<P> The polar analytes diffuse into a stationary water layer associated with the polar stationary phase and are thus retained . The stronger the interactions between the polar analyte and the polar stationary phase (relative to the mobile phase) the longer the elution time . The interaction strength depends on the functional groups part of the analyte molecular structure, with more polarized groups (e.g. hydroxyl -) and groups capable of hydrogen bonding inducing more retention . Coulombic (electrostatic) interactions can also increase retention . Use of more polar solvents in the mobile phase will decrease the retention time of the analytes, whereas more hydrophobic solvents tend to increase retention times . </P> <P> Normal--phase chromatography was one of the first kinds of HPLC that chemists developed . Also known as normal - phase HPLC (NP - HPLC) this method separates analytes based on their affinity for a polar stationary surface such as silica, hence it is based on analyte ability to engage in polar interactions (such as hydrogen - bonding or dipole - dipole type of interactions) with the sorbent surface . NP - HPLC uses a non-polar, non-aqueous mobile phase (e.g. Chloroform), and works effectively for separating analytes readily soluble in non-polar solvents . The analyte associates with and is retained by the polar stationary phase . Adsorption strengths increase with increased analyte polarity . The interaction strength depends not only on the functional groups present in the structure of the analyte molecule, but also on steric factors . The effect of steric hindrance on interaction strength allows this method to resolve (separate) structural isomers . </P> <P> The use of more polar solvents in the mobile phase will decrease the retention time of analytes, whereas more hydrophobic solvents tend to induce slower elution (increased retention times). Very polar solvents such as traces of water in the mobile phase tend to adsorb to the solid surface of the stationary phase forming a stationary bound (water) layer which is considered to play an active role in retention . This behavior is somewhat peculiar to normal phase chromatography because it is governed almost exclusively by an adsorptive mechanism (i.e. analytes interact with a solid surface rather than with the solvated layer of a ligand attached to the sorbent surface; see also reversed - phase HPLC below). Adsorption chromatography is still widely used for structural isomer separations in both column and thin - layer chromatography formats on activated (dried) silica or alumina supports . </P> <P> Partition - and NP - HPLC fell out of favor in the 1970s with the development of reversed - phase HPLC because of poor reproducibility of retention times due to the presence of a water or protic organic solvent layer on the surface of the silica or alumina chromatographic media . This layer changes with any changes in the composition of the mobile phase (e.g. moisture level) causing drifting retention times . </P>

A gradient elution in hplc is one in which the composition of the solvent