Electricaldriven Separations

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• Electrical-driven Separations
• Electrophoresis
• Capillary
• Slab
• Electrochromatgraphy

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Electrophoresis Inject small band of sample into
aqueous buffer solution contained in a narrow
tube (capillary) or on a flat porous support
medium (slab) like a gel. High dc potential
applied across length of buffer by means of pair
of electrodes located at either end of
buffer. Potential causes ions to migrate toward
one of the electrodes. Only one phase involved in
separation. Separation based on charge-to-size
ratio of analytes.
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Schematic of CE system.
Source Skoog, Holler, and Nieman, Principles of
Instrumental Analysis, 5th edition, Saunders
College Publishing.
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Electrochromatography Both neutral and ionic
analytes. Electroosmotic flow of buffer solution
acts as a pump to move analyte molecules past a
second phase that is capable of retaining the
analyte molecules to various extents. Separation
is based on differences in distribution
equilibria, as in LC.
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• Migration Velocity
• meE where u is migration velocity (cm per
  second),
• me is the electrophoretic mobility (cm2V-1s-1)
  and E is the
• electric field strength (V cm-1).
• Electrophoretic mobility proportional to ionic
  charge and
• inversely proportional to frictional retarding
  factors. These
• frictional retarding factors are related to size
  and shape of
• ion and viscosity of medium.

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Efficiency in CE No mass-transfer
considerations, just longitudinal diffusion. N
meV/2D where D is diffusion coefficient of
solute. Plate number does not depend on column
length, only on applied potential. Much higher
applied potentials (20,000-60,000 V)
possible with capillaries compared to about 500 V
with slabs.
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Electroosmotic Flow Capillary wall is negatively
charged due to ionization of surface silanol
(Si-OH) groups. Buffer cations congregate
adjacent to capillary surface. Cations are
attracted toward cathode. Since the cations are
solvated, they draw bulk solvent with them.
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Electroosmotic Flow Profile
Source Skoog, Holler, and Nieman, Principles of
Instrumental Analysis, 5th edition, Saunders
College Publishing.
Flow profile of electroosmotic flow (left) is
flat, compared with parabolic flow profile
(right) found with pressure-induced flow (as in
GC, LC, or SFC). Since profile is flat,
electroosmotic flow does not contribute to band
broadening as it does in chromatography.
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Total Flow Rate of electroosmotic flow gt rate of
electrophoretic migration Electroosmotic flow
sweeps all solutes (anions, cations,
and neutrals) in same direction.
Source Skoog, Holler, and Nieman, Principles of
Instrumental Analysis, 5th edition, Saunders
College Publishing.
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• Instrument Components
• Injection
• Electrokinetic
• Pressure
• Detectors
• Similar to LC
• Should not be flow-dependent

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Source Skoog, Holler, and Nieman, Principles of
Instrumental Analysis, 5th edition, Saunders
College Publishing.
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• Modes of Electrophoretic Separation
• Capillary Zone Electrophoresis (CZE)
• Buffer composition constant, similar to
  chromatography
• Separation of small ions, some proteins, amino
  acids, and
• carbohydrates
• Capillary Gel Electrophoresis (CGE)
• Porous gel polymer matrix containing buffer
  mixture in
• pores. Provides additional sieving mechanism.
• Useful for size separations, like macromolecules
  including
• proteins, DNA fragments, and oligonucleotides.

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• Modes of Electrophoretic Separation (cont.)
• Capillary Isotachophoresis (CITP)
• All bands migrate at same velocity.
• Separation of cations or anions, not both.
• Sample injected between two buffers. Leading
  buffer with ions
• with higher mobility than analyte. Trailing
  buffer with ions of
• lesser mobility than analyte.
• After band formation (as in CZE), bands travel at
  same velocity.
• Capillary Isoelectric Focusing (CIEF)
• Used to separate amphiprotic compounds (like
  amino acids).
• No net migration occurs at pH where concentration
  of cation
• and anion forms are identical. This is the
  isoelectric point (pI).
• Create continuous pH gradient in column.
  Analytes separate to bands
• according to pI. Note this separation is based
  on equilibria, not rates
• of migration.
• Migration by applied pressure or change in
  solution at one end of column.

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Modes of Electrophoretic Separation
Source Skoog, Holler, and Nieman, Principles of
Instrumental Analysis, 5th edition, Saunders
College Publishing.
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• Capillary Electrochromatography
• Hybrid of CE and LC.
• Applicable to neutral species.
• Highly efficient, like CE, due to electroosmotic
  flow, etc.
• CEC
• Used packed columns like LC.
• Gaining in popularity.
• Micellar Electrokinetic Capillary Chromatography
  (MECC)
• Uses surfactant micelles to solubilize nonpolar
  compounds.
• Micelles act as pseudostationary phase, so
  chromatographic
• separation based on distribution is achieved.