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chromatograhy! using acetone... - (Oct/02/2004 )

the protocol:

Vg = volume not accessable to solvent (volume of the gel matrix)

Vg cannot be measured directly. However, it should apparant that:
Vg = Vt - volume accessable to solvent

We can measure the volume accessable to the solvent, using a small molecule such as acetone which can be easily visualized by a UV detector.

Pharmacia Superose 6 column - volume accessable to solvent = 19.5 mL (measured with acetone)
Vg = Vt - 19.5 mL = 24.4 - 19.5 mL = 4.9 mL


hi guys!
i just find this calculation very unreasonable, since as we know Vt=Vi+Vg+Vo,
where Vt =total column volum, Vo= void volum due to EXTREMELY huge substances, Vg= volum of matrix and Vi=volum inside the beads due to EXTREMELY small substances.

my question is, how come we use acetone (small molecule) to determine both Vo and Vi, since Vg= Vt- (Vo+Vi)?

hopes for replies!



by the way here is the link;