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

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Methods of Protein Microcharacterization

Part of the book series: Biological Methods ((BM))

Abstract

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 (1–4) or a fluorogenic reagent (5–9). 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.

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Author information

Authors and Affiliations

  1. Unigene Laboratories, Inc., Fairfield, New Jersey

    Barry Nelson Jones

Authors
  1. Barry Nelson Jones
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Editor information

Editors and Affiliations

  1. Beckman Research Institute of the City of Hope, Duarte, California

    John E. Shively

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© 1986 The Humana Press Inc.

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Jones, B.N. (1986). Amino Acid Analysis by o-Phthaldialdehyde Precolumn Derivatization and Reverse-Phase HPLC. In: Shively, J.E. (eds) Methods of Protein Microcharacterization. Biological Methods. Humana Press. https://doi.org/10.1007/978-1-59259-436-8_5

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  • DOI: https://doi.org/10.1007/978-1-59259-436-8_5

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-090-9

  • Online ISBN: 978-1-59259-436-8

  • eBook Packages: Springer Book Archive

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