Chromatography is a technique used across scientific disciplines to separate chemical mixtures. This method involves passing a mixture through a system consisting of two fundamental components: a mobile phase and a stationary phase. The mobile phase is a liquid or gas that carries the sample, while the stationary phase is a fixed solid material within a column or on a surface. The polarity of the stationary phase is the most important factor determining how a complex mixture is resolved into its individual components.
The Fundamental Rule of Chemical Separation
Chromatography depends on a competition between the mobile and stationary phases for the sample components. This competition is governed by the principle of “like attracts like,” meaning substances with similar polarities preferentially interact. For example, a polar compound is strongly attracted to a polar stationary phase, while a nonpolar compound prefers a nonpolar mobile phase.
Components spend more time interacting with the phase whose polarity they match, slowing their progress through the system. This retention causes separation, as different components travel at different speeds. The relative time a compound spends in the stationary phase determines its retention time, the key measurement used to identify separated substances.
Normal Phase Chromatography
Normal Phase (NP) chromatography uses a highly polar stationary phase. Materials like bare silica or alumina are used, featuring hydroxyl or other polar groups on their surface. The mobile phase is chosen to be comparatively nonpolar, often consisting of solvents such as hexane or nonpolar organic liquids.
In this setup, polar analytes form strong interactions, such as hydrogen bonds, with the polar stationary phase. These attractions cause polar compounds to be retained longer and elute later. Conversely, nonpolar analytes have little affinity for the stationary phase and are rapidly carried through the system by the nonpolar mobile phase, eluting first. This technique is effective for separating compounds of low to intermediate polarity that are not water-soluble.
Reversed Phase Chromatography
Reversed Phase (RP) chromatography uses a nonpolar stationary phase and a polar mobile phase. This configuration is the most widely used form of liquid chromatography today, especially in high-performance liquid chromatography (HPLC). The nonpolar stationary phase is created by chemically bonding hydrophobic carbon chains, such as C18 or C8, onto a silica backbone.
The mobile phase is polar, often a mixture of water or an aqueous buffer combined with a polar organic solvent like methanol or acetonitrile. Nonpolar analytes are retained longer because they are strongly attracted to the nonpolar stationary phase through hydrophobic interactions. Polar analytes prefer the polar mobile phase, passing through the column quickly and eluting earlier. This method is highly versatile, offering superior reproducibility for analyzing a vast range of compounds, including pharmaceuticals and biomolecules.
Choosing Between Polar and Nonpolar Stationary Phases
The decision to use a polar (Normal Phase) or nonpolar (Reversed Phase) stationary phase depends primarily on the chemical characteristics of the mixture. The chromatographer selects the phase that provides the best separation. For instance, if the compounds are highly water-soluble and polar, the nonpolar stationary phase of Reversed Phase is the preferred choice.
If a mixture contains less polar compounds or isomers requiring specific resolution, Normal Phase chromatography may be selected. Other practical factors, such as the sample’s solubility, compound stability in the mobile phase, and compatibility with the detection method, also influence the final selection.