Chromatography Forum

Hello,

There are several topics on the effect of pore size, and I see the recommendation for Uwe Neue's book, so I will pursue that. However, is it possible to answer the following question simply? No problem if not, just let me know.

We are supplied with a compound by a company who use and HPLC chromatogram as their method of choice for purity estimation. The compound is the TFA salt of a cyclodextrin, and the column is a 4.6 x 150mm Vydac C18 run at room temperature. They run a gradient of acetonitrile in 0.1% TFA (10-50% ACN over 30mins) and calculate the area under the curve. I can't give any more information on the method, sorry, I don't know it.

The last two batches (of what should be the same material) have eluted at 20mins and 24mins respectively. The only difference I can see between methods is that the first column has a pore size of 100A and the second has a pore size of 300A.

So the question is, can the pore size make that much difference to a small molecule separation?

Overall though, I think the better approach will be to go back and ask them to run the same column each time.

Thanks in advance.

larger pore size generally results in lower surface area for the silica. Lower surface area results in smaller amount of stationary phase.
Retention is governed by phase volume ratio so larger pore size>>less stationary phase>>lower phase volume ratio goes down>>retention goes down.

So yes, larger pore size usually results in less retention.

You say that this is a cyclodextrin, so maybe the mol-weight is not that big but it could be already a bit too "bulky" to access the small pores very well.

So I would say, such effects are possible.
Even if the phase ration is worse, it could be possible that some molecules could be better retained by in big pores (with less loadability).
Also some effects from the electric charge could have some influence, in the way that the density of the ions (you say it's a salt) is higher in the small pores, so it restrains new (charged) molecules to access the pores.

I once found this paper from SGE on the web:
http://www.sge.com/uploads/b8/4c/b84c77 ... 0136-H.pdf