Method development on HPLC

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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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?
Try 3 microliter or 5 microliter injection. 10 is very large by today's standards.

We assayed lactic acid using an "organic acid column" and conductivity detector.
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-modes
https://helixchrom.com/compounds/dimethylmalonic-acid/

Contact me through our website if you have questions.
Vlad Orlovsky
HELIX Chromatography
My opinions might be bias, but I have about 1000 examples to support them. Check our website for new science and applications
www.helixchrom.com
The polar character of lactic acid that makes it poorly retained in reversed-phase chromatography makes it well-retained in HILIC. In fact, the fourth paper in the literature on HILIC (S.E. Pike et al., PNAS USA 88 (1991) 11081) concerned the isolation and identification of a compound that promoted growth of B cells. It turned out to be lactic acid. The chromatogram in the paper shows that lactic acid is nicely retained in HILIC. The other polar solutes in your sample will be as well.
PolyLC Inc.
(410) 992-5400
aalpert@polylc.com
… and if you're not sure where to start with Hilic, the nice thing about lactic acid is that it's not obscure, so probably nearly all the manufacturers of columns suitable for Hilic will have a method in their library, and they'll be happy to advise. My only add-on would be that in Hilic, solvents we think of as weak (water) are strong, so you'll need to be careful about your sample. If you inject a large volume of an aqueous sample, you will get poor peak-shape, and it might be sensible to try to get your sample into 50% acetonitrile or similar (Andy will know far, far more about this).
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