Postcolumn Derivatizations of Amino Acids

Even today, the postcolumn derivatization with ninhydrin introduced by Spademan, Stein, and Moore [29] represents the most common detection method for quantitative amino acid analysis. As a strong oxidant, ninhydrin reacts with the a-amino groups of eluting amino acids at temperatures around 130 °C, according to Eq. (5.3), releasing carbon dioxide. 

To generate the ninhydrin chromophore, primary amino acids are condensed with a molecule of ninhydrin to give a Schiff base. The ketimine formed undergoes decarboxylation yielding an aldehyde and an intermediate amine. Condensation of this intermediate amine with another molecule of ninhydrin follows to form a red dye called Ruhemann s Purple. This dye has an absorption maximum at 570 nm (see Eigure 5.33). 

Secondary amino acids, the so-called imino acids such as proline and hydroxyproline, do not possess an a-amino group and react with ninhydrin to form a yellow product that is usually detected at 440 nm. Therefore, amino acid analyzers are equipped with a photometric detector capable of measuring at two different wavelengths (570 and 440 nm). The sensitivity of this detection method for primary amino acids is about 30 pmol. 

The subsequent fluorescence detection is carried out at an excitation wavelength of 340 nm the emission is measured at 455 nm. The addition of thiols such as 2-mercaptoethanol or A/ ,Af-dimethyl-2-mercaptoethylamine (Thiofluor , Pickering Laboratories, Mountain View, CA, USA) increases the fluorescence yield. However, OPA only reacts with primary amino acids. Secondary amino acids can be detected only after their oxidation (e.g., with hypochlorite or chloramine T) [50]. In practical applications, this creates significant difficulties. 

An interesting derivatization method was described by Jenke and Brown [51]. They mixed the column effluent with a buffered solution of 5,5 -dithiobis(2- 

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OPA reacts with thiols and amines to form highly fluorescent isoindole compounds as described in the section on Amino group . The analytical methods using OPA have been reported for pre- and postcolumn derivatization of amino acids. These methods are very sensitive and afford detection as low as 25 fmol. 

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