Gel filtration chromatography

In gel filtration chromatography (molecular sieve chromatography or size exclusion chromatography), molecules are divided on the basis of their shapes and sizes. The protein sample in a small volume is applied to the top of a column of porous  beads  (diameter  0.1  mm)  which  are  made  of  an  insoluble  but  highly hydrated   polymer   like   as  polyacrylamide Bio-Gel or  the   carbohydrates dextran  (Sephadex)  or agarose  (Sepharose) in  Figure. Larger or more elongated molecules cannot whereas Small molecules can enter the pores in the beads. The smaller  molecules  thus  have a larger volume  of liquid accessible  to them; both  the  liquid  surrounding  the  porous  beads  and  which  inside  the  beads.  In compare, the larger molecules have only the liquid surrounding the beads accessible to them, and therefore move by the column faster, emerging out of the bottom (eluting) first. The smaller molecules move more slowly by the column and elute later. Beads of different pore sizes are available allowing proteins of various sizes to be efficiently separated. The Gel ?ltration chromatography   is  often  used  to  de-salt  a  protein  sample  for  instance  to erase the ammonium  sulfate after ammonium  sulfate precipitation,  because the salt  enters  the  porous  beads  and  is eluted  late,  while  the  protein  does  not enter the beads  and is eluted  before.  The Gel filtration chromatography can also be used to estimate the molecular mass of a protein.

Figure: Separation of molecules on the basis of size by dialysis. (a) Starting point, (b) at equilibrium.

Figure: Gel ?ltration chromatography. (a) Schematic illustration of gel ?ltration chromatography; (b) elution diagram indicating the separation; (c) a plot of relative elution volume versus the logarithm of molecular mass for known proteins, indicating how the molecular mass of an unknown can be read off when its relative elution volume is known.

There is a linear connection among the relative elution volume of a protein (V_e or V_o where V_e   is the elution volume of a provided protein and V_o   is the void volume of the column, which is the volume of the solvent space surrounding the beads) and the logarithm of its molecular mass.  Therefore  a standard  curve  of  V_e or V_o   against  log₁₀    molecular mass  can  be  determined of  known  mass  for  the  column  using  proteins .  The elution volume of any sample protein then permits its molecular mass to be estimated through reference to its position on the standard curve.