Chromatography Forum

I realize that our field of work is gradually (or predominantly) phasing in the use of Ultra High Pressure Liq. Chrom (psi ~ 10,000) in lieu of conventional chromatography (psi ~ 3000). And, that this evolution is being in large part driven by the column manufacturers (i.e., making stationary phases of very very small particle sizes) and "green" concerns (i.e., less organic solvent usage/waste). My question is: if an analytical method is developed and validated on a conventional HPLC, would a switch to an ultra high pressure LC system protend any problems with acceptance of the method developed in terms of regulatory/litigationals?
Now, I fully realize that there are no lawyers online here, yet the insight of professional chromatographers is a valuable guideline/starting point. Thanks!

It all depends on how many things you are changing.

If you take the same column and the same mobile phase at teh same flow rate, the change from an HPLC system to a UPLC system is no different than going from one LC system to another. You need to verify the method in the same way in both cases, following your standard procedures.

This, however, does not gain you anything to speak of. If you want to gain in analysis time, there are ways to scale a method between UPLC and HPLC. I have outlined how to do this in a straighforward protocol that has been published in the US Pharmacopeial Forum (U. D. Neue, D. McCabe, V. Ramesh, H. Pappa, “Transfer of Chromatographic Methods to Suitable Columns of Reduced Dimensions and Particle Sizesâ€

Thanky for the reply Uwe. Please email me the reference you authored if possible.
So, a C-8 column in UHPLC is shorter and comprised of smaller packing material than a comparator C-8 column in conventional HPLC. If I understand your comment correctly, then they are in essence "equivalent" phases? Albeit, the UHPLC column would probably show a shorter retention time for the analytes than the HPLC column--given that the order of elution is the same.

...and your separation/resolution between the compounds of interest can be the same

The article in the Pharmacopeial Forum gives you the details of the scaling. I need your e-mail address, if you want me to send you the article. You can contact me at either the e-mail address below or at uwe_neue@waters.com.

In brief: to get the same separation, the column length is scaled with the particle size (e.g. 15 cm 5 micron will scale with 5 cm 1.7 micron), and the flow rate (at equal diameter) is changed inversely to the particle size (1 mL/min on a 2.1 mm 1.7 micron column translates to 0.33 mLmin on a 2.1 mm 5 micron column and vice versa). If you are running a gradient, the gradient volume is changed in proportion to the column volume.

Chromatograms demonstrating the scaling in both isocratic and gradient separations are shown in this publication.

Uwe, can you please send me your article? My email is mail/@/jiriurban.cz

Thank you

Hi Jumpshooter -

Thank you for your discussion. I would like to add that column manufacturers are not
driving U-HPLC sales, (certain) instrument manufacturers are.

Also, an alternative to U-HPLC is semi-micro LC. These instruments are lower in price and green
(i.e. solvent consumption is reduced by reducing column diameter).