I use this slide for my weekly presentation during my study to present the fundamental study of capillary electrophoresis. This slide also highlighted the use of ionic liquid to modify cyclodextrin as chiral selector.

2. Epimers?
Meso compound?

3.  Separation is based on differences in solute mobilities
when a strong electric field is applied across a buffer
 Has the ability to separate chiral compounds or
enantiomers.
WHY WE NEED TO SEPARATE CHIRAL COMPOUNDS?
 Majority of bioorganic molecules are chiral. Living
organisms show different biological response to one of
enantiomers.
 The desired pharmacological activity is exhibited by only
one enantiomer, while the other enantiomer may be toxic,
less active, and/or possess undesirable effects.
 For example, R-ibuprofen is inactive in the body but S-
enantiomer is 160 times more active as pain killer.

4. HPLC CAPILLARY ELECTROPHORESIS (CE)
Expensive column, short
lifetimes
Inexpensive fused-silica capillary, long
lifetimes
High consumption of solvent and
sample
Low consumption of solvent and
sample; typical injection volume range
from picoliters to nanoliters
Faster analysis time, high efficiency
Several modes and ability of using free
form of chiral selectors

5. Figure 1 Figure 2
E=V/d
Buffer
Buffer
Anode Cathode
Detector

6. Migration Velocity:
Where:
v = migration velocity of charged particle in the potential field (cm sec -1)
ep = electrophoretic mobility (cm2 V-1 sec-1)
E = field strength (V cm -1)
V = applied voltage (V)
L = length of capillary (cm)
Electrophoretic mobility:
Where:
q = charge on ion
 = viscosity
r = ion radius Frictional retarding forces
L
V
E epep  
r
q
ep


6


7. Where:
v = electroosomotic mobility
o = dielectric constant of a vacuum
 = dielectric constant of the buffer
 = Zeta potential
 = viscosity
E = electric field


4
0
eo
 Net flow becomes is large at higher pH:
 Key factors that affect electroosmotic mobility: dielectric
constant and viscosity of buffer (controls double-layer
compression)
 EOF can be quenched by protection of silanols or low pH
 Electroosmotic mobility:
EEv eo 









4
0

8.    
L
V
E eoepeoep  
Figure from R. N. Zare, Stanford

9.  Separation capacity factor, k’
K’=
(𝑡1−𝑡0)
𝑡0
 Resolution, Rs
Rs=2
(𝑡2−𝑡1)
𝑊1+𝑤2
 Selectivity, α
α=
𝑘′2
𝑘′1

10. The efficiency of the separation is indicated using this
formula:
Van Deemter Equation:
relates the plate height H to the velocity of the carrier gas
or liquid
CuuBAH  /
Where A, B, C are constants, and a
lower value of H corresponds to a
higher separation efficiency

11.  Enantiomeric resolution occurs due to the different
interactions between enantiomers and chiral
selectors.
 The interaction of chiral selector and analytes before
or during the separation process forming stable
diastereoisomers or labile diastereomeric complexes.
 The most common: cyclodextrins and their
derivatives.
 Hydrophobic interaction between analytes and CD
cavity and hydrogen bonds with hydroxy or modified
groups can lead to the formation of labile
diastereoisomeric complexes with different stability
constants.
 The most stable complex formed moves with a lower
effective mobility.

12.  Reasons:
 Weak UV absorption
 Excellent chiral discrimination towards wide range of
enantiomers.
 Most popular is β-CD as its cavity size matches most of
hydrophobic groups of analytes, however, its weakness is
low aqueous solubility.
 The straightforward way to modify CD is by exchanging
the hydroxy group.
 The advantages of modified CD:
 To improve β-CD solubility in aqueous.
 Improve resolution to opposite charge of analyte
 Ability to form strong electrostatic interaction between
CD and analytes other than inclusion complaxation.
 Ability to separate anionic and ampholytic analytes.

13.  Ionic liquids have been explored in CE to be
modifier, background electrolyte, additives and also
as chiral selectors.
 Ionic liquids have the ability to assist the separation
of hydrophobic analytes while maintaining the
background current.
 Ionic liquids have the advantages of soluble in water,
good electric conductivity, can act as good
electrolytes, more viscous than organic solvent, thus
the amount used is small, less volatile and also
green solvent.

15. AUTHOR/
YEAR
TITLE ANALYTES
Ong et al.,
2005
Synthesis and Application of Single-isomer 6-
mono(alkylimidazolium)-β-cyclodextrin s as Chiral
Selectors in Chiral Capillary Electrophoresis
Dns-amino acids such as Dns-DL-
leucine, Dns-DL-serine, Dns-DL-
valine etc.
Tang et al.,
2007
Effect of alkylimidazolium substituents on
enantioseparation ability of single-isomer
alkylimidazolium-β-cyclodextrin derivatives in capillary
electrophoresis
Dansyl amino acids
Tang et al.,
2007
Chiral separation of dansyl amino acids in capillary
electrophoresis using mono-(3-methyl-imidazolium)-
β-cyclodextrin chloride as selector
Dansyl amino acids
Ong et al.,
2007
Synthesis and application of mono-6-(3-
methylimidazolium)-6-deoxyperphenylcarbonyl-β-
cyclodextrin chloride as chiral stationary phase for high-
performance liquid chromatography and supercritical fluid
chromatography
Racemic aryl alcohol; p-
fluorophenylethanol, p-
bromophenylehtanol etc.
Jia et al., 2013 Synthesis and application of a chiral ionic liquid
functionalized β-cyclodextrin as a chiral selector in
capillary electrophoresis
Racemic drug; chlorpheniramine,
brompheniramine, pheniramine,
tropicamide, etc
Huang et al.,
2010
Ionic cyclodextrin in ionic liquid matrices as chiral
stationary phases for gas chromatography
2-(bromomethyl)tetrahydro-2H-
pyran, borneol, butyl lactate

16. 1. INFLUENCE OF LENGTH OF ALKYL LENGTH SIDE
CHAIN OF IMIDAZOLE
 As the alkyl length longer, the selectivity and
resolution ability reduced.
 Due to the steric hindrance
2. ANION TYPE OF CHIRAL SELECTOR
 The desired anion should not have the ability to
absorb UV region.
 Will resulting in high background noise and low
detection level of analytes.
 E.g: as -OT and Cl- anions were compared, -OT
anion was found to absorb UV region.

17. 3. pH OF BUFFER
 The choose of pH is depend on the pka of the
analytes.
 However, higher pH will increase EOF, thus reduce the
migration time and might affect the resolution.
4. TEMPERATURE OF CAPILLARY
 As the temperature increases, the migration time
becomes shorter.
 The increased temperature decreased the stability of
the labile disasteromeric complexes between CD and
analytes.
 Temperature dependency might be due to change in
bulk liquid structure and viscosity, thus affecting the
rotational and transitional degrees of freedom of the
host-guest associates, and in conductivity of running
buffer, and solvation status of the CD.

18. 5. CONCENTRATION OF CHIRAL SELECTORS
 As the concentration increased, the resolution
improved and migration time shorten. This due to
stable inclusion complex of solute with CD.
 An increase of chiral selectors can lead to a general
decrease of effective mobility as BGE more viscous.
 As the concentration above optimum, the CD tend to
precipitate and clog the column.
6. ORGANIC MODIFIER USED IN BGE
 Generally, the addition of modifier, eg; methanol
increase the chiral resolution and selectivity.
 The addition of modifier decrease the effective
mobility, thus increase in migration time.
 The increase of modifier also decrease the EOF, via
the interaction of modifier with capillary wall by
reducing the adsorption of cationic CD and thus
changing the driving force of EOF.

19. 7 CONCENTRATION OF IONIC LIQUID
 Low concentration of IL will causes the IL to move
with buffer without coating onto the capillary wall.
 The addition of IL will allow the IL to be coated onto
the capillary wall and cause ionic interaction
between the cationic of imidazole and anionic
analytes. Thus will improve the resolution of the
enantiomers.
 However, the excess of IL will causes the decrease of
EOF although there is enhancement of current.
 This will shorten the migration time and poor peak
efficiency.
8 CONCENTRATION OF BUFFER
 Improve the resolution as the migration time is
increased.
 High ionic concentration of buffer solutions improve
the resolution and peak shape.
 However, increase in concentration also enhance the
current and lead to Joule heating.

20. EEv eo 









4
0
Variable Result Notes
Electric Field Proportional change in EOF Joule heating may result
Buffer pH
EOF decreased at low pH,
increased at high pH
Best method to control EOF, but may
change charge of analytes
Organic Modifiers
Decreases  and EOF with
increasing modifier
Complex effects
Surfactant
Adsorbs to capillary wall through
hydrophobic or ionic interactions
Anionic surfactants increase EOF
Cationic surfactants decrease EOF
Neutral hydrophilic
poymer
Adsorbs to capillary wall via
hydrophobic interactions
Decreases EOF by shielding surface
charge, also increases viscosity
Temperature Changes viscosity Easy to control

21.  Study on capillary electrophoresis is
interesting.
 A lot more to explore.