Hi everyone,
I'd like to better separate two close peaks coming from a bioconversion sample, I'm currently using a C18 KromaPhase 4,6mm Ø.I. SCHARLAU, particle size (µm): 10, pore size (Å): 100, length (mm): 250, internal Ø (mm): 4,6, with 10/90% acetonitrile/water (0.1% formic acid) as mobile phase at the temperature of 20 °C. I've also tried with 5/95% ACN/water as mobile phase obaiting a better separation, but I've read that is not very good to work with a organic percentage lower than a 10%. I'm open to any suggestion to improve my hplc method. Thank you
It's helpful to know what sort of compounds you are running because this will allow us to give better suggestions to you. Generally, running at 5% organic is fine on C18. You are worried about "phase collapse" where the concentration of organic solvent becomes so low that the C18 chains interact with themselves rather than the analyte. This can be reversed by washing the column with acetonitrile. A C18AQ type column is resistant tp phase collapse:
https://www.teledynelabs.com/en-us/resources/Documents/Application-Notes/RediSep-Gold-C18Aq-for-Highly-Aqueous-Mobile-Phases.pdf
An alternative is to use another column such as silica, diol, alumina, or amine in HILIC mode (aqueous mobile phase). The column is run with acetonitrile with 1-2% water, and a gradient run to 50 to 100% water. You may need to dissolve the sample in DMSO or DMF since dissolving it in water may cause the sample to be unretained. Run an injection solvent blank because these columns can sometime retain DMSO- the blank allows you to see where the DMSO elutes! If using silica based columns, the mobile phase should be neutral or acidic.
i’m trying to separate the peak corresponding to L-DOPA from an unknown peak derived from the bioconversion, maybe an adduct of the product or a ionized form or a decarbo product. Maybe using methanol instead of acetonitrile can help me? yeah I am worried of phase collapse running with 5% ACN
We really do not have enough information to provide specific suggestions for analysis. Does your sample have mild to strong chromophores ? If so, use a scanning diode-array detector inline for detection. Change the column to one that has a particle size of 5 microns for better resolution. Your isocratic mobile phase is VERY weak. To improve resolution of actual sample peaks, proper HPLC method development must be undertaken. Generic advice may include: Use a gradient HPLC method starting at 5% organic increasing to 95% organic (i.e. ACN) over time. Hold at 95% organic to insure all samples have eluted off the column. Review the results. Is retention found for the compound(s)? Does the K prime of the sample show adequate retention? Is the peak shape Gaussian? HOw many peaks were detected? What do their UV/VIS spectra look like? Etc. Do this following good chromatography fundamentals Please contact a local, experienced chromatographer to assist you.
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