chromatography....using acetone!

[question]

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.

 

example:

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

 

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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!

 

thanks!

[question]

by the way here is the link; http://itsa.ucsf.edu/%7Ehdeacon/Stokesradius.html