Chromatography Forum

conditions given:
solvents water/ACN (0.1/0.085 % TFA)
CV~120µl (1x150, perfusion resin)
flow 0.1--0.5 ml/min
multistep gradient with 1--2 % / CV in the range of interest

question:
Shall I stick to the 350 µl static mixer or replace it with a smaller one?

Thanks in advance!

Generally speaking, you are using one of the most challenging mixing compositions in HPLC. Although the default UltiMate mixer is highly efficient, the mixing performance might not be sufficient for this scenario. I doubt that a smaller mixing volume will be good enough. Suggestion: Start with the default mixer; if mixing is incomplete, consider a larger mixing volume.

Thanks Hfranz,
can you recommend some literature dealing with the TFA mixing issue? I thought it would be related to the retention of TFA on C18 columns and therefore less pronounced with C<=4 resins.

The origin to TFA mixing ripple is improper mixing; but you are right, it is often emphasized by TFA retention on the stationary phase. Also important is the UV detection wavelength. I have an overview article which I can mail you.

I tested mixing using the standard mixer and the 150 µl mixer at flow rates between 0.1 and 2 ml/min. Generally speaking: using the standard (350 µl+50) mixer at low flow rates (<0.5 ml/min) I observed intolerable instability of the solvent composition and UV baseline, both using 0.1% acetone at 254 nm and 0.1 % TFA at 214 nm in MeCN, more pronounced at 10% and 90 % B compared to 50 % B, indicating incomplete mixing or diffusion effects due to the large mixer volume and delay time. Results were better using the SMALLER mixer (150 µl+50), but at flow rates of 0.2 ml/min and less, mixing and stability of the solvent composition was still unsatisfying. So I discussed with my contact at Dionex they will send me a 100 µl kit and the 35 µl kit for testing purposes. Due to the special dependence of the capacity factor on the concentration of organic modifier in case of proteins, fluctuations of o.m. concentration not only effects the UV baseline but also leads to severe peak broadening.... I wonder why a laminar mixing system was chosen for the RS-System. Turbulent mixing systems are much more efficient. They have to be adjusted to the different flow regimes but this is the same for the laminar mixing (see above). I hope Dionex comes up with a more universal solution, like Agilent did with the 1290 mixer. They should have a closer look in the microfluidics literature; they would find lot of interesting mixing concepts.