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Advanced Analytical Chemistry – CHM 6157
Updated on 10/17/2006
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Chapter 9
Florida International University
Capillary Electrophoresis
Chapter 9 Capillary Electrophoresis (CE)
References:
• Dale R. Baker, Capillary Electrophoresis,
John Wiley & Sons, 1995.
• M.G. Khaledi, Ed., High-Performance
Capillary Electrophoresis, John Wiley &
Sons, 1998.
• Colin F. Poole, The Essence of
Chromatography, Elsevier, 2003.
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
3. Modes of CE
3.1
Capillary Zone Electrophoresis (CZE)
CZE or Free solution capillary electrophoresis (FSCE).
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
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Fill capillary with a buffer of constant composition
Fill source and destination vials with same buffer
Analyze cations and anions simultaneously
Nor for neutral species
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2008
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2008
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2008
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
3.2 Micellar electrokinetic capillary
chromatography (MEKC)
(Electrochromatography)
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Developed by Shigeru Terabe et al. (Anal. Chem. 1984, 56, 111)
Provides a method for separation of electrically neutral
compounds
Combines the separation mechanism of chromatography with
the electrophoretic and electroosmotic movement of solutes and
solutions
Separation is based on the partitioning of solutes between the
surfactant micelles and the run buffer.
The detector output is referred to as electrokinetic
chromatogram
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
Three factors affecting apparent electrophoretic
mobility of an anlyte in MEKC:
 The system µEOF
 The fraction of analyte in the electrolyte solution and its
µEP
 The fraction of analyte in the pseudo-stationary phase
and the µEP of the micelle
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
3.2.1 Principles of MEKC
This mode of CE is based on the partitioning of solutes
between micelles and the run buffer.

Detergents (surfactants)
Molecules that have a hydrophilic, water soluble moiety
on one end of the molecule and a hydrophobic, water
insoluble moiety on the other.
e.g. Sodium dodecyl sulfate (SDS), [CH3-(CH2)11-OSO3-], also called sodium lauryl sulfate
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
 Critical micelle concentration (CMC)
The concentration of a detergent present in
solution when micelles begin to form.
 Micelles
Aggregations of individual detergent molecules.
 Aggregation number
The number of detergent that make up the a
micelle.
 Kraft point
The temperature at which the solubility of the
detergent = the critical micelle concentration
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2005
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
Differences between chromatography and
MEKC:

Chromatographic separations are based differences in distribution
of sample molecules between a stationary phase and a mobile
phase. However, in MEKC, there are two phases, aqueous and
micelle, both of which move.

In chromatography, the solutes and mobile phase are moved
through the column by pumped flow, whereas in MEKC, they are
moved through the capillary by EOF.
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
Retention Parameters in MEKC
• Capacity factor (retention factor) of an electrically neutral
solute is defined the ratio of the umber of moles of solute
in the micelles, nmc, to the number of the moles in the
aqueous phase, naq:
k’ = nmc/naq
[3-1]
k’ = (tR – t0)/t0(1 – tR/tmc)
[3-2]
(Terabe et al. Anal. Chem. 1984, 56, 111)
This is similar to the equation in Chromatography
k’ = (tR – t0)/t0
[3-3]
Advanced Analytical Chemistry – CHM 6157
® Y. CAI
Updated on 10/27/2008
Chapter 9
Florida International University
Capillary Electrophoresis
k’ = (tR – t0)/t0(1 – tR/tmc)
(3-2)
k’ = (tR – t0)/t0
(3-3)
(1 – tR/tmc) is due to the retention properties of MEKC.
When Tmc is very large, eq (3-2) is same as (3-3).
k’ is related to T0, TR, and Tmc in MEKC
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2008
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
Retention Parameters in MEKC
• Because tR = l/s, t0 = l/0, tmc = l/mc:
k’ = (tR – t0)/t0(1 – tR/tmc)
[3-2]
Insert tR = l/s, t0 = l/ 0, tmc = l/mc and rearrange
k’ = (0/s – 1)/(1 - mc/s)
Since  = E
k’ = (EOF/s – 1)/(1- mc/ s)
[3-4]
[3-5]
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
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Chapter 9
Resolution in MEKC
Florida International University
Capillary Electrophoresis
Baker, 1995
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
3.2.2 Separation of ionic solutes by MEKC

Neutral molecules:
Differences in their distribution between the aqueous
buffer and the micelles

Ionic molecules:
Differences in their electrophoretic mobilities or
because of interactions with micelles, depending on the
charges of the ionic solutes and the micelles.
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
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Chapter 9
Florida International University
Capillary Electrophoresis
For negatively charged micelles are used, such as SDS:
neutral
cations
Baker, 1995
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
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Chapter 9
Florida International University
Capillary Electrophoresis
3.2.3 Using Modifiers in MEKC

Modifiers affecting the charge on the micelle or the
solute and changing the solubility of a solute in the
micelles.
e.g. addition of tetraalkylammonium (TAA) to an SDS
buffer to improve the separation of carboxylic acids
(formation of neutral ion pairs ).
Affects also the retention of positively charged solutes
(decreases when TAA salts were added).
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis

Modifiers serving as a second pseudophase
•
Cyclodextrin-modified MEKC has been used to separate
very hydrophobic solutes.
Cyclodextrins (CD’s) are water-soluble oligosacchrides.
CD’s have a characteristic toroidal shape, with a
hydrophobic cavity and a hydrophilic external surface.
CD’s are electrically neutral, they migrate with the
velocity of the EOF
Hydrophobic solute is not separated if no CD is added.
When a CD is added to the buffer,
•
•
•
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
k’ = nmc/nCD
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Chapter 9
Florida International University
Capillary Electrophoresis
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
3.3 CAPILLARY ISOELECTRIC
FOCUSING (CIEF)
3.3.1 Properties of Amphiprotic Compounds
•
An amphiprotic compound is a species that in solution is
capable of both donating and accepting a proton. A
typical amino acid, such as glycine, is an amphiprotic
compound.
•
CIEF is used to separated amphiprotic species, such as
amino acids and proteins that contains a weak carboxylic
acid group and a weak base amine group.
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
When glycine is dissolved in water, three important
equilibrium:
NH2CH2COOH  NH3+CH2COO-
[1]
Internal acid/base reaction proceeds far to the right, with
product being the predominant species in the solution.
NH3+CH2COO- + H2O  NH2CH2COO- + H3O+
[2]
Ka = 2 x 10-10
NH3+CH2COO- + H2O  NH3+CH2COOH + OH-
[3]
Kb = 2 x 10-12
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
•
•
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Chapter 9
Florida International University
Capillary Electrophoresis
zwitterion: The amino acid product
bearing both a positive and a negative
charge, is called a zwitterion.
Isoelectric point (PI): The isoelectric point
of an amphiprotic compounds is the pH at
which the compound has a net charge of
zero. No net migration of amino acid
occurs in an electric field when the pH of
the solvent is such that the concentrations
of anionic and cationic forms are
identical.
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
The PI is readily related to the ionization concentrations for
the species. For glycine:
Ka = [H3O+][NH2CH2COO-]/[NH3+CH2COO-]
Kb = [OH-][NH3+CH2COOH]/[NH3+CH2COO-]
At isoelectric point,
[NH2CH2COO-] = [NH3+CH2COOH]
Thus,
Ka/Kb = [H3O+]/[OH-]
Substitution of Kw/[H3O+]for [OH-], then
[H3O+] = (KaKw/Kb)2
[H3O+] = 1 x 10-6
pI = 6
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
Three Steps involved in CIEF:
 Formation of a pH gradient in the capillary
 Performing Isoelectric Focusing
 Mobilization of the focused zones
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
3.3.2 Formation of a pH gradient in the
capillary
In isoelectric separation of amphiprotic
species, the separation is performed in a buffer
mixture that varies in pH continuously along its
length. The pH gradient is prepared from the
mixture of several different ampholytes in an
aqueous solution.
.
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
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Chapter 9
Florida International University
Capillary Electrophoresis
H3PO4
NaOH
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
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Chapter 9
Florida International University
Capillary Electrophoresis
To form a pH gradient in a capillary:
(1) The capillary is filled with a mixture of ampholytes
that will produce a certain pH gradient (e.g. 3-10).
(2) One end of the capillary is then inserted in a solution
of strong base (NaOH) (cathode). The other end is
immersed in a solution of strong acid (phosphoric)
(anode).
(3) When an electric field is applied, hydrogen ions begin
to migrate from the anode toward the cathode, while
hydroxide ions begin to move in the opposite direction.
(4) The ampholytes in the buffer mixture migrate also
depending on their net charge. Ultimately they reach the
pH where their net charge is zero (pI).
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
3.3.3 Isoelectric Focusing

CIEF is performed by filling the capillary with a mixture
of ampholytes and SAMPLE.

Similar to the migration of ampholytes, analyte ions also
migrate until they reach their pI.

No EOF in the capillary. Large volumes of sample are
injected.
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
3.3.4 Mobilization of the focused zones
 Hydrodynamic flow: to pressure the
capillary.
 Electrophoretic mobilization
•
•
•
•
Add NaCl into the NaOH solution after focusing.
Both Cl- and OH- migrate into that end of the
column, and sum of these two concentrations is
balanced by H+.
The pH gradient is no longer stable
Analytes change to positively charged, and moves
toward the cathode.
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
Advanced Analytical Chemistry – CHM 6157
Updated on 10/27/2006
® Y. CAI
Chapter 9
Florida International University
Capillary Electrophoresis
3.4 Capillary Gel Electrophoresis
• Dr. Bruce McCord
November 6, 2006
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