Submarine Gel Electrophoresis Purification of a Small Molecule:  99.9998 Fluorescently Pure IMI2

• Published in: Analytical chemistry (Washington) Vol. 70; no. 18; pp. 3978 - 3980
• Main Authors: Lan, Zhang-Hua, Shen, Xiaohang, Giese, Roger W
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15.09.1998
• Subjects: Analytical chemistry; Chemistry; Chromatographic methods and physical methods associated with chromatography; Exact sciences and technology; Fluorescence; Molecules; Other chromatographic methods; Imidazole derivatives; Separation method; Ultraviolet detector; Analysis method; Purification; Organic dye; Gel electrophoresis
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac980521t

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 5000× more concentrated sample than the original. The amount of dye purified on a 87- × 67- × 10-mm (l × w × 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, 3518−3520). More generally, we conclude that submarine gel electrophoresis can be a convenient and useful separation technique for purifying small molecules.