column capacity

[Pistek]

Hello,
I would like to use a 4,6 mm ID * 250 mm, 5um C18 column for semipreparative separation of a small peptide (cca MW 2500). What is the approximate capacity (in mg) of this column format? Is it possible to use a column with 100 A pore diameter for this compound (MW 2500,hepcidin) or should I order a 300 A column?
Thanks..

[tom jupille]

The Practical HPLC Method Development book by Snyder, Glajch, and Kirkland (http://www.lcresources.com/resources/resbooks.html) suggests 150 - 400 mg as a rough approximation for small molecules on a 100A column (page 631 if you have access to the book). You'd probably have to cut that by a factor of 3-5 for peptides on a larger-pore column. That said, there are so many variables that you really have to do a loading study on a specific column to get any kind of accurate answer.

My guess is that MW=2500 would be on the ragged edge of workability for a 100A column.

[Alex Buske]

150 -400 [b]mg[/b] on a 4.6mm column? Even if it was pure water that would be 150-400-µl. I wouldnt call this chromatography, its more like filtration or in the best case SPE.
A little bit more than 1 mg would be o.k.

[Victor]

I agree with Alex. Tom- think Pistek wants to perform a separation-he is perhaps less interested in the more academic measure of loading capacity that Snyder is referring to in his book.

[Pistek]

Thank you all for your replies.
1mg is in agreement with the value I found for RP columns (0,1-2,5 mg) in Phenomenex catalogue...

[tom jupille]

The rule of thumb that I've always used is that you can get 50-100 micrograms of total sample on a 250 x 4.6 mm ID column before it starts to go into overload.

The trick in prep is to push the overload as far as possible. If you have barely adequate resolution between product and impurities on the analytical scale separation, then there's not a lot of room to overload. On the other hand, if you have good resolution, then you can push the loading a lot more than many people suspect (that's why I said you have to do a loading study with your particular system).