ELECTROPHORESIS

1
ELECTROPHORESIS
CHAPTER 23
2
Electrophoresis

• Separation of solutes based on different rates of
  migration though an electric field (through
  background electrolyte running buffer. Anions
  move toward the anode.

Note that charge and size influence the movement
of charged particles, in opposite ways.
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Electrophoresis

• Moving Boundary applied electric field that
  causes charged particles to move and produces
  broad areas separated by moving boundaries of
  buffer
• Zone uses smaller samples and provides narrow
  zones of separated analytes (shown here)

4
Electrophoresis Gel

• In gel electrophoresis, small samples are used.
  Samples travel through a running buffer when an
  electric field is applied.
• Separation is stopped before analytes come to end
  of the support.
• A series of bands represent separated analytes.
  Distance traveled is called migration time (dm),
  which can be compared to standards.

5
Electrophoresis Capillary

• With capillary, all analytes travel the same
  distance, but the migration time (tm) for that
  distance is measured.
• Relate time to identity
• Relate peak area or height to amount

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Electrophoretic Mobility

• Movement depends on interaction between two
  competing forces, F (attraction of opposite ion
  for electrode) and F (resistance to movement).
• When the electric field is applied, the solute
  will accelerate until forces become equal. Then a
  steady state is achieved and the solute migrates
  at constant velocity.
• F 6prhv F Ez
  
• (r solvated radius h viscosity)
• The velocity for the steady state will be as
  follows
• Electrophoretic mobility m z/6prh
• Now, v mE See exercise 23.1 note the slight
  modification.

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Electrophoretic Mobility m

• The charge and/or apparent size of the analyte
  can be advantageously modified by secondary
  interactions.
• For weak acids/bases the pH will affect the A/HA
  (or BH/B) ratio and, therefore, affect the net
  charge.
• Using sodium dodecyl sulfate polyacrylamide
  electrophoresis (SDS-PAGE) denatures large
  proteins and then coats them with negatively
  charged SDS, which converts proteins into
  rod-shaped particles.

8
Electroosmosis

• Fixed charges present in the system (i.e., on the
  support of the system) will attract charged ions
  from the running buffer, creating a double layer
  at the interface.
• This causes net movement of the buffer toward the
  oppositely charged electrode versus the fixed
  ionic charge. This process is called
  electroosmosis. The extent of this effect is
  electroosmotic mobility.
  meo meo ezE/h (z
  is zeta potential, the
  
  charge on the support.)
• For analyte, mnet m meo
• Net electrophoretic mobility analytes
  electorphoretic mobility electroosmotic flow
  of the running buffer

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Factors Affecting Band Broadening

• Longitudinal Diffusion The size of the solvated
  radius has influence in this problem. Larger
  analytes have slower diffusion, so that reduces
  the problem (as does lower temps and increased
  viscosity).
• Lower electric fields cause slower analyte
  movement.
• If electroosmosis moves in the same direction as
  the analyte, then longitudinal diffusion will
  decrease. If electroosmosis is in the opposite
  direction of the analyte, then analyte movement
  is decreasedso there is more time for
  longitudinal diffusion.
• If the analyte must move through pores in the
  support, movement through diffusion is
  inhibited.

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Factors Affecting Band Broadening

• Joule Heating According to Ohms Law, voltage
  encountering resistance requires a current to
  maintain the voltage.
• This generates heat, which will increase
  longitudinal diffusion.
• Heat will lead to band broadening and may even
  degrade the analyte and/or system components.
• The ionic strength of the running buffer also
  affects heating. Lower ionic strength means more
  resistance, which means less current and less
  heat.
• Lower voltage may lower heat too, but going too
  low can increase separation timesuse better
  cooling.

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Factors Affecting Band Broadening

• If a support is used to reduce the Joule heating
  effects it is possible to have multiple flow
  paths, which create an eddy diffusion.
• Band broadening may also be a problem with some
  secondary reactions.
• Wick flow happens when a gel is kept in contact
  with the electrodes and buffer reservoirs through
  the use of wicks. Joule heating will lead to
  evaporation of some solvent. This loss is
  replaced by flow from wicks so a net movement of
  buffer from reservoirs toward the center of the
  support takes place.

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Gel Electrophoresis

• The sample is placed on the gel support, which is
  then subjected to an electric field. Usually
  multiple samples are applied and allowed to
  separate as they migrate toward the ends of the
  gel (analytes are stopped before reaching the end
  of the support).
• The location of the migration band (and
  intensity) is determined. The size and charge of
  the analyte is influential.
• The support may be horizontal or vertical with
  a running buffer that
  carries current.
• The applied field causes migration
  of
  analytes for a
  set time, then
  
  migration is
  measured.

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Gel Supports

• Agarose Electrophoresis nonspecific binding for
  biologicals, low inherent charge, wide pores
  allow for work with large molecules. i.e. DNA
  sequencing
• Polyacrylamide is the most common support in gel
  electrophoresis (PAGE). Pore size can vary but
  polyacrylamide is typically smaller than agarose
  and is more suitable for proteins. It has low,
  nonspecific binding. Polyacrylamide does not
  have charged groups within its structure.

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Applying the Sample (Gel)

• Small wells are prepared in the gel where
  micropipette is used to introduce 10-100 mL
  samples. To avoid band broadening two types of
  gel may be used.
• Running gel the support used for separation in
  the sample.
• Stacking gel has a lower degree of cross-linking
  (has larger pore size). This is where the wells
  are located (stacked on top of running gel).
• With the applied field the analytes travel
  quickly to the boundary of the running gel. Then
  they slowly travel and other parts begin to catch
  up, resulting in narrower bands and formation of
  more contentrated bands.

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Detection (Gel)

• One method is direct examination of the analytes
  on the gel after treatment with a dye or stain
  (for proteins, Amido black, Coomassie Brilliant
  blue). Silver nitrate may be used in silver
  staining for lower concentrations. Ethidium
  bromide is used in DNA detection. Fluorescent
  NAD(P)H works will for enzymes.
• Another detection method is transferring a
  portion of the bands to a second support
  (nitrocellulose) where they react with a labeling
  agent (blotting).
• Southern Blot specific DNA sections react with
  a radioactive tag (32P) or a chemiluminescence
  tag.
• Northern Blot this is used to detect specific
  sequences of RNA by using a labeled DNA probe.
• Western Blot the transfer support is treated
  with labeled antibodies that react with specific
  proteins (used in blood screening for HIV).

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Mass Spectroscopy with Gels

• Can be used to find the molecular mass of a
  protein in a specific band
• Remove a portion of the band, then use
  matrix-assisted laser desorption/ionization
  time-of-flight mass spectrometry (MALDI-TOF MS).
  See Box 23.1.

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Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis

• SDS-PAGE Proteins are denatured, which converts
  them to single-stranded polypeptides.
• Then they are treated with SDS, which coats each
  protein, forming rod-like negative structures
  with similar mass/charge.
• The rods are passed through a porous
  polyacrylamide gel near an electric field. Small
  rods travel more quickly. See Exercise 23.2.

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Isoelectric Focusing

• IEF Separates zwitterions (substances with both
  acidic and basic groups) using isoelectric points
• Compounds migrate in an electric field across a
  pH gradient and stop when pH the isoelectric
  point (pI).
• pH is high near the negative electrode. There is
  minimal diffusion due to the focusing of ions.
  As an analyte gains a charge it will refocus back
  to its original charge. IEF provides high
  resolution of bands! (Useful for paternity suits,
  forensics)

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2-D Electrophoresis

• A high-resolution technique for complex proteins
• First, apply IEF to the sample (based on pI),
  then apply SDS-PAGE (based on size).
• Apply the first process at the top. Then turn 90o
  to apply the second
  process.
• Allows analysis for
  a larger number of
  proteins
  
  

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Capillary Electrophoresis

• Using narrow bore tubes, CE removes the Joule
  heating effect, which decreases band broadening,
  giving faster separations than gel.
• CE uses tubes 20-100mm in diameter and 20-100 cm
  in length.
• Often, CE is used with no gel (so there is no
  eddy diffusion). Longitudinal diffusion is the
  main source of band-broadening.

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Capillary Electrophoresis

• Higher electric fields result in high efficiency
  and narrow peaks (analyte migrates faster).
• Theoretical plates (N) can be determined
• m is electrophoretic mobility D is the diffusion
  coefficient E is electric field strength L is
  total capillary length Ld is the length from
  the injection to the detector V voltage.
• Migration times( tm) can decrease with more V (up
  to a limit, due to instigating Joule heating).
• See Exercise 23.3 (electric field effects)

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Capillary Electrophoresis

• The tube in CE is typically fused silica, which
  may be coated or uncoated.
• Uncoated silica can lead to electroosmosis when
  run at neutral or basic pH due to deprotonation
  of silanol groups.
• In normal polarity mode, a sample with many
  types of ions can be injected (at the end), and
  they then travel in the same direction toward the
  negative electrode as they through a dectector.
• Observed mobility will be the sum of inherent
  electroosmosis plus electrophoretic mobility.
  These affect time, efficiency, and separation.

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Capillary Electrophoresis

• If an analyte has a migration rate faster than
  electroosmosis, it may flow in the opposite
  direction of the electoosmotic flow. This is
  known as the reverse polarity mode.
• Changing the degree of deprotonation (altering
  the pH) of the silica will alter electroosmotic
  flow. Analysis is done by injecting at the
  negative electrode.
• Using a neutral coating in the tube reduces
  electoosmosis, while a positive coating will
  reverse direction of flow toward the positive end.

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Capillary Electrophoresis

• CE tubes have only a small amount of running
  buffer (0.5mL). Coupled with high efficiency,
  only a small sample size can be injected (lt 10 nL
  ).
• Hydrodynamic injection pressurize the sample
  container after connecting it to the capillary
  tube, then release and replace the capillary tube
  in the running buffer.
• Electrokinetic injection the electrode and
  capillary are in contact with the samplethe
  applied field causes the sample to enter the tube.

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Capillary Electrophoresis

• Concentrating samples helps provide narrow bands.
• Sample packing occurs when the sample ionic
  strength is less than the ionic strength of the
  running buffer.
• With an applied field, analytes will migrate
  through the matrix until reaching the ionic
  boundary between the sample and the buffer.
  Analytes will slow down there, concentrate, and
  produce narrow bands.

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Capillary Electrophoresis

• Detection in CE requires techniques for small
  samples.
• In CE, analytes with different migration times
  also spend different times in the detector.
  Comparing peak areas requires a correction to
  obtain the corrected peak area Ac
  See
  Exercise 23.4.

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Capillary Electrophoresis

• Laser-induced Fluorescence the laser is focused
  as an intense, selective narrow beam. This works
  well with small-bore electrophoresis capillaries
  (the analyte must be naturally fluorescent, or
  the derivatized product must be).
• LIF is used in DNA sequencing (using fluorescent
  dyes).

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Capillary Electrophoresis

• Electrophoresis on a chip See Box 23.2. The
  narrow channels on a microchip can be used for
  electrophoresis-based separations.
• Capillary sieving electrophoresis uses an agent
  that separates analytes based on size as analytes
  pass through a porous gel in the tube. This
  sieving can be accomplished by placing a large
  entangling polymer in the running buffer.

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Capillary Electrophoresis

• Electrokinetic chromatography place a charged
  agent in the running buffer that can interact
  with neutral analytes.
• Micellar electrokinetic chromatography (MEKC)
  uses SDS to form a micelle particle that can be
  attracted to the positive electrode of CE.
  Nonpolar analytes enter the micelle interior in a
  partitioning process where the micelle is the
  stationary phase.
• Separation is based
  on the degree of
  entering
  micelle.

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Capillary Electrophoresis

• Capillary isoelectric focusing (CIEF) uses a pH
  gradient across the capillary.
• As shown here, electrodes are in contact with the
  basic catholyte and acidic anolyte solutions.
• The capillary is coated inside with an ampholyte
  that, when the field is applied, will create a pH
  gradient.
• Zwitterions will migrate until
  reaching their pI. Pressure
  is then used to
  push the
  bands to the detector.

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Capillary Electrophoresis

• Affinity Capillary Electrophoresis uses a
  biologically active compound in the running
  buffer.
• Using cylcodextrans or proteins, ACE can separate
  chiral analytes.