I am working in Supercritical Fluids Chromatography. I use two columns to separate polyphenols. When I use only anyone of these two columns I can detect the compounds, but when I use both I can't detect anything.
Which 2 columns? Are they the same column (stacked)? what is your solvent system, besides CO2?
Thank you for your reply. I use a Diol column 5um particle size, 4x250 mm and after that I use a Zorbax SB-C18 3.5um particle size, 4,6x150. My mobile phase is CO2 +ethanol or ethyl acetate or a combination of both cosolvents.
Thank you.
Over the years there has been few separations on C-18 columns using SFC-conditions. Guess that you want to have some additional chromatographic selectivity. Silica perhaps. Before disconnecting the two columns from the system you can try methanol as modifier; reasonable high % or a decent gradient.
Diol can be run in normal phase or reverse phase, depending on the polarity of the compound and the solvents used- with this solvent system, I expect it to run as normal phase for all compounds, with ethyl acetate or ethanol being the strong solvent that elutes the compound. Diol can run as reverse phase with high concentrations of water, not the case here. C18 is reverse phase for all compounds and solvents, and CO2 is the strong solvent, causing compound elution, for C18 with more polar solvents being weaker solvents.
I suspect the strong solvent that allows elution from the diol column carries your compound to the C18 column, but now this same solvent is the _weak_ solvent for C18, so your compound hangs up on the column. As the concentration of ethyl acetate or ethanol increases in your gradient (becoming a stronger solvent for diol), the solvent becomes a weaker solvent for C18. To test this hypothesis, you can do a run with both columns, then do a run without adding anymore sample to the C!8 column, using only the C18 column, using the gradient method that worked for your C18 column- your compound should then elute from the C18
Like Olle mentioned, suggest you try another column chemistry- silica, alumina, cyano, amine, and many others. Or you can try other solvents with your CO2 such as acetonitrile, chloroform, dichloromethane, or tetrahydrofuran. But don't run a normal phase and reverse phase column back-to-back.
Thank you all for your replies. They are very useful. I will try with different columns and different solvents and I will change my column configuration as you suggest me.
Once again, Than you so much.
Acetonitrile is not that popular modifier in packed column SFC systems. Selectivity ok but peaks are not that good. Recently it has become popular to have acetonitrile mixed with an alcohol modifier such as methanol. A brief glance in the recent literature may help. Also, some hints can perhaps be found on "Past conferences" at http://www.greenchemistrygroup.org/index.html#
This abstract to a recent paper in JCA 2014
should be useful even if the analytes are
enantiomers. A lecture was given in Basel
(cf above) butno file available.
Evaluation of non-conventional polar modifiers on immobilized chiral stationary phases for improved resolution of enantiomers by supercritical fluid chromatography.
http://europepmc.org/abstract/MED/24456706
(link to abstract)
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