Why do we use a gradient in centrifugation?

[red65]

Why do we use a gradient of ions(Caesium chloride )in centrifugation, I expect if we add in a test tube the molecule that we want to know its density and a gradient of caesium chloride, that the caesium chloride will accumulate in the region that corresponds to its density so as our molecule what is the purpose of this gradient ???

 

[TeethWhitener]

We don’t put in a gradient of CsCl; we put in a homogeneous solution of CsCl and subject it to ~100 000 g, and the gradient forms naturally. This happens to be convenient for us if we want to separate a mixture based on density, as the different density components of the mixture are buoyant at different locations in the centrifuge tube.

 

[red65]

We don’t put in a gradient of CsCl; we put in a homogeneous solution of CsCl and subject it to ~100 000 g, and the gradient forms naturally. This happens to be convenient for us if we want to separate a mixture based on density, as the different density components of the mixture are buoyant at different locations in the centrifuge tube.

why would a gradient form, shouldn't all molecules of CsCl assume the location corresponding to their density, and why would it be more convenient to have a gradient in centrifugation?

 

[TeethWhitener]

why would a gradient form, shouldn't all molecules of CsCl assume the location corresponding to their density,

Why don’t all molecules of CsCl assume the location corresponding to their density even before you centrifuge the mixture? CsCl is far more dense than water; shouldn’t all the CsCl sink to the bottom? What else do you think could be going on?

 

[red65]

Why don’t all molecules of CsCl assume the location corresponding to their density even before you centrifuge the mixture? CsCl is far more dense than water; shouldn’t all the CsCl sink to the bottom? What else do you think could be going on?

you previously said "We put in a homogeneous solution of CsCl" , which suggests that the concentration of CsCl is the same anywhere in the solution, well if CsCl is way denser than water, you can continuously mix the solution so that it stays homogeneous

 

[TeethWhitener]

The point I’m trying to nudge you towards is that if gravity were the only force acting on the molecules, then yes, all the CsCl would immediately sink to the bottom of the vessel, regardless of how weak that gravity is. However, gravity is not the only force at work. Intermolecular and ionic forces, along with thermal fluctuations, are orders of magnitude larger than the gravitational force at 1g. However, at ~100 000 g, the gravitational force is strong enough to begin separating the CsCl from the water by density, at least a little bit. This is what gives you the gradient in density gradient ultracentrifugation.