Reversed-phase chromatography
[1][2][3] The vast majority of separations and analyses using high-performance liquid chromatography (HPLC) in recent years are done using the reversed phase mode.
The mobile phases are mixtures of water and polar organic solvents, the vast majority of which are methanol and acetonitrile.
[6] In the 1970s, most liquid chromatography runs were performed using solid particles as the stationary phases, made of unmodified silica gel or alumina.
At the same time hydrophobic molecules experience less affinity to the polar stationary phase, and elute through it early with not enough retention.
This was the reasons why during the 1970s the silica based particles were treated with hydrocarbons, immobilized or bonded on their surface, and the mobile phases were switched to aqueous and polar in nature, to accommodate biomedical substances.
Surface functionalization of silica can be performed in a monomeric or a polymeric reaction with different short-chain organosilanes used in a second step to cover remaining silanol groups (end-capping).
[22] Recent developments in chromatographic supports and instrumentation for liquid chromatography (LC) facilitate rapid and highly efficient separations, using various stationary phases geometries.
[23] Various analytical strategies have been proposed, such as the use of silica-based monolithic supports, elevated mobile phase temperatures, and columns packed with sub-3 μm superficially porous particles (fused or solid core)[24] or with sub-2 μm fully porous particles for use in ultra-high-pressure LC systems (UHPLC).
[25] A comprehensive article on the modern trends and best practices of mobile phase selection in reversed-phase chromatography was published by Boyes and Dong.
This is a crucial property for common reversed phase chromatography since sample components are typically detected by UV detectors.
Acetonitrile is more transparent than the others in low UV wavelengths range, therefore it is used almost exclusively when separating molecules with weak or no chromophores (UV-VIS absorbing groups), such as peptides.
Conversely, using a mobile phase at a pH lower than 4[31] will increase their retention, because it will decrease their ionization degree, rendering them less polar.
The same considerations apply to substances containing basic functional groups, such as amines, whose pKa ranges are around 8 and above, are retained more, as the pH of the mobile phase increases, approaching 8 and above, because they are less ionized, hence less polar.
[32] The choice of buffer type is an important factor in RP-LC method development, as it can affect the retention, selectivity, and resolution of the analytes of interest.
