Submarine gel electrophoresis purification of a small molecule: 99.9998% fluorescently pure IMI2

HPLC and related techniques failed to raise the fluorescence purity of a BODIPY-imidazole dye (IMI2) above 99.5%, based on assessment of dye purity by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). This difficulty was overcome by employing submarine gel electrophoresis, which raised the fluorescence purity level to about 99.9998%. This estimate of final dye purity was made by using immobilized metal affinity chromatography to selectively remove the dye from a sample that was then analyzed by CE-LIF as a 5000x more concentrated sample than the original. The amount of dye purified on a 87- x 67- x 10-mm (I x w x h) gel in a 40 min run was at the 0.5-mg level. The dye was recovered by soaking the sliced-out electrophoretic band in methanol, We are now in a position to employ this dye for labeling of trace phosphomonoester analytes without being hampered by dye impurities, by further developing methodology that we reported previously (Wang, P.; Giese, R. W. Anal. Chem. 1993, 65, 3518-3529). More generally, we conclude that submarine gel electrophoresis can be a convenient and useful separation technique for purifying small molecules.