ANALYTICAL CHEMISTRY CHEM 3811 CHAPTER 23

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ANALYTICAL CHEMISTRY CHEM 3811CHAPTER 23
DR. AUGUSTINE OFORI AGYEMAN Assistant professor
of chemistry Department of natural
sciences Clayton state university
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CHAPTER 23 CAPILLARY ELECTROPHORESIS
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ELECTROPHORESIS
- Migration of ions in an electric field -
Cations are attracted to the negative electrode
(cathode) - Anions are attracted to the
positive electrode (anode)
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ELECTROOSMOSIS
- The application of electric field to drive
fluid in a capillary tube from anode towards
cathode Capillary Electrophoresis - Is a
combination of electrophoresis and electroosmosis
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CAPILLARY ELECTROPHORESIS
- High-resolution separation method - Separates
charged and neutral analytes in a narrow
capillary tube - Capillary tube wall is usually
fused silica Good technique for the separation
of - Small ions (Na, K) - Proteins - DNA
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CAPILLARY ELECTROPHORESIS
Background Electrolyte (run buffer) - A buffer
solution in the electrode reserviors - Controls
pH and ionic strength
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CAPILLARY ELECTROPHORESIS
- Both ends of the capillary tube is first
immersed in a background electrolyte - One end
of tube is dipped in vial containing the
sample - Pressure or electric field is applied
to introduce 10 nL of sample into the
capillary - Capillary is placed back into the
electrolyte - 20 to 30 kV is applied and causes
ions in capillary to migrate
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CAPILLARY ELECTROPHORESIS
- Different ions migrate at different speeds -
This results in separation of ions - Ions reach
a detector and an electropherogram (response
versus time) is recorded - Very narrow bands are
usually seen - Terms A and C in the van Deemter
equation are reduced (no multiple paths and no
stationary phase)
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CAPILLARY ELECTROPHORESIS
- Electroosmosis sweeps analyte molecules towards
the detector - Detector is placed near the
cathode - Detector is set to a wavelength of
about 200 nm - Cations reach the detector
first - Neutral molecules reach the detector
after cations - Anions reach the detector after
neutral molecules
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CAPILLARY ELECTROPHORESIS
At High pH - Electroosmosis is faster than
electrophoresis - Net flow of anions is towards
the cathode At Neutral pH - Electroosmosis is
faster than electrophoresis - Net flow of anions
is towards the cathode
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CAPILLARY ELECTROPHORESIS
At Low pH - Electroosmosis is slower than
electrophoresis - Net flow of anions is towards
the anode - Anions may not reach the detector -
Polarity can be reversed to separate anions
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CAPILLARY ELECTROPHORESIS
Detectors - Ultraviolet absorbance (most
common) - Conductivity - Electrochemical -
Fluorescence
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CAPILLARY ELECTROPHORESIS
Types Capillary Ozone Electrophoresis -
Separation is based on different velocities of
different ions Micellar Electrokinetic Capillary
Chromatography - Used to separate different
neutral molecules - Also used for ions - Micelles
are added to the background electrolyte - An
example is sodium dodecyl sulfate (a surfactant)
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CAPILLARY ELECTROPHORESIS
Types Capillary Gel Electrophoresis -
Separation is based on size of molecules -
Molecules are separated upon migrating through a
gel in the capillary column - The process is
called sieving - Small molecules travel faster
than large molecules