Chromatography Forum

Hi,
I am facing some problem with my RP-HPLC (C-18) analysis. I have conducted the TLC (hexane: ethyl acetate) and scrapped off the different bands and then dissolve them in methanol. But when I run them in RP-HPLC, my peaks are eluting at the end of the chromatogram. I use methanol: water mobile phase. May I know what can I do to incorporate TLC and HPLC analysis. Please do advice. I have been repeating this numerous time with similar results.

Please advice. Thank you.

Regards,
Jamie

You're doing a 2-d separation? If a single RP step does not get you situated, I'd sooner look into changing selectivity by using a different column/mobile phase, or else use the column you already have (but with MeCN instead)... I'd frankly be shocked of you could pull apart two co-eluting components in TLC where HPLC failed.

Your normal phase TLC has somewhat orthogonal selectivity to RP-HPLC, meaning that substances that elute fast on the TLC plate (the "high spots") will elute late on RP-HPLC and vice versa.

For speeding up elution in RP-HPLC there are several possibilities you may consider:
- Increase content of organic compound (methanol) in your mobile phase
- Change Methanol for a stronger eluent, i.e. acetonitrile or even THF
- If by now you're running isocratic, consider going to gradient elution
- Increase column temperature
- Increase flow-rate, if possible (pressure increase, peak area decrease!)
- If your analytes are ionazible, you may tune retention times by varying pH of your mobile phase
- Use a different column. C8 instead of C18 should give you generally less retention.

Thank you so much for the reply. I think I will try using a different column for HPLC, perhaps C8. But to clear my doubt, is it possible to purify my compounds from TLC?