King Saud University Riyadh Saudi Arabia Dr. Gihan Gawish Assistant Professor 1

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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 Collodion bag 3

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. 4

 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. 5

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 6

Disadvantage of dialysis  Slow process several hours for completion, and thus, has been replaced by gel filtration for most applications.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 7

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 8

Dialysis tubing Bed of powdered polyethylene glycol 9

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 inter the membrane) such as polyethylene glycol. 10

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. 11

Modified Dialysis A. Pressure Dialysis Modified Dialysis A. Pressure Dialysis  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. Air 12

 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 (N 2 ) pressure, centrifugation, or mechanical forces.  They have very thin polymer membranes. (  m). 13

 Their pore size range from 2A-100A  The flow rate through these membranes is very low, so they are operated under pressure.  Either small or large molecules can be purified in this way Pressure Dialysis 14

Modified Dialysis B. Flow Dialysis Centrifugal Ultra filtration units 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. 15

◦ Simple, easy, and rapid ◦ No stirring or foaming by N 2 ◦ High quality materials to minimize non-specific binding 16

◦ Concentrating and desalting of biological samples, especially small-volume samples ◦ Buffer exchange 17

Semi permeable glass fibers are valuable devices for both :  Dialysis  And concentration. See Physical Biochemistry book (Application to Biochemistry and Molecular Biology), David Freifelder page;157-Fig

 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, thus providing a very large surface area. 19

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 A solvent flows through the fibers, a small molecules enter the fibers  Thus reducing the concentration of small molecules in the sample (purification ) 21

 Vacuum is applied to the filter bundle and the solvent and small molecules inter the fibers  Thus, concentrating any macromolecules in the sample 22