Amino Acid Analysis by o-Phthaldialdehyde Precolumn Derivatization and Reverse-Phase HPLC

  • Barry Nelson Jones
Part of the Biological Methods book series (BM)


The ability to separate and quantitate amino acids is essential for the characterization and structural elucidation of polypeptides and proteins. However, there are several factors associated with amino acid analysis that present formidable problems for the protein chemist. In many instances, only a small amount of the polypeptide sample is available for analysis. Most amino acids possess no significant chromophores or fluorophores and must be quantitated in the presence of other substances that may interfere with the analysis. In addition, mixtures of amino acids with diverse functional groups and polarities have to be resolved and quantitated. Therefore, the chromatographic system used for amino acid analysis must possess high sensitivity, specificity, and chromatographic selectivity, as well as be simple to operate and yield reproducible results. For more than 20 yr the method of choice for amino acid analysis has been classical ion-exchange chromatography followed by postcolumn derivatization with either ninhydrin (1, 2, 3, 4) or a fluorogenic reagent (5, 6, 7, 8, 9). This method resolves most amino acids with good detection limits, especially if either o-phthaldialdehyde (OPA) or fluorescamine is used as the post-column derivatizing reagent. However, this procedure generally requires a dedicated liquid chromatography system and an analysis time of greater than 1 h.


High Performance Liquid Chromatography Amino Acid Analysis Amino Acid Derivative Derivatization Procedure Amino Acid Mixture 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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Copyright information

© The Humana Press Inc. 1986

Authors and Affiliations

  • Barry Nelson Jones
    • 1
  1. 1.Unigene Laboratories, Inc.Fairfield

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