Chromatography Forum

Hello everyone,
I have a gradient method developed in a quaternary LC that once transferred to a binary LC has retention times much faster and now the first peak is in an area that I do not want and all other peaks have shifted too. What do you normally do to correct for the difference in dwell volume of the binary system to go back to the original retention times obtained. Initial hold time?, decrease initial % organic? If I know the dwell volume of both systems can I use this information to adjust my gradient? Any help would be really appreciated.

Check first the volume of your mixing chamber, than reduce % organic modifier. Hope that will help.

I'm in Pharma, so modifying mobile phase composition is, well, challenging .
I'd try the initial hold method first, as this is the approach suggested by the pharmacopoeias (e.g. Ph. Eur. 2.2.46). It's considered a method "adjustment", not a "modification", so no revalidation needed. If you know the dwell volumes of both systems (and the determination is quite easy), this adjustment is usually rather straightforward.

Just to expand a bit on the previous posts, assuming bothe systems are functioning well, what you need to do is to normalize the time offset between injection and arrival of the gradient at the column. The easiest way, as suggested, it to use an isocratic hold (or a delay-to-injection if you are going to a smaller dwell volume) equal to the difference in dwell times. Note that you may still have some issues with early peaks due to differences in mixing/washout characteristics between the systems.

I work in Pharma and ran the same gradient method on two different HPLC brands, the dwell volume was slightly different. I adjusted the temperature within the method requirements and was able to achieve similar results.

My supervisor and I don't often see eye-to-eye about interpreting cGMP and FDA stuff. For example, I take USP <621> as it is, that procedures can be modified without revalidation. And FDA-ORA states similar for house-validated procedures.

So I'd modify the gradient a little to account for different HPLC systems, like one may need to do with systems from different brands, and maybe run some sample vials on both systems and document the agreement. So what should agreement be to be considered equivalent to the FDA/cGMP? I haven't seen any guidance on that.....

Thanks everyone for the great advice, I will start trying them all. Yes Tom, the system dwell volume is smaller for the binary than the quaternary, could you please expand on the delay-to -injection advice? Thank you all

If I'm correct, I think Tom mixed it up. Delayed injection should be used if you go from lower to higher dwell volume.

I think Tom mixed it up. Delayed injection should be used if you go from lower to higher dwell volume.

Yes, I did!

thank all of you for your help, I managed to correct for dwell volume and the Rt as well as the specificity remained the same.