magee wrote:
Hi,
I am trying to develop my method on HPLC. The interesting molecule is lactic acid and the solvent is SVF( simulated vaginal fluid). The pH of the SVF is 4.2. I have tried the MQ: ACN 80:20, with C8 column and the peak, eluted sometime between 3.2 to 3.5 min. I have run it in isocratic mode, the UV wavelength is 200nm and the running time is 5min. the injection volume is 10ul and the flowrate is 0.500ml/min. However, the problem is that in lower concentration, I mean lower than 2.5 mg/ml its not possible to have an acceptable resolution.
Does anybody have any experience?
Your solution is mixed-mode column which retains compounds by combination of anion-exchange and reversed-phase mechanisms. You can get much longer retention. IN addition to this any components of biofluids will elute closer to the void due to electrostatic repulsion. You also need to address issue of dimerization/condensation of lactic acid (and forming dimer, trimer, etc.).
lactic acid will retain similar to these organic acids. It has pKa around 3.8 and you will need to use a buffer with pH above 4. You can move peak far away from the void
https://helixchrom.com/compounds/ascorb ... ange-modeshttps://helixchrom.com/compounds/dimethylmalonic-acid/Contact me through our website if you have questions.