Hi.

I'm doing some RP HPLC of some messy plant extracts, and would like to add column washing (100% meCN) to the end of my gradient method. I am curious about the rate of change of mobile phase composition. Various more experienced chromatographers around me have suggested a maximum of 10% change per minute. Before the column washing step, my mobile phase is 30% meCN. This would mean that my washing step would take at least 15 extra minutes.

I recently went to a chromatography seminar where the presenter suggested using step gradients; he would program a wash step that changes from 30% to 100% meCN in, say, one second.

Does anyone know of a good reason for avoiding these faster (step) gradients?

thanks!

Jonathan

Of course you need to make sure there is not any buffer in the system that will precipitate at 100% ACN, but otherwise steping up to 100% should be just fine. Over the last 10 years I haven't had a problem with doing so. I think most RP columns can take the rapid increase.

I have found no problem with rapid increase of organic solvent to the aqueous mobile phase, BUT ONLY AFTER PRECIPITATION HAS BEEN CHECKED FOR. I use 25mM KH2PO4 with methanol, starting with 7% MeOH, going to 60% MeOH in one step at 15min for 15min to enable flushing of the RP column.

Hope that helps some

Thanks, y'all. I'll check it out.

Hi Jonathan,
Normally you won´t get any problem if you´re changing from 30% organic phase to 100% using a steep linear gradient. If there are buffer salts in your aqueous phase, you can switch to a AcN/water mixture and then go to 100% organic phase in about 1-2 min, or, if this makes necessary a prolongued equilibration afterwards, just wash your column with only 80% organic phase, this worked quite well for me with plant and yeast extracts. Otherwise rethink your sample preparation procedure and make sure that there are no traces of fat or macromolecular contaminants in the sample.

Jonathan,

One additional point: you started this thread with the comment that experienced chromatographers recommended a maximum solvent rate change of 10% per minute. Of course, an eluent step change is not a gradient at all but can be thought of as an extremely steep gradient. Accordingly, if an eluent step change is okay then any other slope is okay as well since an eluent step is a % change which is well above the "recommended" rate change. There is nothing "magic" about using an eluent step that somehow avoids damage that might otherwise have been caused by a true gradient. In fact, the most demanding test of column bed integrity is to expose the column to repeated cycles of eluent step changes. So, it all depends on how careful you would like to be. If you're OK with using the most demanding method, go ahead with using an eluent step change but you will most likely get longer column life using steep gradients. One reason for this is that not only buffers but also samples can cause column bed disruption. If the solution of highly retained sample components produces a highly viscous slug at the solvent zone boundary, this will also increase the opportunity to damage the column bed.