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:
- 3 l Dialysis buffer (NaCl
150mM, Tris-HCl 20mM, glycerol 20%) was prepared and stored at 40C.
- 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.
- Tied the bottom of the dialysis
bag using metal clip or rubber band to seal it.
- 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)
- 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.
- Dialysis bag with the sample
was tied on a glass rod and completely immersed in the beaker with
dialysis buffer.
- 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).
