Advertisement

Amino Acid Analysis by High Performance Liquid Chromatography of Phenylthiocarbamyl Derivatives

  • Tomas Bergman
  • Mats Carlquist
  • Hans Jörnvall

Abstract

Reliable and sensitive amino acid analyses are important steps in studies of protein structures. In this respect, high performance liquid chromatography (HPLC) has greatly increased speed and sensitivity. The use of ortho-phthalaldehyde [1] and subsequent fluorimetric detection is applicable also to HPLC. Thus, separation of underivatized amino acids by ion exchange HPLC and subsequent detection by post-column derivatization [2], as well as pre-column derivatization and subsequent separation of amino acid derivatives by reverse phase HPLC have been widely used. We have tested both methods and find, like others, that they are suitable. However, base line drift due to ammonia contamination is a serious problem when maximal sensitivity is attempted in the post-column mode of the ortho-phthalaldehyde method. Similarly, the lack of direct detection of proline requires one further step in this derivatization procedure [3] both in the pre- and post-column derivatization mode. Therefore, additional methods for analysis are of value. In this respect, pre-column derivatization with phenylisothiocyanate (PITC) to produce the phenylthiocarbamyl (PTC)-amino acids for subsequent separation by reverse phase HPLC has proved highly efficient and valuable [4, 5]. We have tested this method extensively and find it reliable, sensitive, and easy to use.

Keywords

High Performance Liquid Chromatography Amino Acid Derivative Reverse Phase High Performance Liquid Chromatography Coagulation Factor VIII Artifact Peak 
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.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Roth M (1971) Anal Chem 43:880–882.PubMedCrossRefGoogle Scholar
  2. 2.
    Klapper DG (1982) In: Elzinga M (ed) Methods in protein sequence analysis. Humana, Clifton, NJ, pp 509–515.CrossRefGoogle Scholar
  3. 3.
    Bohlen P, Mellet M (1979) Anal Biochem 94:313–321.PubMedCrossRefGoogle Scholar
  4. 4.
    Bidlingmeyer BA, Cohen SA, Tarvin TL (1984) J Chromatogr 336:93–104.PubMedCrossRefGoogle Scholar
  5. 5.
    Heinrikson RL, Meredith SC (1984) Anal Biochem 136:65–74.PubMedCrossRefGoogle Scholar
  6. 6.
    Koop DR, Morgan ET, Tarr GE, Coon MJ (1982) J Biol Chem 257:8472–8480.PubMedGoogle Scholar
  7. 7.
    Chang J-Y, Knecht R, Braun DG (1981) Biochem J 199:547–555.PubMedGoogle Scholar
  8. 8.
    Jörnvall H, Kalkkinen N, Luka J, Kaiser R, Carlquist M, von Bahr-Lindström H (1983) In: Tschesche H (ed) Modem methods in protein chemistry, de Gruyter, Berlin, pp 1–19.Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1986

Authors and Affiliations

  • Tomas Bergman
    • 1
  • Mats Carlquist
    • 1
  • Hans Jörnvall
    • 1
  1. 1.Departments of Chemistry I and Biochemistry IIKarolinska InstitutetStockholmSweden

Personalised recommendations