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DIALYSIS AND
ULTRAFILTRATION
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Dialysis is an
operation to separate dissolved molecules based on molecular weight.
◦ In practice, a biological sample is placed inside a tube of semi permeable membrane, and placed inside a much bigger container.
Buffer
Concentrated
solution
Dialysis
bag
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1. Only small molecules diffuse through the collodion
membrane.
2. At equilibrium, the concentration of small molecules is
the same inside and outside the membrane.
3. Macromolecules remain in the bag.
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The only two variables in this method are:
1. The type of membrane (most common are
cellophane & cellulose)
2. The size of pores or the molecular weight cut off.
Only molecules or ions smaller than MWCO will move out of the dialysis bag.
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Advantage of dialysis
1. Dialysis is still in use today for it is very simple and is still the only way to deal with large-volume samples.
2. characterization of a candidate drug in serum binding assays or detailed study of antigen-antibody interactions
3. proves to be the most accurate method available.
4. inexpensive and easy to perform
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Disadvantage of dialysis
Slow process several hours for completion, and thus, has
been replaced by gel filtration for most applications.
Other forms of dialysis includes flow-dialysis and
pressure-dialysis
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10mm = 6.4mm Dia; 0.32 ml/cm Vol/L
16mm = 10mm Dia; 0.79 ml/cm Vol/L
24mm = 16mm Dia; 1.8 ml/cm Vol/L
31mm = 20mm Dia; 3.1 ml/cm
1. Removal of salts and low molecular weight compounds
2. Buffer exchange
3. Concentration of macromolecules
4. Purification of biotechnological products
5. Medical applications: kidney dialysis and Haemodialysis
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Dialysis tubing
Bed of powdered
polyethylene glycol
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concentrate the material inside the dialysis tubing.
polyethylene glycol and macromolecules can’t bath through the membrane solution is concentrated
Water then leaves the bag to equilibrate which the dry external phase.
The filled bag is packed in a dry, water-soluble polymer (which can't enter the membrane) such as polyethylene glycol.
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We must be careful when using reverse dialysis, this is because:
1. Equilibrium is never reached.
2. Water and salts are continually removed until the sample is totally dry.
3. Most macromolecules become irreversibly bound to the dialysis tubing and hence, for all practical purposes they are lost.
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An important modification of dialysis tubing is the Diaflo or Pellicon membrane
Pressure dialysis is a common technique for concentrating samples.
Other applications of pressure dialysis include: desalting, buffer exchange, and purification of macromolecules.
Support Media
Sample
outlet
Pressure
Membrane
Air
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The basic design is ultra filtration cell.
There are a wide variety of filters to choose from
(materials and cut off limits).
The applied pressure can be gas (N2) pressure,
centrifugation, or mechanical forces.
They have very thin polymer membranes. (0.1-1.0
m).14
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Their pore size range from 2A-100A
The flow rate through these membranes isvery low, so they are operated underpressure.
Either small or large molecules can bepurified in this way
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It is an example for flow dialysis, tubes containing polypropylene filter,
◦ Tubes comes in variety of sizes suitable for samples
◦ (Tubes+ samples) are centrifuged to concentrate the samples.
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◦ Simple, easy, and rapid
◦No stirring or foaming by N2
◦High quality materials to minimize non-
specific binding
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◦Concentrating and desalting of biological
samples, especially small-volume
samples
◦ Buffer exchange
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Semi permeable glass fibers are valuable devices for both :
Dialysis
And concentration.
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They are fibers whose glass walls contain pores of
controlled size
Molecules smaller than the pores pass freely
through the wall of the fiber .
These fibers are usually used in bundles, thusproviding a very large surface area.
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A solvent flows through the fibers, a small
molecules enter the fibers
Thus reducing the concentration of smallmolecules in the sample (purification)
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Vacuum is applied to the filter bundle and the
solvent and small molecules enter the fibers
Thus, concentrating any macromolecules in the
sample
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