Hydrophobic interaction chromatography was first developed in the early 1970s for the separation of proteins by using columns packed with appropriately modified agarose beads. The hydrophobic interaction parameter was found to be a linear function of the protein surface area and the molal surface tension increment of the salt in the eluant, in accordance to the theory. The silanol groups at the surface of silica serve as convenient anchor groups for covalent binding of almost any desired functional groups via siloxane bridges by using appropriate silanizing agents. The mobile phases in Reversed-phase chromatography are typically hydro-organic mixtures containing methanol, isopropanol, acetonitrile, or tetrahydrofuran as the organic “modifier.” The influence of residence time on bovine and human growth hormone conformation in reversed-phase gradient elution was studied by online spectroscopic analysis and significant time-dependent changes were observed in the spectroscopic properties of the protein.