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Reversed-phase chromatography (also called RPC, reverse-phase chromatography, or hydrophobic chromatography) includes any
chromatographic In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it through a system (a ...
method that uses a hydrophobic stationary phase. RPC refers to liquid (rather than gas) chromatography.


Stationary phases

In the 1970s, most
liquid chromatography In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it through a system (a ...
was performed using a solid support stationary phase (also called a column) containing unmodified
silica Silicon dioxide, also known as silica, is an oxide of silicon with the chemical formula , most commonly found in nature as quartz and in various living organisms. In many parts of the world, silica is the major constituent of sand. Silica is ...
or alumina resins. This type of technique is now referred to as
normal-phase chromatography High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pa ...
. Since the stationary phase is
hydrophilic A hydrophile is a molecule or other molecular entity that is attracted to water molecules and tends to be dissolved by water.Liddell, H.G. & Scott, R. (1940). ''A Greek-English Lexicon'' Oxford: Clarendon Press. In contrast, hydrophobes are ...
in this technique, molecules with hydrophilic properties contained within the
mobile phase In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent; as in washing of loaded ion-exchange resins to remove captured ions. In a liquid chromatography experiment, for exa ...
will have a high affinity for the stationary phase, and therefore will adsorb to the column packing.
Hydrophobic In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water. Hydrophobic molecules tend to be nonpolar and, t ...
molecules experience less of an affinity for the column packing, and will pass through to be
eluted In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent; as in washing of loaded ion-exchange resins to remove captured ions. In a liquid chromatography experiment, for exam ...
and detected first. Elution of the hydrophilic molecules adsorbed to the column packing requires the use of more hydrophilic or more polar solvents in the mobile phase to shift the distribution of the particles in the stationary phase towards that of the mobile phase. Reversed-phase chromatography is a technique using
alkyl In organic chemistry, an alkyl group is an alkane missing one hydrogen. The term ''alkyl'' is intentionally unspecific to include many possible substitutions. An acyclic alkyl has the general formula of . A cycloalkyl is derived from a cycloa ...
chains covalently bonded to the stationary phase particles in order to create a
hydrophobic In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water. Hydrophobic molecules tend to be nonpolar and, t ...
stationary phase, which has a stronger affinity for hydrophobic or less polar compounds. The use of a hydrophobic stationary phase is essentially the reverse of normal phase chromatography, since the polarity of the mobile and stationary phases have been inverted – hence the term reversed-phase chromatography. Reversed-phase chromatography employs a polar (
aqueous An aqueous solution is a solution in which the solvent is water. It is mostly shown in chemical equations by appending (aq) to the relevant chemical formula. For example, a solution of table salt, or sodium chloride (NaCl), in water would be re ...
) mobile phase. As a result, hydrophobic molecules in the polar mobile phase tend to adsorb to the hydrophobic stationary phase, and hydro''philic'' molecules in the mobile phase will pass through the column and are eluted first. Hydrophobic molecules can be eluted from the column by decreasing the polarity of the mobile phase using an organic (non-polar) solvent, which reduces hydrophobic interactions. The more hydrophobic the molecule, the more strongly it will bind to the stationary phase, and the higher the concentration of organic solvent that will be required to elute the molecule. Many of the mathematical and experimental considerations used in other chromatographic methods also apply to RPC (for example, the separation
resolution Resolution(s) may refer to: Common meanings * Resolution (debate), the statement which is debated in policy debate * Resolution (law), a written motion adopted by a deliberative body * New Year's resolution, a commitment that an individual mak ...
is dependent on the length of the column). It can be used for the separation of a wide variety of molecules. It is typically used for separation of proteins, because the organic solvents used in normal-phase chromatography can denature many proteins. Today, RPC is a frequently used analytical technique. There are a variety of stationary phases available for use in RPC, allowing great flexibility in the development of separation methods.


Silica-based stationary phases

Any inert polar substance that achieves sufficient packing can be used for reversed-phase chromatography. The most popular column is an octadecyl carbon chain (C18)-bonded silica (USP classification L1). This is followed by C8-bonded silica (L7), pure silica (L3), cyano-bonded silica (L10) and phenyl-bonded silica (L11). Note that C18, C8 and phenyl are dedicated reversed-phase resins, while cyano columns can be used in a reversed-phase mode depending on analyte and mobile phase conditions. Not all C18 columns have identical retention properties. 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). While the overall retention mechanism remains the same, subtle differences in the surface chemistries of different stationary phases will lead to changes in selectivity. Modern columns have different polarity. PFP is pentafluorphenyl. CN is cyano. NH2 is amino. ODS is octadecyl or C18. ODCN is a mixed mode column consisting of C18 and nitrile. SCX is strong cationic exchange (used for separation of organic amines). SAX is strong anionic exchange (used for separation of carboxylic acid compounds).


Mobile phases

Mixtures of water or aqueous buffers and organic solvents are used to elute analytes from a reversed-phase column. The solvents must be miscible with water, and the most common organic solvents used are acetonitrile, methanol, and
tetrahydrofuran Tetrahydrofuran (THF), or oxolane, is an organic compound with the formula (CH2)4O. The compound is classified as heterocyclic compound, specifically a cyclic ether. It is a colorless, water- miscible organic liquid with low viscosity. It is ...
(THF). Other solvents can be used such as ethanol or 2-propanol (isopropyl alcohol). Elution can be performed isocratically (the water-solvent composition does not change during the separation process) or by using a solution gradient (the water-solvent composition changes during the separation process, usually by decreasing the polarity). The pH of the mobile phase can have an important role on the retention of an analyte and can change the selectivity of certain analytes. Charged analytes can be separated on a reversed-phase column by the use of ion-pairing (also called ion-interaction). This technique is known as reversed-phase ion-pairing chromatography.


See also

* Aqueous normal-phase chromatography


References


External links


Tables
summarizing different types of reverse phases, and information on the functionalization process {{chromatography Chromatography