How is Acetone Used in Chromatography Calculations?

AI Thread Summary
The discussion centers on the use of acetone in chromatography to determine the volume accessible to solvent (Vg) in a gel matrix. The protocol outlines that Vg can be calculated by subtracting the volume accessible to solvent from the total column volume (Vt). An example using a Pharmacia Superose 6 column illustrates that with acetone, the volume accessible to solvent is measured at 19.5 mL, leading to a calculated Vg of 4.9 mL.A key point of contention arises regarding the validity of using acetone, a small molecule, to determine both the void volume (Vo) and the volume inside the beads (Vi). The original poster questions how acetone can effectively measure these volumes, given that Vg is derived from the equation Vt = Vi + Vg + Vo. The inquiry seeks clarification on the methodology and the implications of using acetone for accurate volume measurements in chromatography.
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chromatography...using acetone!

the protocol:

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

Vg cannot be measured directly. However, it should apparent 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

---------------------------------------------------------------------------------------

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!
 
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