Mixed-mode HPLC uses several retention mechanisms within a single column; reversed phase, ion exchange, and HILIC. Mixed-mode HPLC can be incredibly useful when separating mixtures of hydrophobic, hydrophilic, and charged compounds that may not be separated using other modes of chromatography. This module discusses the primary modes of retention which must be understood to successfully develop mixed-mode separations. Each method parameter, how it is altered, and the effect it has on the resulting chromatography is discussed. We will also provide you with a method development strategy which can be applied to separations.