Dialysis lecture 3

1

DIALYSIS AND

ULTRAFILTRATION

2

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

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been replaced by gel filtration for most applications.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

8

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

9

Dialysis tubing

Bed of powdered

polyethylene glycol

10

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.

11

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.

12

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

13

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

15

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

16

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.

17

◦ Simple, easy, and rapid

◦No stirring or foaming by N2

◦High quality materials to minimize non-

specific binding

18

◦Concentrating and desalting of biological

samples, especially small-volume

samples

◦ Buffer exchange

19

Semi permeable glass fibers are valuable devices for both :

Dialysis

And concentration.

20

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.

21

22

A solvent flows through the fibers, a small

molecules enter the fibers

Thus reducing the concentration of smallmolecules in the sample (purification)

23

Vacuum is applied to the filter bundle and the

solvent and small molecules enter the fibers

Thus, concentrating any macromolecules in the

sample

24

25