Salt-dependent purification of green fluorescent protein via hydrophobic interaction chromatography

Green fluorescent protein, GFP, is a widely used fluorescent reporter, and its purification provides a clear model for studying downstream bioprocessing. This thesis investigates how binding-buffer salt strength affects GFP retention and elution during hydrophobic interaction chromatography, HIC, by comparing three conditions (1.0×, 0.8×, and 0.6×). GFP-expressing E. coli were lysed and clarified, and the lysate was purified on HIC columns; collected fractions were evaluated qualitatively under UV illumination to track fluorescence distribution. Results showed a salt-dependent trend in which higher salt improved retention and shifted fluorescence toward later elution fractions, while reduced salt weakened hydrophobic interactions and increased earlier release. These findings demonstrate how a single controllable parameter can tune HIC separation behavior and will be used to develop a graduate-level protein purification laboratory protocol at the University of Tennessee at Chattanooga.