1.2.3. HPLC of amino acids as o-phthalaldehyde derivatives

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Summaryo1.Based on stoichiometric and mass spectrometric studies of the OPA derivatives of AAs the nightmare of their instability has been solved.2.It was confirmed, for the first time, that the common characteristic of the believed to be less stable OPA derivatives is associated with their initial molecular structure: they do contain the -NH 2 -CH 2 -R moiety and all of them furnish more than one OPA derivative.3.In the light of the common structure of the multiple OPA derivatives providing compounds a plausible reaction pathway has been described. According to the proposed mechanism hydrogen atoms of the -CH 2 - group, neighbor to the primary amino group, play the key role in the reaction with an additional molecule of OPA: resulting in consecutive OPA-derivatives transformed from the initially obtained species.4.As analytical consequences, the author of this review, based on recent experience, takes the responsibility to state: OPA derivatives of AAs are very much more stable than they are generally believed to be, considering that some of them are providing more than one reaction product (For the time being the successively formed products have not been taken into account). Consequently, in the HPLC analysis of AAs the following requirements are to be followed: (i) the molar ratios of the OPA/SH-group additive/AA should be close to 20/60/1, (ii) the reagents, saved in the refrigerator at ≤4°C, should be at least 90 min and maximum 9 days old, (iii) the reaction time between the OPA reagent and the AAs should be at least 7 min, (iv) the OPA derivatives should not be acidified before loading them onto the chromatographic column, (v) the HPLC elution procedure should be proper for the separation and quantitation of all components formed, including the more than one OPA derivative providing AAs, (vi) last but not least, in order to be able to get reliable AA contents, in particular when working in the low pM level, the blank value of the reagent should be carried out at least every day and the impurity values are to be substracted from the coeluting AA derivative.

Original languageEnglish
Title of host publicationJournal of Chromatography Library
Number of pages36
Publication statusPublished - 2005

Publication series

NameJournal of Chromatography Library
ISSN (Print)0301-4770


ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Filtration and Separation
  • Clinical Biochemistry

Cite this

Molnár-Perl, I. (2005). 1.2.3. HPLC of amino acids as o-phthalaldehyde derivatives. In Journal of Chromatography Library (C ed., pp. 163-198). (Journal of Chromatography Library; Vol. 70, No. C). Elsevier. https://doi.org/10.1016/S0301-4770(05)80008-9