I tried to purify a highly water soluble compound using RP-HPLC. Unfortunately, no UV peak was detected, however, when a same compound was run on a normal C18 column, there were three different bands with better separation was achieved. In a normal C18 column the mobile was chosen as 5:95 CH3OH: H20 mixture. The same or higher polarity was employed for HPLC. But I was failed to detect any peak in the HPLC chromatograph.
Kindly help me with your suggestions.
Are you sure you have a correct way of detection? Could it be that your substance absorbs insufficiently in UV? What is your HPLC column, is it the same C18?
Andrei Gavryushin: Same C18 column has been used in HPLC. I could detect the UV absorption in spectrometer but not in HPLC detection system.
Possibly the amount of your compound taken for HPLC is not enough to give a response of the detector? When you take it in larger amounts, as for normal chromatography, final concentration of the compound could be much higher than in the stream passing through the detector in HPLC. You can try to connect the autosampler directly to the detector without column, if you see your mixture by the detector, it means the compounds stay in the column. Otherwise this is the detection problem.
The retention can be decreased by adding instead of MeOH, ACN into the mobile phase to reduce the surface tension of water.
Very large molecules can have problems entering the pores of the stationary phase.
As mentioned by others, first make sure your compound does show up on the appropriate UV wavelength, load the appropriate amount of sample into the column (you don't need much for analytical column but more for semi prep or prep HPLC), and finally change the solvent system accordingly
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