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

  • Barry Nelson Jones
Protocol
Part of the Biological Methods book series (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, 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.

Keywords

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.

References

  1. 1.
    Moore, S., Spackman, D. H., and Stein, W. H. (1958) Anal Chem. 30, 1185–1206.CrossRefGoogle Scholar
  2. 2.
    Hamilton, P. B. and Anderson, R. A. (1959) Anal. Chem. 31, 1504–1510.CrossRefGoogle Scholar
  3. 3.
    Hamilton, P. B. (1963) Anal. Chem. 35, 2055–2064.CrossRefGoogle Scholar
  4. 4.
    Murayama, K. and Sugawara, T. (1981) J. Chromatogr. 244, 315–320.Google Scholar
  5. 5.
    Roth, M. (1971) Anal. Chem. 43, 880–882.PubMedCrossRefGoogle Scholar
  6. 6.
    Udenfriend, S., Stein, S., Bohlen, P., and Dairman, W. (1972) Science 237, 871–872.CrossRefGoogle Scholar
  7. 7.
    Benson, J. R. and Hare, P. E. (1975) Proc. Natl. Acad. Sci. USA 72, 619–622.PubMedCrossRefGoogle Scholar
  8. 8.
    Bohlen, P. and Mellet, M. (1979) Anal. Biochem. 94, 313–321.PubMedCrossRefGoogle Scholar
  9. 9.
    Bohlen, P. and Schroeder, R. (1982) Anal. Biochem. 126, 144–152.PubMedCrossRefGoogle Scholar
  10. 10.
    Schmidt, G. J., Olson, D. C., and Slavin, W. (1979) J. Liq. Chromatogr. 2, 1031–1042.CrossRefGoogle Scholar
  11. 11.
    Tapuhi, Y., Miller, N., and Karger, B. L. (1982) J. Chromatogr. 205, 325–336.CrossRefGoogle Scholar
  12. 12.
    Weidmeier, V. T., Porterfield, S. P., and Hendrich, C. E. (1982) J. Chromatogr. 231, 410–417.CrossRefGoogle Scholar
  13. 13.
    Dejong, C., Highes, G. J., Van Wieringen, E., and Wilson, K. J. (1982) J. Chromatogr. 241, 345–359.CrossRefGoogle Scholar
  14. 14.
    Lindroth, P. and Mopper, K. (1979) Anal Chem. 51, 1667–1674.CrossRefGoogle Scholar
  15. 15.
    Hodgin, J. C. (1979) J. Liq. Chromatogr. 2, 1047–1060.CrossRefGoogle Scholar
  16. 16.
    Gardner, W. S. and Miller, W. H. (1980) Anal. Biochem. 101, 61–65.PubMedCrossRefGoogle Scholar
  17. 17.
    Hill, D. W., Walters, F. H., Wilson, T. D., and Stuart, J. D. (1979) Anal Chem. 51, 1338–1341.PubMedCrossRefGoogle Scholar
  18. 18.
    Jones, B. N., Paabo, S., and Stein, S. (1981) J. Liq. Chromatogr. 4, 565–566.CrossRefGoogle Scholar
  19. 19.
    Larsen, B. R. and West, F. G. (1981) J. Chromatogr. Sci. 19, 259–265.Google Scholar
  20. 20.
    Turnell, D. C. and Cooper, J. D. H. (1982) Clin. Chem. 28, 527–531.PubMedGoogle Scholar
  21. 21.
    Hogan, D. L., Kraemer, K. L., and Isenberg, J. I. (1982) Anal. Biochem. 127, 17–24.PubMedCrossRefGoogle Scholar
  22. 22.
    Umagat, H., Kucera, P., and Wen, L. F. (1982) J. Chromatogr. 241, 324–334.Google Scholar
  23. 23.
    Burback, J. P. H., Prias, A., Leboville, J. L. M., Verhoef, J., and Witter, A. (1982) J. Chromatogr. 237, 339–343.CrossRefGoogle Scholar
  24. 24.
    Jones, B. N. and Gilligan, J. P. (1983) J. Chromatogr. 266, 471–482.PubMedCrossRefGoogle Scholar
  25. 25.
    Cooper, J. D. K, Lewis, M. T., and Turnell, D. C. (1984) J. Chromatogr. 285, 484–494.CrossRefGoogle Scholar
  26. 26.
    Jones, B. N. and Gilligan, J. P. (1983) Am. Biotech. Lab. 1, 46–51.Google Scholar
  27. 27.
    Fleury, M. O. and Ashley, D. V. (1983) Anal. Biochem. 133, 330–335.PubMedCrossRefGoogle Scholar
  28. 28.
    Cooke, N. H. C., Archer, B. C., Olsen, K., and Berick, A. (1982) Anal. Chem. 54, 2277–2280.CrossRefGoogle Scholar
  29. 29.
    Jones, B. N., Shively, J. E., Kilpatrick, D. L., Stern, A. S., Lewis, R. V., Kojima, K., and Udenfriend, S. (1982) Proc. Natl. Acad. Sci. USA 79, 2096–2100.PubMedCrossRefGoogle Scholar
  30. 30.
    Moore, S. (1963) J. Biol. Chem. 238, 235–240.Google Scholar
  31. 31.
    Lice, T. Y. and Chang, Y. H. (1971) J. Biol. Chem. 246, 2842–2848.Google Scholar
  32. 32.
    Jones, B. N., Vigna, R. A., Dwulet, F. E., Bogardt, R. A., Lehman, L. D., and Gurd, F. R. N. (1976) Biochemistry 15, 4418–4422.PubMedCrossRefGoogle Scholar
  33. 33.
    Rothgeb, T. M., Jones, B. N., Hayes, D. F., and Gurd, R. S. (1977) Biochemistry 16, 5813–5818.PubMedCrossRefGoogle Scholar
  34. 34.
    Garner, M. H., Garner, W. H., and Gurd, F. R. N. (1974) J Biol Chem. 249, 1513–1518.PubMedGoogle Scholar
  35. 35.
    Light, A. (1972) Methods Enzymol. 25B, 253–262.CrossRefGoogle Scholar
  36. 36.
    Simons, S. S. and Johnson, D. F. (1978) J. Org. Chem. 43, 2886–2891.CrossRefGoogle Scholar
  37. 37.
    Simpson, R. C., Spriggle, J. E., and Veening, H. (1983) J Chromatogr. 261, 407–414.CrossRefGoogle Scholar
  38. 38.
    Strobaugh, J. F., Repta, A. J., Sternson, L. A., and Garren, K. W. (1983) Anal Biochem. 135, 495–504.CrossRefGoogle Scholar
  39. 39.
    Cooper, J. D. H. and Turnell, D. C. (1982) J. Chromatogr. 227, 158–161.CrossRefGoogle Scholar
  40. 40.
    Venema, K., Leever, W., Bakker, J. O., Haayer, G., and Korf, J. (1983) J Chromatogr. 260, 371–376.PubMedCrossRefGoogle Scholar
  41. 41.
    Winspear, M. J. and Oaks, A. (1983) J. Chromatogr. 270, 378–382.CrossRefGoogle Scholar
  42. 42.
    Turnell, D. C. and Cooper, J. D. H. (1983) J Automatic Chem. 5, 36–39.Google Scholar
  43. 43.
    Simons, S. S. and Johnson, D. F. (1977) Anal Biochem. 82, 250–254.PubMedCrossRefGoogle Scholar
  44. 44.
    Kilpatrick, D. L., Jones, B. N., Lewis, R. V., Stern, A. S., Kojima, K., Shively, J. E., and Udenfriend, S. (1982) Proc Natl. Acad. Sci. USA 79, 3057–3061.PubMedCrossRefGoogle Scholar
  45. 45.
    Birnbaum, R. S., O’Neill, J. A., Muszynski, M., Aron, D. C., and Roos, B. A. (1982) J. Biol. Chem. 257, 241–244.PubMedGoogle Scholar
  46. 46.
    Birnbaum, R. S., Mahoney, W., and Roos, B. A. (1983) J Biol. Chem. 258, 5463–5466.PubMedGoogle Scholar
  47. 47.
    Wassner, S. J. and Li, J. B., (1982) J Chromatogr. 227, 497–502.PubMedCrossRefGoogle Scholar
  48. 48.
    Alfredsson, G. and Sedvall, G. (1983) J Chromatogr. 274, 325–330.PubMedCrossRefGoogle Scholar
  49. 49.
    Gray, W.R. (1972) Methods Enzymol 25B, 121–138.CrossRefGoogle Scholar
  50. 50.
    Levina, N. B. and Nazimov, I. V. (1984) J Chromatogr. 286, 207–216.CrossRefGoogle Scholar
  51. 51.
    Wolfram, J. K., Feinberg, J. I., Doerr, R. C., and Fiddler, W. (1977) J Chromatogr. 132, 37–43.PubMedCrossRefGoogle Scholar
  52. 52.
    Ahnoff, M., Grundavik, I., Arfwidsson, A., Fonselius, J., and Persson, B. (1981) Anal Chem. 53, 484–489.CrossRefGoogle Scholar
  53. 53.
    Imai, K. and Watanabe, Y. (1981) Anal Chim. Acta 130, 377–383.CrossRefGoogle Scholar
  54. 54.
    Watanabe, Y. and Imai, K. (1981) Anal Biochem. 116, 471–472.PubMedCrossRefGoogle Scholar
  55. 55.
    Watanabe, Y. and Imai, K. (1982) J Chromatogr. 239, 723–732.CrossRefGoogle Scholar
  56. 56.
    Einarsson, S., Josefsson, B., and Lagarkvist, S. (1983) J Chromatogr. 282, 609–618.CrossRefGoogle Scholar
  57. 57.
    Chang, J.-Y., Knecht, R., and Braun, D. G. (1981) Biochem J. 199, 547–553.PubMedGoogle Scholar
  58. 58.
    Lin, J.-K. and Lai, C.-C. (1982) J Chromatogr. 227, 369–377.PubMedCrossRefGoogle Scholar
  59. 59.
    Heinrikson, R. L and Meredith, S. C. (1984) Anal Biochem. 136, 65–74.PubMedCrossRefGoogle Scholar
  60. 60.
    Bidlingmeyer, B. A., Cohen, S. A., and Tarvin, T. L. (1984) J Chromatogr. 336, 93–104.PubMedCrossRefGoogle Scholar

Copyright information

© The Humana Press Inc. 1986

Authors and Affiliations

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

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