Not all C18 columns have identical retention properties. 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. This is followed by C8-bonded silica (L7), pure silica (元), cyano-bonded silica (L10) and phenyl-bonded silica (L11). The most popular column is an octadecyl carbon chain (C18)-bonded silica (USP classification L1). There are a variety of stationary phases available for use in RPC, allowing great flexibility in the development of separation methods.Īny inert polar substance that achieves sufficient packing can be used for reversed-phase chromatography. Today, RPC is a frequently used analytical technique. It is typically used for separation of proteins, because the organic solvents used in normal-phase chromatography can denature many proteins. It can be used for the separation of a wide variety of molecules. Many of the mathematical and experimental considerations used in other chromatographic methods also apply to RPC (for example, the separation resolution is dependent on the length of the column). 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. 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. 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. Reversed-phase chromatography employs a polar ( aqueous) mobile phase. 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 is a technique using alkyl chains covalently bonded to the stationary phase particles in order to create a hydrophobic stationary phase, which has a stronger affinity for hydrophobic or less polar compounds. 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. Hydrophobic molecules experience less of an affinity for the column packing, and will pass through to be eluted and detected first. Since the stationary phase is hydrophilic in this technique, molecules with hydrophilic properties contained within the mobile phase will have a high affinity for the stationary phase, and therefore will adsorb to the column packing. This type of technique is now referred to as normal-phase chromatography. Saving Earth Britannica Presents Earth’s To-Do List for the 21st Century.In the 1970s, most liquid chromatography was performed using a solid support stationary phase (also called a column) containing unmodified silica or alumina resins.Britannica Beyond We’ve created a new place where questions are at the center of learning.100 Women Britannica celebrates the centennial of the Nineteenth Amendment, highlighting suffragists and history-making politicians.
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