Separation of molecules that involves two phases, the mobile phase and the stationary phase.

Choice of mobile and stationary phases determines extent of separation Common criteria for separation: > Polarity > Size > Charge

 Where solute molecules are dissolved Can be liquid or gaseous Carries solute across the stationary phase

 Matrix where separation occurs Interactions of molecules with stationary phase reduces movement speed through matrix Can be paper or column type

 Separates molecules based on charge Solute is bound on the matrix due to electrostatic interactions with immobilized ionic groups Binding is reversible Became popular in biochemistry because it is simple, controllable, has high resolving power and capacity

 Stationary phase: ion exchange resins Mobile phase : buffer solution

Column and Gel Preparation

Anionic: DEAE-cellulose

Cationic: Dowex 50W

Loading Samples

Sample: 5 mg/ml each of invertase, albumin and casein

Elution

Gradient: 0.1 M, 0.2 M, 0.3 M, 0.5 M, 1.0 M KCl in buffer

Regeneration

Washing column and re-equilibrating with buffer

Protein Detection

Absorbance

Bradford reagent

 Has ionic groups covalently bound on surface Attracts oppositely charged molecules Can be cationic or anionic Synthetic resins are unsuitable for biomolecules Cellulose, agarose, and dextran became popular

Source: Voet, Voet. Biochemistry 4th ed.

Source: H. U. Khan. The Role of Ion Exchange Chromatography in Purification and Characterization of Molecules

Stable below pI: anionic exchanger Stable above pI: cationic exchanger

 Components must have same charge as matrix If not, it will take part in ion exchange pH also important (dependent of pKa)

Source: D. Voet, J. Voet. Biochemistry 4th edition

0.03

Absorbance versus salt concentration for DEAE-cellulose

~0.2 M KCl Source of Error: pH of the Buffer Instrumental Error

0.02

0.01

Absorbance

0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1

-0.01

-0.02

-0.03

[KCl], molL-1

Absorbance vs salt concentration

0.05 0.04 0.03 0.02 0.01 0 0 -0.01 -0.02 -0.03 0.2 0.4 0.6 0.8 1 1.2

~0.3 M KCl Source of Error: pH of the Buffer Instrumental Error

Absorbance

[KCl], molL-1

Dowex 50 fractions with Bradford reagent (start from left)

 Separation was not achieved using both exchangers Possible sources of error: > pH of buffer > Instrument

 Use only DEAE-cellulose for IEC of protein samples with pI below 7 Prepare reagents (esp. buffers) on the day of the experiment Spectrophotometric assay must be done on the same day