Chromatography Forum

I inheritted an HPLC method using a Poroshell 2.1 x 75mm 300 C-8 running 19-28% n-propanol/0.1%TFA gradient over 10 minutes at 2 mL/min and 75C column oven. I need to transition this analysis to a conventional porous column for both LC-MS and preparative LC for fraction collections that probably don't allow this high temp, and flow will also be much less on a similar size column with porous particles. Not a lot of good guidance, it seems, for transitioning these fast column analysis back to typical HPLC columns, and visa versa.

Any guidance on the following:
1. Current HPLC column selectivity comparisons using good probe mixtures that include shell and porous technology C-8 columns available?

2. Good, systematic development for adjusting all parameters (flow, gradient time, temperatures, etc) from shell to porous columns?

Appreciate guidance and/or referrals.

First of all, the flow rates used will depend on the column dimensions (and particle size, if pressure is an issue) and not on whether the particles themselves are totally or superficially porous. There are a number of on-line calculators that take the pain out of the calculations (you can even download one from our web site: http://www.lcresources.com/sandbox/UHPLC-HPLC.xls ).

The other thing you have to match is chemistry, and that's not as straightforward. The place to start is with the USP column equivalents database: http://www.usp.org/app/USPNF/columns.html . Scroll down to the "PQRI" database, since that includes more columns. My guess is that your best bet would be a 300A pore size column from Agilent.

Even with close surface chemistry, you will almost certainly have to adjust the mobile phase and gradient. I suspect your compounds (proteins ? ) will elute at a higher % organic.

Bottom line is that you are essentially developing a new method.