Difluorocarboxyfluorescein NHS Ester, 6-isomer, is a fluorescent analogue of fluorescein that reacts with amines. It addresses several limitations of fluorescein, offering enhanced photostability and a lower pKa (~4.7 compared to fluorescein's 6.4), resulting in fluorescence that remains largely pH-insensitive within physiological ranges. Although mixed isomers of difluorocarboxyfluorescein NHS ester are often used as routine fluorescent dyes for labeling proteins, peptides, and nucleotides, purifying peptides and nucleotides labeled with the 5(6) isomer can be challenging due to signal broadening during HPLC purification. Peptides and nucleotides labeled with a single isomer typically show improved resolution in HPLC purification, which is often necessary during conjugation processes.