Additional Separations Methods

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Additional Separations Methods
As we have discussed many times, there are
thousands of different separations methods.
Todays lecture will briefly describe several
different methods to give you and idea of the
breadth of methods and how various driving
forces are combined to create interesting and
powerful separations methods.
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Electrodialysis
Feed solution
Electrode rinse solution
Electrode rinse solution
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Cathode

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Anode
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Concentrate
Diluate
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Entropic Traps for DNA Separation
Baumgartner and Muthukumar, 1987. Han and
Craighead, 1999.
G
Thick Region
Thin Region


DNA Motion
Thick region consists of spaces larger than the
radius of gyration of DNA, whereasthe thin
regions consist of spaces smaller than the Rg for
longer DNA molecules. All DNA flows to the the
anode, although DNA must fold more tightly to
enter thin region, and thus lose entropy. This
creates a difference in residence times in the
entropic traps leading to differential transport.
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Microchip Electrophoresis
Microchip fluidic devices can be made using
lithography methods from microelectronics
fabrication. Microfluidic techniques for fluid
handling are combined with voltage supply by
metallization of lines.
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Electrospray High Resolution Ion Mobility
Spectroscopy
Electrospray high resolution ion mobility
spectroscopy takes advantage of the different
ion mobilities in a buffer gas at atmospheric
pressure to create differential transport.
Buffer gas inlet
Sample inlet
drift
Ionizationchamber
V
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Continuous-Flow Electrophoresis
Protein Separation by Continuous-Flow
Electrophoresis Microgravity Clifton et al.
AICHE Journal 42, 1996
Sample injection
Carrier Solution
Continuous flow operation High sensitivity
Preparative quantities  Natural
convection  Electrohydrodynamic spread Joule
heating
Anode
Cathode
Fraction collector
Electrode buffer
Electrode buffer
Ion exchange membranes
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Dielectrophoretic Field-Flow Fractionation
The polarizability of living cells depends
strongly on their composition, morphology and
phenotype and on the frequency of the applied
electric field.
FDEP
FDEP
Fg
Fg
FDEP depends on the polarizability of the
particle, in this casehuman leukocytes. Fg
depends on the density of the particle.The net
force determines the average levitation height
and thus the average velocity. This is a
chromatographic methodand is operated in a batch
mode where separated cells are elutedfrom the
chamber at different times.
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High Gradient Magnetic Separation
These methods rely on the ability of magnetic
fields to attract diamagnetic particles, and to
repel paramagnetic particles. Large magnetic
field gradients are used to obtain higher
separation factors.
Fractionator
B
Flow
diamagnetic
paramagnetic
This method can be used to separate heavy metals,
radioactive waste (in slurry form), water
purification, and the separation of blood
(reduced and methemoglobin are paramagnetic
while oxyhemoglobin is diamagnetic).
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Gradient Magnetic Separation Using Magnetic
Tagging
The gradient magnetic separation method depends
on bacteria or cells that have beenmagnetically
tagged. Tagging can be done using antibody
antigen binding/recognition where the antigen is
attached to ferromagnetic particles, beads or
functional groups. The antigens bind selectively
to their antibody counterpart on cells or
bacteria which can then be separated in a
magnetic separator.
Bacteria or cell
Antibody
antigen
antigen
Magnetic particle
antigen
antigen
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Mass Spectrometry
Mass spectrometry involves separating species
based on their size to charge ratios. Species
are vaporized, ionized and accelerated by an
accelerating field. A magnetic field is applied
orthogonal to the velocity of the cations which
causes their paths to curve, depending on their
charge and mass.
B
Inlet for sample gas
Ionizer


Detector
Coil
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Field-Flow Fractionation
Field flow fractionation exploits the fact that
larger particles in a flowchamber on average are
further away from the chamber walls. In
parabolic flow this creates a higher average
velocity for larger particles, separating them
from smaller ones.
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Sedimentation
Sedimentation relies on the variation in density
between materials to be separated. This process
is often used in crystallization of
pharmaceuticals, waste water treatmentplants
using settling ponds, and refining of metals
(removal of slag).
Crystallized
Slag (fused silica, etc)
Molten metal
Liquid
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Centrifugation
Denser material
Less dense material
Centrifugation is similar to sedimentation,
although it creates larger forces using high
angular velocity rotating units. Denser material
moves towards radially outward.
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Colloid Precipitate Flotation
This technique involves creating a colloid
containing a specific species using a coagulant
and a surfactant to stabilize the colloid. The
coagulant which forms the colloid binds to
one solute, but not the other which creates the
partitioning. The colloid will be less dense
thanthe solution and the difference in densities
causes the colloid to float where it can be
collected off.
surfactant
Colloid
Species toseparate
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Capillary Gel Electrophoresis
This is a combination of CE and size exclusion
chromatography.
Inner bore filled with Gel
Resin Coating
Fused silica
Solutes move through the capillary due to their
electrophorectic mobility. However, the gel slows
down the larger species. This technique can be
used to separate species with similar charge to
size ratios, but different sizes (for example
DNA and other oligonucleotides).