Chromatography Forum

Hi! We are planning a method transfer from one chromatographic system to another (HPLC to UPLC). Purpose of the system is screening for pharmaceutically active substances.

The method on the old system is quite time-consuming (45 min), we use a gradient going from acidic phosphate buffer to acidic phosphate buffer / acetonitrile 30:70, then the buffer is replaced by water and the gradient goes further up. Unluckily this is not possible on the UPLC system (Acquity) because it only has a binary pump. To catch the lipophilic substances as well, we need to make some changes. We already thought of the following, not being entirely satisfied with either of these approaches:

• - Changing the buffer (needs to be miscible with acetonitrile and UV-transparent down to 200 nm)
  - Changing to methanol (here UV-transparency would suffer as well as the peak shapes)
  - Using a separate method with acetonitrile / water to elute the lipophilic substances (this approach requires three methods in total as the column has to be flushed and washed prior to the method for the second analysis.)

Any ideas how can we keep the method quick and also keep UV-transparency down to 200 nm? Comments and help are most welcome!

What pH and concentration of buffer?

And perhaps a bigger issue, is your present stationary phase available in a sub-2 micron particle size?

My buffer is adjusted to pH 5.6 and its concentration is 25 mM.
The stationary phase from the old system is not available for UPLC conditions,
so I actually need to rebuild a method more than just transferring it.
This is also the reason I can consider bigger changes to the system like changiong the buffer.
I am really glad the old system is still running, so I can plan the new one thoroughly.

When rebuilding the method, make sure you're actually using a buffer (pH 5.6 is outside the buffering range of phosphate).
Is a true buffer really needed? What about just using a "standard" acidic mobile phase (water, ACN and TFA/phosphoric acid/whatever)? If it's just for screening purposes...
Regarding the use of methanol, consider that it will bring you a lot more backpressure than acetonitrile. Especially with such a wide gradient, it's use might not such a good idea (maximum backpressure for methanol/water is at ~50% MeOH).