE
but with a DIFFERENCE of SIZE.
Anode
++
–
–
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Principle of separation under electric fields
Principle of separation under electric fields
Voltage
30 kV
Voltage
30 kV
Detector
+
Detector
µep
+
Cathode
Cathode
Anode
Cathode
Cathode
++
–
++
µep
Anode
–
µep
–
µep
–
Electropherogram
+
Time
Principle of separation under electric fields
Principle of separation under electric fields
Important question to ask
Voltage
30 kV
o So.. How it works?
Detector
o How to see neutrals and anions ?
+
µep
o How to calculate electroosmotic and electrophoretic mobilities ?
Cathode
Cathode
Anode
++
o How to detect all anions and cations during the separtation ?
–
µep
Electropherogram
–
+
How to separate neutrals and anions
o What are the parameters to optimize the separation ?
Time
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Electroosmotic mobility
Electroosmotic mobility
Bare Fused Silica Capillary
Voltage
30 kV
Detector
Detector
Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si
OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH
Cathode
Background Electrolyte = Salted solution
Anode
Cathode
Anode
+
Conductivity
–
+
–
OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH
Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si
Electroosmotic mobility
Electroosmose
Bare Fused Silica Capillary
Acidic condition
 Protonation of silanols Si-OH
 Surface net charge = 0
Basic condition
 Deprotonation of silanols Si-O Surface net charge < 0
Si Si Si Si Si Si
OH OH OH OH OH OH
Si Si Si Si Si Si
Charge capillary surface
+
Neutralization of opposite
charge of electrolyte
DOUBLE LAYER
O- O- O- O- O- Odisplacement of the solvent that occurs under the effect of the
electric field
Surface net charge depending the pH value of Background Electrolyte
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Electroosmotic mobility
Bare Fused Silica Capillary
At basic conditions
Electroosmotic mobility
Bare Fused Silica Capillary
At basic conditions
Detector
Detector
-----------------------------
Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si
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-
Cathode
Anode
Cathode
Background Electrolyte = Salted solution
Anode
+
–
+
Conductivity
–
-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
Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si
Electroosmotic mobility
Bare Fused Silica Capillary
At basic conditions
Electroosmotic mobility
Bare Fused Silica Capillary
At basic conditions
Detector
----------------------------Cathode
+
Background Electrolyte = Salted solution
Anode
Cathode
Conductivity
–
+
Detector
----------------------+ – + – + – + – + – + –
–
–
–
–
–
–
+
+
+
+
+
+
+
+
+
+
+
+
---+ –
–
+
– + –
–
–
–
–
–
–
–
–
–
–
–
–
+
+
+
+
+
+
+
+
+
+
+
+
+
– + –
–
–
–
–
–
–
–
–
–
–
–
–
-+
–
+
–
Anode
–
+
–
Voltage
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DOUBLE LAYER: STERN Model
Electroosmotic mobility
Creation of an ion movement due to the formation
of double ionic layer = electroosmotic mobility
Bare Fused Silica Capillary
At basic conditions
+
----------------------------+ + + + + + + + + + + + + + +
– + – + – + – + – + – + – + +
–
+
–
+
–
–
+
–
–
–
+
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
+
+
–
–
+
–
+
–
+
–
+
–
+
–
+
–
+
+
– + –
–
--
+
--
+
+ +
+
+
+ + -+ + +
--
+
+
---
+
electroosmose
---
+
+
--
Anode
silanols
–
+
distance
–
1/x

Voltage
potential
The decrease of the double layer potential determines the
velocity of solvent.
E
0
s

Influence of pH on silica group of the capillary
Electroosmotic flow
4
In solution
Diffuse layer
-- + - +
+ +
+
+
Electroosmotic mobility
µeo
Veo =
-
+
- - - - - - - - - - - - - - - - - - - - - - -
Cathode
+
Bare fused silica
pI  2

µeo =
4
 : Zeta potentiel zéta
 : dielectric constant of electrolyte
 : viscosity
Increase of  (Charge density)
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Principle of separation under electric fields
Bare Fused Silica Capillary
At basic conditions
Principle of separation under electric fields
Bare Fused Silica Capillary
At basic conditions
Voltage
30 kV
Voltage
30 kV
Detector
Detector
-----------------------------
-----------------------------
+
+
Cathode
Cathode
Anode
Cathode
Cathode
++
–
++
Anode
–
µep
–
Principle of separation under electric fields
Bare Fused Silica Capillary
At basic conditions
–
Principle of separation under electric fields
Bare Fused Silica Capillary
At basic conditions
Tension
30 kV
µeo
µep
Detector
- - - - - - - -µep- - - - - - - - - - - - - - - - - - - - +
Cathode
Cathode
++
µep
–
What compounds will I be able to detect ?
µeo
µeo
µeo
Apparent mobility µapp = µep + µeo
+
Anode
µep and µeo same direction µapp > 0
Detection
µep = 0 then µapp = µeo
Detection
µep et µeo opposite direction
µep < µeo
Detection
µep < µeo
No detection
–
Electropherogram
+
–
–
Time
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Principle of separation under electric fields
Apparent mobility
µapp = µeo + µep
Important question to ask
 Bare fused silica capillary:
o So.. How it works?
+
o How to see neutrals and anions ?
ep
-
- - - - - - - - - - - - - - - - - eo
-
eo
+
eo
ep
eo
o How to calculate electroosmotic and electrophoretic mobilities ?
o How to detect all anions and cations during the separtation ?
+
+
o What are the parameters to optimize the separation ?
-
time
Measurement of mobilities
Ld
Principle of separation under electric fields
+
Lt
détecteur
Important question to ask
-
+
+
-
o So.. How it works?
o How to see neutrals and anions ?
tapp
Electroosmotic mobility
Apparent mobility
t0
Electrophoretic mobility
µapp=µep + µeo
veo/E
vapp/E
o How to calculate electroosmotic and electrophoretic mobilities ?
o How to detect all anions and cations during the separtation ?
o What are the parameters to optimize the separation ?
µep=µapp - µeo
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The modification of the capillary surface, Why?
 To modulate the electroosmotic flow whatever
the pH value
 To cancel or reverse the electroosmotic flow
 To avoid adsorption phenomenon in the
capillary wall
The modification of the capillary surface, Why?
Anion separation:
+
ep
- - - - - - - - - - - - - - - - - eo
ep
eo
app
app
Dynamic coating
Presence of additives in the electrolyte
détecteur
+
 To realize short time analyses
The modification of the capillary surface, How?
-
-
eo
-
Amino « quenchers » : polycationic polymers
CH3
N+
CH3
2Br
(CH2)6
CH3
N+
(CH2)3
CH3
polybrene
Permanent coating
Chemical linkage
1- Silica activation by a reaction of silylation
2- Coating by functional group
+ - - - +- - - - +- - - - +- - - - +- - +
+
+
+
+
Thermal immobilization
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Separation on coated capillary
Other additives
Positive coated capillary
Neutral polymers
Voltage
- 30 kV
HPMC
PEO
µeo
PVA
Detector
+ + + + + + + +µep+ + + + + + + + + + + + + + + + + + + + +
Neutral surfactants
Triton X-100
Brij-35
Tween 20
µeo
–
Anode
Cathode
µeo
–+
Zwitterionic surfactants
n=9,11,15
CH 3(CH2)n
N+
SO3-
µep
+
µeo
Cathode
+
Electropherogram
–
+
SB-n+1
Apparent mobility µapp = µep + µeo
Separation on coated capillary
Time
The modification of the capillary surface, How?
Positive coated capillary
What compounds will I be able to detect ?
–
+
µep et µeo same direction
µapp > 0
Detection
µep = 0 then µapp = µeo
Detection
µep et µeo opposite direction
µep < µeo
Detection
µep < µeo
No detection
Rapid Commun. in Mass Spectrom., 1997, 11, 307
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Efficacity of the separation
Flow profiles
HPLC
Peak distortion following Van Deemter model
In terms of height of theoretical plate (H) :
x
x
Hydrodynamic flow: parabolic profile
POMPE
H = A + B/u + C.u
Hopt
Turbulence (A):
CE
uopt
Electroosmotic flow: flat profile
-
+
Molecular diffusion (B) :
Mass transfert (C) :
INCREASE OF EFFICACITY
Voltage issue
Separation factors
1
Limitation: Joule effect
2
P
L
t
HPLC
CE
Retention or migration
k’
µep
 = k’1/k’2
 = µep,1/µep,2
=
 C r2 V2
L2
L : capillary length
r : capillary radius
C : electrolyte concentration
Selectivity
Resolution
Rs =
1
-1
k’
4

k ’+1
N
Rs =
1
µep
N
4 µep,moy + µeo
 : Molecular conductance
Low inner diameter capillary
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Principle of separation under electric fields
Outline
1. Migration phenomenon in CE
Important question to ask
1.1 Electrophoretic mobility
1.2 Electroosmotic mobility
o So.. How it works?
2. The separation in CE
o How to see neutrals and anions ?
2.1 Efficacity
2.2 Resolution
o How to calculate electroosmotic and electrophoretic mobilities ?
3. Improvement of selectivity
o How to detect all anions and cations during the separtation ?
4. Quantitative analysis
o What are the parameters to optimize the separation ?
4.1 Injection
4.2 Detection
5. Capillary isoelectric focalisation (CIEF)
IMPROVMENT OF SELECTIVITY
CZE: Capillary Zone
Electrophresis
Factors which can modifiy electroosmotic mobility
MEKC

µeo =
Non-aqueous CE
SELECTIVITE
4



 : charge density of capillary surface
EKC:
cyclodextrines,…
 : viscosity
 : double layer thickness
CGE
Electrochromatography
(CEC)
Background electrolyte : nature and concentration of ions,
pH, organic solvents
 Nature of capillary
 Temperature
23
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Influence of pH on silica group of the capillary
Factors which can modify electrophoretic mobilities
q
µep =
Bare fused silica
pI  2
6r
 pH : modification of analyte apparent charges
 Nature of the electrolyte
 Addition of organic solvent
 Temperature
Increase of  (Charge density)
Acidic or basic properties: pKa
Usual buffer used in CE
LH ↔ H+ + L-
[L-]
pKA = pH - log
1
[HL]
charge
0,8
0,6
0,4
Buffer
pKA
Phosphate
Citrate
Formate
Succinate
Acetate
Borate
MES
HEPES
TRIS
2.12 - 7.21 -12.32
3.06 - 4.74 - 5.40
3.75
4.19 - 5.57
4.74
9
6.15
Weak
7.55
conductivity
8.30
0,2
pK
-6 A-6
pK-4A-4
pK
-2A-2
0
pK
0A
pKA2+2
pKA+4
4
pKA6+6
24
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Influence of ionic strength
Influence of ionic strength
 = K.(T/Cizi2)1/2

µeo =
4

Increase of concentration

 decrease of 

Phosphate buffer
 : charge density of capillary surface
 : viscosity
 : double layer thickness
 = K.(T/Cizi2)1/2
Tech. Prot. Chem. II, 3-19 (1991).
Influence of organic modifier
Influence of organic modifier
q
Polaires ( >30)
Protiques
Eau
MeOH
Apolaires ( <30)
Aprotiques
ACN
DMF
DMSO
Protiques
Aprotiques
EtOH
THF
PrOH
Dioxane
 Influence on mobility and/or constant of dissociation (pKA, ion
pairing,…)
µep =
 Influence on viscosity
6r
 Influence on pH
 Influence on solvation effect
Solvent
Cations
Anions
Water
++
++
Methanol
+/-
++
Ethanol
-
++
Acetonitrile
--
--
25
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General rules:
Influence of organic modifier
veo (10-3
m.s -1)
2

1,8
1,6
µeo =
1,4
1,2
4



To DECREASE the electroosmotic mobility:
1
0,8
 When the pH decreases
 decrease of 
0,6
0,4
0,2
0
0
20
40
60
80
% v/v
 Influence on viscosity
 Influence on zeta potential
(10-4kg.m-40
1 -1
s )
Polar Solvents (ex : water): zeta potential  which can
be up to 100mV.
35
30
Non-polar Solvents (ex : heptane): No zeta potential ,
except in the presence of additives.
25
20
15
 When the concentration of electrolyte increases
 decrease of 
 When the percentage of organic modifier increase
 decrease of 
ACN < acetone < MeOH < EtOH, PrOH < DMSO
10
5
•Increased percentage of organic modifier
0
0
20
40
60
80
 decrease of 
100
% v/v
DMSO
acetone
ACN
ACN < acetone < MeOH < EtOH, PrOH < DMSO
IMPROVEMENT OF SELECTIVITY
CZE: Capillary Zone
Electrophresis
Non-aqueous capillary electrophoresis NACE
 weak electric currents
MEKC
increase of inner diameter of capillaries
semi-preparative
increase of efficacity (N/t /2)
Non-aqueous CE
SELECTIVITE
EKC:
cyclodextrines,…
Water
methanol
CGE
Electrochromatography
(CEC)
/
/2
88,2
6924
22,7
552
NMF
110,3
20075
acetonitrile
110,3
4136
 Changes on selectivity
 Better compatibility with MS detection
 Increase of solubility (ex: cyclodextrins)
26
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Non-aqueous capillary electrophoresis NACE
Non-aqueous capillary electrophoresis NACE
3
1
10
11
9
5
1
A
8
67
3
24
12
9
5
24
10
8
6
B
11
12
7
Separation of a 12 compounds mixture
Bare fused capillary 58;5cm x 50µm i.d. - 30kV
electrolyte : (A) ethanol/acetonitrile/acetic acid (50:49:1) in 20mM CH 3COO-, NH4+
(B) methanol/acetonitrile/acetic acid (50:49:1) in 20mM CH 3COO-, NH4+
1 amphétamine, 2 éphédrine, 3 levorphanol, 4 dextromoramide, 5 morphine, 6 hydrochlorothiazide, 7 acide benzoïque, 8 acide
meso-2,3-diphénylsuccinique, 9 probenecid, 10 chlorothiazide, 11 acide phénylènediacétique, 12 acide éthacrynique.
Chromatographia, 2000, 52, 403-407
J. Chromatogr. A, 1997, 792, 13-35.
IMPROVMENT OF SELECTIVITY
CZE: Capillary Zone
Electrophresis
Non-aqueous CE
SELECTIVITE
CGE
Micellar eletrokinetic chromatography
MEKC
EKC:
cyclodextrines,…
Electrochromatography
(CEC)
= separation technique that combines type of phenomena :
 electroosmose
 electrophoresis
 chromatography
Partition between mobile phase and pseudo stationary
phase
No instrumental development
Separation of neutral molecules
27
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Separation of neutral molecules
Micellar eletrokinetic chromatography
 sufficiently soluble electrolyte to form micelles
 No electrophoresis mobility under electrical field
 UV transparent
 Co-elution of all de toutes ces molécules avec le flux électroosmotique
Homogeneity of micelles
 Low viscosity
Strategies:
1. Formation of charged complexes
CMC(10 -3 M) à 25°C dans l ’eau
Surfactant
ex :
S + THA+
S(THA)+ + THA+
Sodium dodecylsulfate (SDS)
Sodium tetradecylsulfate (STS)
Sodium N-lauroyl-N-methyltaurate (LMT)
Sodium cholate
Cetyltrimethylammonium bromide (CTAB)
S(THA)+
S(THA)22+
2. Ionic micelles
8.1
2.1 (50°C)
8.7
13-15
0.92
 The most commonly used
Micellar eletrokinetic chromatography
Micellar eletrokinetic chromatography
+
-
- - - - - - - - - - - - - - - - - vep,mc
-
-
-
-
-
-
-
-
veo
-
vep,mc
-
-
vep,mc
-
-
-
-
-
-
-
-
-
= Neutral compound
-
-
-
-
-
-
- = micelle
-
-
28
16/04/2020
injection
detection
Free
micelle
Analyt
e
Micellar eletrokinetic chromatography
water
analyte
Free micelle
eau
Migration time
t0
tR
tmc
t0< tR < tmc  Window of detection
Different type of Pseudo-phases used in MEKC
IMPROVMENT OF SELECTIVITY
CZE: Capillary Zone
Electrophresis
Non-aqueous CE
SELECTIVITE
CGE
MEKC
EKC:
cyclodextrines,…
Electrochromatography
(CEC)
29
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Chiral separation (identical charge density)
Chiral separation (identical charge density)
= separation technique that combines type of phenomena :
 electroosmose
 electrophoresis
On-line separation
Off-line separation
- - - - - - - - - - - - A-
A+
AA-
A-
A+
A+
A+
A-
A+
- - - - - - - - - - - - Principle based on chiral recognition by addition of chiral
selector
Formation of
diastereoisomers
= chiral selector
A-
A-
A+
A+
No instrumental development
Chiral recognition
Electrophoretic separation
Separation of enantiomers
cyclodextrins
Chiral selectors
Complexation by inclution
 , , -cyclodextrins
 éthers-crowns
Complexation by chelation
 -hydroxy ou -aminoacids
et metals (Cu)
Association with chiral polymers
 maltodextrins
 heparin, sulphated dextran
Micellar separation
 -hydroxy ou -aminoacides
with alkyl chain
Formation of ion pairing
 camphrosulfonates
 quinine and derivatives
 -CD
Interaction by affinity
 proteins
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Characteristics of cyclodextrins
 -CD
-CD
Cyclodextrine
Number of glucose
Molecular weight
Inner diameter of cavity/nm
Diameter/nm
6
972.9
0.47–0.52
1.46
Height of cavity/nm
Solubility in water at 25°C
-CD
-CD
7
1135.0
8
1297.2
Large number of modified cyclodextrins:
0.62–0.64 0.75–0.83
1.54
1.75
Reaction with OH in position 2,3 et 6
neutral
0.79–0.80 0.79–0.80 0.79–0.80
140mM
16mM
140mM
Positively charged
Negatively charded
OH
O
O
COO-
H
N
Hydroxy-CD
in 4M urea : 89mM
Amino-CD
NH2
OO
Carboxy-CD
in 8M urea : 226mM
Sulfonato-CD
-O
3S
OO
SO3- -O3S SO3-
Allow separation of netraul compounds
4,3Å
Chiral selector
IMPROVMENT OF SELECTIVITY
5,0Å -CD
8,0Å -CD
HS--CD
HS- -CD
CZE: Capillary Zone
Electrophresis
6,2Å -CD
Non-aqueous CE
Rs = 2.2
Rs = 2.9
SELECTIVITE
10.0
temps (min)
alprenolol
2.0
4.0
temps (min)
isoproterenol
EKC:
cyclodextrines,…
Rs = 1.8
CGE
5.0
MEKC
HS--CD
5.0
Electrochromatography
(CEC)
10.0
temps (min)
atenolol
Capillary 50 µm i. d. x 31.5 cm - separation under -15 kV, T = 22°C
5%HS--CD in 25mM tetraethylammonium phosphate, pH 2.5
31
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Influence of cyclodextrins in MEKC
IMPROVMENT OF SELECTIVITY
CZE: Capillary Zone
Electrophresis
Non-aqueous CE
MEKC
CGE
Electrochromatography
EKC:
cyclodextrines,…
SELECTIVITE
Electrochromatography
(CEC)
Electrochromatography
Advantages
= separation technique that combines type of phenomena :
 electroosmose
 electrophoresis
 chromatography
Partition between mobile phase and stationary phase
o
o
o
o
o
o
o
Selectivity
Injection capacity
Low dispersion in mobile phase and stationary phase
Low diffusion effect
Allow the use of very low particle size
High efficiency
Hyphenation with MS detection
o
o
o
o
o
o
Manufacture of columns
Fragility of columns
Control of mobile phase flow rate
Nature of the stationary phases
Difficult to perform gradient
Increase of the analysis time
Drawbacks
No instrumental development, but
modification of capillary
Separation of neutral molecules
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Comparison LC/CEC
Comparison LC/CEC
HPLC
CE
Classical column volumes
4 mL
2 µL
Classical injection volumes
1-10 µL
1-10 nL
Decrease of efficacity
Limits of detection
10-7-10-8 M
10-5-10-6 M
(UV detection)
Electrochromatography
IMPROVMENT OF SELECTIVITY
CZE: Capillary Zone
Electrophresis
Non-aqueous CE
SELECTIVITE
CGE
MEKC
EKC:
cyclodextrines,…
Electrochromatography
(CEC)
33
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Capillary Gel Electrophoresis (CGE of CE-SDS)
Capillary Gel Electrophoresis (CGE of CE-SDS)
= separation technique that combines type of phenomena :
 electroosmose
 electrophoresis
Separation due to the presence of polymer in the
electrolyte: Sieving gel
No instrumental development
Addition of polymer in the electrolyte to create a sieve
with a control grid
Separation of compounds which have a uniforme
distribution of electric charge.
Separation in function of mass
gain in selectivity
Separation by size exclusion
Capillary Gel Electrophoresis (CGE of CE-SDS)
Capillary Gel Electrophoresis (CGE of CE-SDS)
34
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Outline
INJECTION
1. Migration phenomenon in CE
1.1 Electrophoretic mobility
1.2 Electroosmotic mobility
Most classical injection modes
2. The separation in CE
 Electrokinetic injection
 Hydrodynamic injection
2.1 Efficacity
2.2 Resolution
3. Improvement of selectivity
Injected sample quantity Q is defined by:
4. Quantitative analysis
Q = l.r2.C
4.1 Injection
4.2 Detection
avec
5. Capillary isoelectric focalisation (CIEF)
l, lenght of the sample zone
r, capillary radius
C, sample concentration
Hydrodynamic Injection
By difference of pressure,
Electrokinetic injection
Realized by the application of an electrical field in the sample vial.
The injected volume Vinj is proportional to:
l = tinj (veo+ vep)
Time of injection tinj,
Difference of pressure ΔP0
Vinj =
Qinj =
r4.Po.tinj
8.L
(eo+ ep)V.r2.C.tinj
L
Due to electrophoretic mobility of analytes, injected quantity is diferent in funtion
of sample nature.
Equation is not valid if sample conductivity is different to electrolyte conductivity.
Electrokinetic mode is always used in CGE.
35
16/04/2020
Outline
DETECTION
1. Migration phenomenon in CE
Most often used:
1.1 Electrophoretic mobility
1.2 Electroosmotic mobility
 UV detection
2. The separation in CE
 Mass spectrometry detection
 Fluorescence detection
2.1 Efficacity
2.2 Resolution
OFF-COLUMN
Detection
ON-COLUMN
Detection
3. Improvement of selectivity
4. Quantitative analysis
4.1 Injection
4.2 Detection
+
-
+
-
5. Capillary isoelectric focalisation (CIEF)
ON-COLUMN: DIRECT MODE
ON-COLUMN: DIRECT MODE
Detection directly on the capillary, near the capillary tip
Detection directly on the capillary, near the capillary tip
 operated through a window formed by removing the polyimide protection of the capillary.
 operated through a window formed by removing the polyimide protection of the capillary.
 UV Detection
 Fluorescence detection
 Generaly well adapted to bare fused capillary which present a weak luminescence
 require the use of transparent capillaries up to 170 nm
Generaly need a derivation step of the sample:
Equiped all commercial apparatus
 limited sensitivity because of the low capillary loading capacity and low
diameter :  10-5 mol.L-1
 dansyl/fluorescein-thiocarbamyl for amino acids
 Fluorescamine for amino acids or peptides
ex : phenol, LOD = 67 fmol
ex : -chymotrypsinogen, LOD = 2 fmol
 Develpment of capillary with a bubble cell of a Z cell to increase the optilcal
lenght
Linearity: 10-3 - 10-7 M
CE-LIF commercialized with an argon laser at 488 nm
36
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OFF-COLUMN
 Mass spectrometry detection
 Need an adapted interface
Méthode
LDD (mol)
LDD (M)
UV- Vis
10-13 - 10-16
10-5 - 10-8
Universel
Possibilité d’information spectrale
Fluorescence
10-15 - 10-17
10-7 - 10-9
Sensible
Requiert souvent une dérivatisation
Fluorescence induite
par laser
10-18 - 10-20
10-14 - 10-16
Extrêmement sensible
Requiert souvent une dérivatisation
Cher
Ampérométrie
10-18 - 10-19
10-10 - 10-11
Sensible
Sélective mais seulement pour
analytes electroactifs
Requiert une électronique spéciale
et des modifications du capillaire
Conductivité
10-15 - 10-16
10-7 - 10-8
Universel
Requiert une électronique spéciale
et des modifications du capillaire
Spectrométrie
de masse
10-16 - 10-17
10-8 - 10-9
Sensible
Informations structurales
 Maintain the electrical field
 No succion effect
 Increase of sensitivity due to ultra-low flow separation
Détection indirecte
10 - 100 moins qu’en direct
(UV, fluorescence, ampérométrie)
Optimization of sensitivity
1. On-line preconcentration before separation
2. Isotachophoresis (ITP)
Avantages/ inconvénients
Universel
Plus faible sensibilité qu’en direct
On-line preconcentration: amplification of the electric field
Conductivity of the
sample zone
(к0)
Conductivity of the
electrolyte
(к)
Electric field
Limitations
• axial dispersion
• Joule effect
Optimal conditions (hydrodynamic injection)
• weak voltage
• 8 < к/к0 <10
• Injected volume: x 8 to 10
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On-line preconcentration: amplification of the electric field
On-line preconcentration: amplification of the electric field (large volume)
Hydrodynamic injection
Elimination of low conductivity zone by reversing the polarity
Separation in classical condition
On-line preconcentration: transient Isotachophoresis
On-line preconcentration: transient Isotachophoresis
 Principe: sample preconcentration in a gradient of conductivity
BGE
Leader
Sample
 Background electrolyte : (HCOOH)
 Sample contain a leader ion (NH4+)
+
1
2
3
4
-
5
Electrical field
Conductivity
Injection of large sample zone
38
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On-line preconcentration: transient Isotachophoresis
On-line preconcentration: transient Isotachophoresis
A
B
Capillaire silice fondue 60 cm x 75 µm d.i. (10 cm au détecteur) avec greffage dynamique HPC; Voltage : - 25 kV ;
Température : 25°C ; Détection UV à 200 nm ; Electrolyte : acide formique 50 mM, pH 2,7 ; Echantillon : digeste de BSA 20
pmol/µL
A : Sample with leader ions in the sample, Vinj = 10% of the capillary
B : Sample without leader ions in the sample, Vinj = 10% of the capillary
Outline
1. Migration phenomenon in CE
1.1 Electrophoretic mobility
1.2 Electroosmotic mobility
2. The separation in CE
CIEF
Characteristics:
• Separation in function of isoelectric point.
• Migration in a pH graditn (presence of ampholytes)
• Two step: Focusing and Mobilization
2.1 Efficacity
2.2 Resolution
3. Improvement of selectivity
4. Quantitative analysis
4.1 Injection
4.2 Detection
5. Capillary isoelectric focalisation (CIEF)
39
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CIEF : Principe
CIEF
Filling the capillary
2D gel electrophoresis
Acidic pH
Basic pH
Detector
CIEF : Principe
CIEF : Principe
Filling the capillary
Acidic pH
Second step: Mobililsation
pH Gradient
Basic pH
Acidic pH
-
Basic pH
-
+
Pressure
+
pH Gradient
Detector
Power supply
Detector
Power supply
40
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CIEF
Separation of proteins
By CIEF
Lab-on-chip
Production and applications
Contact : Yannis FRANCOIS, Lab. de Dynamique et Structure
Moléculaire par Spectrométrie de Masse, institut de Chimie, 1
rue Blaise Pascal, 67000 Strasbourg
email: [email protected]
TOWARDS THE MICROFLUIDIC SCALE
Microfluidics: it typically regulates flows in labs on a chip.
Channels with diameter smaller than 100 microns or less than μL.
1930
1980
1990
2000
Chip
µ-TAS
IMPORTANT SURFACE FORCES AND OFTEN PREDOMINANT
Van der Waals interaction (associated with charged
surfaces in the presence of ionic solutions)
Surface tension (liquid/liquid or liquid/gas interactions)
Electrophoresis
Capillary zone
electrophoresis
MEKC, CEC,..
Detection: LIF,
MS
If you return a microchip, water stays on the surface!
Surface treatments have more influence than gravity!
In these flows: no TURBULENCE and mixtures are made by DIFFUSION.
The drops keep their integrity and the bubbles behave like obstacles in the
channels.
41
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MICRO-CE
« HARD » TECHNOLOGY: SILICIUM
electrolyte
RESIN DEPOSIT
sample
waste
DEVELOPMENT
electrolyte
COLLAGE
ENGRAVING
Cross device
ENGRAVING PROCESS
ENGRAVING PROCESS
HUMID ENGRAVING: Chemical attack in liquid phase
Ex: Spherical cavity
SF6, CF4  SiF4
DRIED ENGRAVING:
ISOTROPIC ENGRAVING: it develops indifferently in 3 directions
HF/HNO3/CH3COOH for silicium
HF for glass
Spray
Chemical
+
Physico-chemical
+
ANISOTROPIC ENGRAVING: it develops preferentially according to certain
crystalline planes
Température°C
KOH for silicium
Structure cristalline
du silicium
Taux de gravure µm/h
Glass = amorphous no anisotropic
engraving
ANISOTROPOUS
100Å/min
Etching by physical action of
the incident ion flux
Etching by chemical action of
reactive species
Target placed on the cathode
Diffusion of reactive species towards
the target and adsorption
Ions accelerated by an electric
field
Ex: faceted cavities
ISOTROPOUS
ANISOTROPOUS
1000Å/min
Engraving by chemical action of
reactive species assisted by ion
bombardment
= RIE (Reactive Ion Etching)
Reaction with the target material and
formation of a volatile compound
42
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PALSTIC TECHNOLOGY: PDMS (polydimethylsiloxane)
REPLICATION
MOLDING
(ex : PDMS)
Polyméthylméthacrylate (PMMA)
Polycarbonate
Crosslinking agent
Polydimethylsiloxane (PDMS)
+ T (70°C)
Polytetrafluoroethylene
Pression + T
(>T° of glassy
transition)
Pression + T (170°C)
MATRICAGE (ex : PMMA)
MICRO-INJECTION
Possibility of multi-dimentional separation
PDMS : hydrophobic material
 O2 Plasma
Surface treatment (polyvinylpyrrolidone, poly-L-lysine,…)
Without surface treatment
With surface treatment
43
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Possibility of multi-dimentional separation
MICRO-Chip: Injection
Fixed volume
= volume of the intersection
« Pinched » injection
Sample
electrolyte E
Esample
Purification
Concentration
Derivation/Reaction
Separation (s)
Detection
electrolyte E
buffer
buffer
Ewaste
sample
waste
Esample
Ewaste
sample
microCE
CE
waste
µ1
µ2
µ3
Eanalysis
electrolyte
Eanalysis
Good injection
Bad injection
electrolyte
Kirchhoff’s law (law of the nodes):
At an intersection, the sum of the currents is zero
Loading
Injection + Analyse
Esample + Ebuffer + Eanalysis= Ewaste
Esample + Ewaste + Eanalysis = Ebuffer
Sample preparation
MICRO-Chip: Injection
Volumes between 10 and 400 pL
Sample treatment
« Gated » injection
sample
sample
Esample
waste
electrolyte
Eanalysis
Esample
Ewaste
Ebuffer
Ebuffer
Eanalysis
electrolyte
waste
electrolyte
waste
electrolyte
Ewaste
Ebuffer
sample
Esample
Eanalysis
electrolyte
On-chip
Off-chip
Ewaste
electrolyte
E1 + E2 = E3 + E4
Loading
Injection
Analyse
Esample  Ewaste ou Ebuffer  Eanalysis
Esample  Ewaste ou Ebuffer  Eanalysis
Esample + Ewaste + Eanalysis = Ebuffer
Grinding
Linked to the separation
Dissolution
Purification
Derivation
Lié à la détection
Elimination of large
particles
Lyse des cellules
Biochemical treatment
(amplification, enzymatic
digestion, …)
Preconcentration
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Sample derivation
Current limits: Detection
Chip design
Chip voltages
Optical detection: A = 
lc
2cell=V2cell
Reactor
Pre-column derivation
Laser induced fluoresence detection
(« gated » injection)
Injection
Detection
Separation
Allows to focus the detection on a width of
5-20μm or less than linj
Injection
Ar+: exc = 488nm
Post-column derivation
(« gated » injection)
He-Cd: exc= 350/ 442nm
Separation
Detection
Reactor
Requires optically transparent substrates: glass, quartz, some plastics
Nitroaromatic and nitrosamines
separation in MEKC
Current limits: Detection
Mass spectrometry
ESI-MS
Agilent
µ-Chip L=65mm
Capillary L=50cm
7
3
4
10
8
9
Electrolyte:10mM borate de sodium,
50mM SDS
Detection: Fluorescence inverse
6
5
1=TNB, 2=DNB, 3=NB, 4=TNT, 5=tetryl, 6=2,4-DNT, 7=2,6-DNT,
8=2-,3-,5-NT, 9=2-Am-4,6-DNT, 10=4-Am-2,6-DNT
2
MALDI-MS
Less popular
Development of the ROACHE « Rapid Open-Access Channel Electrophoresis »
Analytes are separated in open channels.
1
La matrice est ajoutée à l’électrolyte avant la séparation.
Wallenborg S. R. et al. Anal.Chem. 2000, 72, 1872-1878.
Wallenborg S. R. et al., Electrophoresis 2000, 21, 3257-3263.
At the end of the separation, a laser pulse leads to the ionization of the analytes which are
then directly directed to the MS
45
16/04/2020
Current limits: To mix
LOW NUMBER OF REYNOLDS: LAMINARY DISCHARGE
Molecular diffusion mixing
Hydrodynamic focalising
Chaotic advection
trésidence~d
tdiffusion
Flow separation
Creating a chaotic flow
~d2
Division and recombination of flows
46