Reversed-phase chromatography general

The surface of microparticulate silica can be modified by the attachment of different groups to produce bonded phases. Reverse phase chromatography using bonded phases is generally faster and easier than other modes, and consequently has achieved very wide popularity. 

Ellgehausen, H., D Hondt, C., Fuerer, R. (1981) Reversed-phase chromatography as a general method for determining octan-l-ol/ water partition coefficients. Pestic. Sci. 12, 219-227. 

Reverse-phase chromatography may also be used to separate proteins on the basis of differential hydrophobicity. This technique involves applying the protein sample to a highly hydrophobic column to which most proteins will bind. Elution is promoted by decreasing the polarity of the mobile phase. This is normally achieved by the introduction of an organic solvent. Elution conditions are harsh and generally result in denaturation of many proteins. 

The extent to which the ions compete with B for the charged sites (X) will determine their retention. In general, this type of chromatography may be used to separate ionic species, such as organic acids or bases, which can be ionized under certain pH conditions. Besides the reaction with ionic sites on the stationary phase, retention may also be affected by the partitioning of solutes between the mobile and stationary phases, as in reversed-phase chromatography. Thus, even nonionized solutes may be retained on ion-exchange columns. 

Since amino acids and nucleotides are all polar and hydrophilic, they will be eluted quickly by the column. The mobile phase (see below) is also selected on the basis of polarity, with a medium- to high-polarity solvent required. The opposite of reverse phase chromatography is normal phase, where the column packing is medium to high polarity and the mobile phase is nonpolar. This technology is generally not applied to the analysis of polar molecules such as amino acids or nucleotides. Some peptides are more hydrophobic, making this method potentially more useful for peptides than for amino acids or nucleotides. 

General Characteristics of Reversed-Phase Chromatography Separations of... 

What are the general steps in developing an isocratic separation for reversed-phase chromatography with one organic solvent ... 

---