Hydrophilic interaction chromatography has gained tremendously in interest over the last decade and is now routinely used for complex separations in areas as different as medicinal chemistry, marine toxins, proteomics, and metabolomics. However, more fundamental theories explaining the factors that lead to retention and selectivity are still largely lacking. As opposed to reversed phase HPLC, selectivity in HILIC is mainly generated by the stationary phase, and as the number of commercially available columns with hydrophilic packings is continuously increasing, it is important to understand the fundamental mechanisms that lead to retention on stationary phases of various type. We have investigated a rather extensive array of columns with interactive layers of different type, and directly determined their water-binding capacity. This was in turn correlated with the retention for a select set of test solutes, in order to shed light on the mechanisms leading to retention. These and other experiments related to the mechanism of HILIC will be covered in the lecture.