Hello,
I am struggling with separation of porphyrins (i.e. 5,10,15,20-Tetrakis(pentafluorophenyl)porphyrin, protochlorophyllide, chlorophyll a and b) using a hypercarb (porous graphite carbon) column in my HPC system. The retention is so strong that I am virtually unable to elute the analytes from my column (both the UV and fluorescent detectors show no porphyrins). I tried both acid (0,1% TFA) and basic (0,1% triethylamine) modifiers. Does anybody have any experience with this setup or any good hints?
Hi Kamil,
Hypercarb columns are made of graphitized carbon, i.e. there is a huge planar pi-electron system here. Porphyrins also have conjugated double bond systems and are also predominantly planar. This means that the conditions are optimal for pi-pi interactions between stationary phase and analyte. You also write that the porphyrins no longer elute. One possibility would be to increase the column temperature. Hypercarb columns can withstand 150°C-200°C. With this you can possibly increase the desorption rate. I don't know if your porphyrins can withstand this. I would successively increase the temperature and see what happens. Another possibility would be rinsing the column with pure ACN, MeOH or IPA.
Good luck.
Regards
Markus
P.S.: For this type of analyte I would use a PFP-column (pentafluorophenyl phase) and a high content of organic solvent in my solvents.
P.S.: An article as an example
Article Determination of porphyrins in oral bacteria by liquid chrom...
Hi Markus Christ,
I have performed porphyrin separation on C18 columns and it's quite a trivial task, but at the moment I'm working on multiple methods for different analytes using hypercarb and reverting to C18 would be a bit impractical (it will be the option if I'm unable to find good separation conditions using hypercarb).
I have expected strong retention, but not so strong. I have tried a linear gradient from water to ACN and IPA afterwards and it did not helped (I have also tried further gradient from IPA to CH2Cl2, with no effect). Sadly I'm unable to heat the column above ca. 100 deg and it did not help.
Anyway thank for your response
Hi Kamil,
do you know this article?
http://www.us.edu.pl/uniwersytet/jednostki/wydzialy/chemia/acta/ac14/zrodla/15_AC14.pdf
Maybe hexane or dichloromethane will eluate your porphyrins .
Regards
Markus
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