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Reversed-phase chromatography (RLC) column has high efficiency, strong separation ability and clear retention mechanism. It is one of the most widely used separation modes in liquid chromatography. RLC is attracting more and more attention for the separation and analysis of biological macromolecules, proteins and enzymes. Reversed-phase chromatography is a liquid chromatographic separation model in which the surface non-polar carrier is the stationary phase and the solvent with stronger polarity than the stationary phase is the mobile phase. The fixed phase of reversed-phase chromatography is mostly bonded hydrophobic groups on the surface of silica gel. In the separation of biological macromolecules, acidic aqueous solutions with low ionic strength are usually used, and some organic solvents, such as acetonitrile, isopropanol or methanol, are added as mobile phase.