Abstract
The adsorption and elution of proteins in reversed-phase liquid chromatographic (RPLC) systems were investigated by visually observing the retention and migration of colored and non-colored proteins in glass columns, packed with RPLC matrices. This system permits direct observation and measurement of the retention characteristics of colored proteins under various isocratic and gradient conditions. The system vividly illustrates that in organic-lean acidic eluents ( e.g., aq. 0.1 % trifluoroacetic acid), proteins are adsorbed on the closest available hydrophobic surface of the support without displacing any noticeable amount of previously injected proteins. When the eluent contains a moderate amount of organic solvent ( e.g., aq. 20% acetonitrile), proteins displace previously injected proteins of lesser hydrophobicity but do not displace previously injected proteins of greater hydrophobicity. These observations support the view that proteins are adsorbed on RPLC matrices in a monolayer. Observation of color-protein migration in glass columns during isocratic elution provides a practical means for determining very long retention times at very low mobile phase strength as well as the acceleration of protein migration during gradient elution. Our observations under such conditions agree with the retention relationships described by the chromatography theory for small molecules.
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