Transcript Lecture 10

Lecture 10
Modes and Methods in Capillary Electrophoresis
Dr. Rasha Hanafi
© Dr. Rasha Hanafi, GUC
Lecture 10, SS16
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Learning outcomes
By the end of this session, the student should be able to:
1. Enumerate advantages of CE.
2. Compare between different modes of CE.
3. Describe injection and detection in CE.
4. Summarize all information acquired about CE to have a
global picture about this analytical process.
© Dr. Rasha Hanafi, GUC
Lecture 10, SS16
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Advantages of capillary electrophoresis
1. Lower instrument costs compared
to HPLC, GC (starts at 15,000 $).
2. Small sample size (nL) compared
to μL in other separation
techniques.
3. Significantly greater speed than
HPLC (Voltage applied in CE versus
pressure applied in HPLC)
4. High resolution due to the nature
of the flow profile (electroosmotic
vs. hydrodynamic) and the
absence of the A and Cu term
parameters in the Van Deemter
equation as there is no stationary
phase in the capillary.
© Dr. Rasha Hanafi, GUC
Lecture 10, SS16
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Modes of capillary electrophoresis
CZE
Capillary Zone Electrophoresis
CIEF
Modes of
CE
Capillary Isoelectric Focusing
1st used in
“slab electro-
Capillary Isotachophoresis
-phoresis”
(what is it? Personal effort)
CITP
CGE
Capillary Gel Electrophoresis
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Lecture 10, SS16
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I. Capillary Zone Electrophoresis CZE
Buffer composition in the capillary: constant
Mobility: due to electroosmotic flow, either :
1- to cathode (most common, see lecture 9). Small anions with high
mobility and highly negatively charged proteins require very
strong electroosmotic flow or otherwise they will not travel
toward the cathode.
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Lecture 10, SS16
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I. Capillary Zone Electrophoresis CZE
2- To anode: how?
For fast separation of anions, the EOF
can be reversed by treating the
walls of the capillary with an alkyl
ammonium salt (ex: cetyl trimethyl
ammonium
bromide).
NH4+
attaches to the –ve charged surface
of the silica, creating a double layer
attracted to the anode.
N.B.: Neutral compounds would
better be derivatized, via formation
of borate ions that are readily
formed if a borate buffer is used in
the capillary.
© Dr. Rasha Hanafi, GUC
Lecture 10, SS16
Br- Br- Br- Br-
Br- Br- Br-
Br-
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I. Capillary Zone Electrophoresis CZE, contd.
• Usually, the number of theoretical plates (N) achieved in CE= 105!!
• It is the higher voltage (V) rather than the long capillaries (unlike other types
of chromatography) that lead to higher number of plates.
N= µapp V Ld /2D Lt
Ld: capillary length to the detector ; D: diffusion coefficient; Lt: total capillary length
N.B.: Increasing Voltage is restricted to certain values to avoid capillary heating
(joule heating) leading to hydrodynamic (parabolic) flow profile.
• Possible reasons for band broadening:
1. The finite width of the injected band.
2. Occurrence of parabolic flow profile from heating inside the capillary.
3. Adsorption of solute on capillary walls.
4. The finite length of the detection zone.
© Dr. Rasha Hanafi, GUC
Lecture 10, SS16
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II. Capillary Gel Electrophoresis CGE
It is a variation of gel electrophoresis which has been a 1ary tool for biochemists in
early slab electrophoresis. In CE, the gels are contained inside the capillary.
Chemical gels contain cross-linked polymer chains covalently bound deriving
their properties from physical entanglement of the polymers. Porous gel polymer
matrix are: agarose, methyl cellulose, polyethylene glycol and polyacrylamide
(most common, acrylamide is CH2=CH-CO-NH2). The pores of the polymer contain
the buffer mixture required for separation.
Advantages:
1. Reduce analyte dispersion by convection and diffusion.
2. Provide a medium suitable for handling and detection
(in slab electrophoresis).
Increasing the amount of cross linking agents results in smaller pore size. Hence,
molecular sieving retards analytes based on their sizes (useful in separation of
macromolecules, DNA fragments and oligonucleotides that have the same charge
but differ in size)
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Lecture 10, SS16
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III. Capillary IsoElectric Focusing CIEF
• CIEF is commonly used to separate zwitterionic compounds (amphiprotic)
whose charge depends on the functional groups attached to the main chain
and the surrounding pH of the environment (peptides).
• An amphiprotic compound is a specie that in solution is capable of donating or
accepting a proton. Most common example: amino acids!
• When glycine is dissolved in water, 3 important equilibria operate:
N.B.: The isoelectric point (pI) is the pH at which the amino acid is neutral, i.e.
the zwitterion form is dominant. At this point, the amino acid does not migrate
in an electric field.
At a pH < pI, the molecule is positive, and when the pH > pI it is negative .
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Lecture 10, SS16
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III. Capillary IsoElectric Focusing CIEF, contd.
•NaOH and H3PO4 in each of the reservoirs
•what happens when potential is applied? H+ moves to cathode and OH- moves to
anode which creates regions of different pH all along the capillary (pH gradient).
H+
OH-
NaOH
H3PO4
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Lecture 10, SS16
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III. Capillary IsoElectric Focusing CIEF, contd.
Strategy:
Because the charge changes with pH, a pH gradient can be used to separate
molecules in a mixture.
The anodic end of the capillary sits in acidic solution (low pH), while the cathodic
end sits in basic solution (high pH).
During a CIEF separation, the capillary is filled with the sample in solution and
typically no EOF is used (EOF is removed by using a coated capillary to hide the
active silanol groups or any interacting groups).
When the voltage is applied, the ions will migrate to a region where they become
neutral (pH=pI).
Compounds of equal isoelectric points are “focused” into sharp segments and
remain in their specific zone, which allows for their distinct detection.
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Lecture 10, SS16
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III. Capillary IsoElectric Focusing CIEF, contd.
If the analyte is –ve, it migrates to anode, progressively to lower pH regions leading to
continuous protonation giving it less –ve charge until net charge = 0 (isoelectric point)
where it stops moving. Since isoelectric points are different for different analytes,
separation takes place with sharp boundaries.
To move the focused uncharged bands at the end of the experiment, pressure is applied to
force them out.
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Lecture 10, SS16
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Injection in CE
Pressure
Electrokinetic
An electric field is applied to drive the sample
into the capillary.
One end of the capillary and its electrode are
removed from the buffer and put in the sample
cup. A potential is applied causing the sample to
enter the capillary by ? ;-) and ? ;-)..
Disadv.: Because each analyte has a different
mobility, in quantitative analysis, the injected
sample does not have the same composition as
the original sample, i.e. unintentional injection
of larger amounts of highly mobile ions relative
to slower moving ions usually takes place.
Adv: most useful in capillary gel electrophoresis
in which the liquid in the capillary is too viscous
for hydrodynamic injection.
© Dr. Rasha Hanafi, GUC
Lecture 10, SS16
A pressure difference
between the 2 ends
of the capillary is
used to drive the
sample
into
it
(vacuum
at
the
detector end or
pressure
at
the
sample end).
Disadv: can not be
used in gel–filled
capillaries
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Detection in CE
On column detection.
The detector cell: A small section of the protective polyimide coating is removed
from the exterior of the capillary by burning, dissolution or scraping.
Path length (b) = only 50-100 μm !
Possible detectors:
1. UV-VIS Absorbance.
2. Fluorescence.
3. ESI Mass spectrometry.
4. Electrochemical (conductometry and amperometry).
Indirect detection??? Smart 
(ex: indirect detection of Cl- isotopes in presence of UV-absorbing anion chromate),
how??
© Dr. Rasha Hanafi, GUC
Lecture 10, SS16
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How to improve Detection sensitivity?
b
b
1. The bubble cell
2. Right angle bend
Limitation: band broadening  successive peaks must be separated by 3 mm
at least or they will overlap.
3. Silver coating on a section of the capillary
allows multiple internal reflection.
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Lecture 10, SS16
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CE versus LC
Advantages:
1. Higher resolution.
2. Low waste production.
3. Generally simpler equipment.
Drawbacks:
1. Higher Limit of Detection (LOD).
2. Run-to-run irreproducibility of
migration times.
3. Insolubility of some analytes in
common electrolyte solns.
4. Inability to scale up to a
preparative separation.
Although liquid chromatography is 2
decades more mature, CE is preferred
for some forensic ex: analysis of
alkaloids in opium and heroin.
© Dr. Rasha Hanafi, GUC
Lecture 10, SS16
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References
1. Textbook: Principles of instrumental analysis, Skoog
et al., 5th edition, chapter 30, pp. 782 to 795.
2. Textbook: Textbook: Quantitative Chemical Analysis,
Harris, chapter 26.
3. http://bioanimations.blogspot.com/2008/04/capillaryelectrophoresis.html
© Dr. Rasha Hanafi, GUC
Lecture 10, SS16
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