Gel Filtration: Separating by Size Alone

Every separation in this batch so far works because molecules STICK to something — to chalk, to silica, to a grease film — and stick by different amounts. Gel filtration works because they stick to nothing at all. Per Flodin and Jerker Porath, at Uppsala in 1959, made beads of cross-linked dextran riddled with pores of a controlled size. A small molecule wanders into every pore and takes a long, tortuous route through the column. A molecule too big to enter any pore is swept straight past, through the gaps between the beads only, and comes out FIRST. That inversion is what makes it unique and slightly counter-intuitive: in gel filtration the biggest thing leaves first, and everything is off the column by the time one column volume has run through. It is also the gentlest separation there is — nothing binds, so nothing is denatured — which is why it is how a protein gets its buffer changed.