Dialysis

Dialysis

Aim

To purify starch from small dissolved substances by diffusion through a semipermeable membrane.

 

Principle

Dialysis of proteins is a purification method used to separate proteins from small molecules such as from salts, acids and alkalis, urea and other impurities based on the selective diffusion of solutes across a semi-permeable membrane. Dialysis is a separation technique where small molecules (salts, urea, sugars) pass through while large protein molecules are retained.

Dialysis finds applications in protein purification, removal of salts from protein solutions, sample preparation and buffer exchange in biochemical experiments. It is a relatively simple and inexpensive technique which does not damage proteins, maintains biological activity and is effective for mild purification. 

Similarly, dialysis is utilized to separate high-molecular-weight polysaccharides, such as soluble starch (>50 kDa), from small solute molecules. Due to its large polymeric structure of amylose and amylopectin, starch cannot cross the semi-permeable membrane and is completely retained inside the dialysis bag. Meanwhile, small-molecule solutes and dyes freely diffuse outward into the dialysate along their concentration gradient, enabling non-destructive purification of the carbohydrate sample.

 

Materials Required:

NaCl, Tris HCl, Glycerol, Dialysis tubing/membrane/bag, Clips, Starch, Phenol red

 

Procedure:

1. 3 l Dialysis buffer (NaCl 150mM, Tris-HCl 20mM, glycerol 20%) was prepared and stored at 4⁰C.
2. Dialysis membrane/bag (made of cellulose or similar semi-permeable material) was prepared by cutting appropriate length of the dialysis bag and soaking in dialysis buffer.
3. Tied the bottom of the dialysis bag using metal clip or rubber band to seal it.
4. The solution (Starch mixed with Phenol red) is taken inside the dialysis bag without any bubbles. Tied the other end of the bag to seal it well. (The dialysis bag is to be suspended in the dialysis buffer taken in a beaker, so both ends of the bag should be sealed well using clips/rubber band)
5. Dialysis buffer (dialysate) was taken in a large beaker and placed on a magnetic stirrer, ideally at 4⁰C. A magnetic bead was placed in the centre of the beaker to facilitate stirring with a magnetic field.
6. Dialysis bag with the sample was tied on a glass rod and completely immersed in the beaker with dialysis buffer.
7. Dialysis bag was allowed to rotate inside the beaker at a fixed speed. Following incubation, the contents inside and outside the tubing were visually inspected for Phenol red migration and qualitatively tested for the presence of Starch using the iodine method.

Observation

Parameter / Test	Inside the Dialysis Bag (Retentate)	Outside in the Beaker (Dialysate)
Initial Colour	Intense reddish-pink	Clear and colourless
Colour after Dialysis	Fades to clear / cloudy white	Turns red-orange / pink
Iodine Test Result	Solution turns deep blue-black (presence of starch)	Solution remains pale yellow / amber (no starch)

 

Result

The starch sample was successfully separated from dye molecules, as confirmed by the Iodine test. Small, low-molecular-weight solute molecules (Phenol Red, ~354 Da) successfully diffused across the semi-permeable membrane into the outer dialysate. High-molecular-weight polysaccharides (soluble starch, >50 kDa) were completely retained inside the dialysis bag (retentate).