Amino Acids, Proteins and Porphyrins
The following separation technique depends on the molecular size of the protein:
|A||Chromatography on a carboxymethyl (CMC) cellulose column|
|C||Gel filtration chromatography|
|D||Chromatography on a diethylaminoethyl (DEAE) cellulose column.|
1. Gel-filtration chromatography (Molecular sieving)
- Hydrophilic cross-linked gel like acrylamide (Sephacryl), Agarose (Sepharose) and dextran (sephalex) are used for separation of molecules based on their size.
- The gel particles are porous in nature, these pores, allow small molecules to enter into the gel but larger molecules can not enter into pores and so are excluded.
- In short, Larger molecules will come out first while smaller molecules are retained in the column,
- Gel — filtration technique is used for
i. separation of protein molecules
ii. purification of proteins, and
iii. molecular weight determination
2. Ion exchange chromatography
a. the separation is based on electrostatic attraction between charged biological molecules to oppositely charged groups on the ion exchange resins.
b. The resins are either cation exchange resins or anions exchange resins. eg. Amberlite — IRC-50 (week cation; Dowex —3 (weak anion); and DEAE-cellulose (Strong anion)
c. The separation is based on the ionic character of proteins and amino acids.
3. Electrophoresis - refers to the movement of charged particles through an electrolyte when subjected to the electric field. The positively charged particles (cations) move to cathode (negative pole) and negatively charged ones (anions) to anode (positive pole).
Isoelectric focusing - in this technique pH gradient is generated from anode to cathode using ampholytes of different pI (isoelectric pH) values, so that the proteins get concentrated as bands at the points where the local pH is the same as isoelectric pH of these proteins. This is useful for studying iso enzyme and genetic variants of Hb and others tissues proteins.