Indirect separation of chiral proteinogenic α-amino acids using the fluorescence active (1 R,2 R)-N-[(2-isothiocyanato)cyclohexyl]-6-methoxy-4-quinolinylamide) as chiral derivatizing agent : A comparison

Abstract

Enantiomerically pure trans-1,2-diaminocyclohexane (DACH) based (1 R,2 R)- and (1 S,2 S)-N-[(2-isothiocyanato)cyclohexyl]-6-methoxy-4-quinolinylamide (( R, R)- and ( S, S)-CDITC) was designed as a new fluorescence tagging chiral derivatizing agent (CDA) for the separation of amino acids and chiral amines. The derivatization of amino acids with CDITC yielded highly fluorescent diastereomeric thioureas. These derivatives were separated on a non-chiral RP-HPLC system employing C 18 as stationary phase. The respective optimum separation condition were evaluated and the optimum pH was found to be 4.30. The α-value achieved ranged from 1.14 to 3.16 with peak resolutions ranging between 0.59 and 13.94. The fluorescent labeled amino acids were detected at λ ex=333 nm, λ em=430 nm. These fluorescence properties almost matched the nitrogen laser line of 335 nm making laser-induced fluorescence detection (LIF) possible. The HPLC separations were compared to quasi capillary electrochromatographic (CEC) measurements of the same diastereomers using polyvinyl pyrolidone (PVP) as pseudo-stationary phase. All 19 chiral proteinogenic amino acids were separated. The selectivity values ranged between 1.026 and 1.232 with R s values from 1.37 to 16.25. Detection was achieved by a diode array detector. The effect of PVP concentration, pH, temperature and organic modifier on the separations was evaluated. The optimum CEC separation conditions were found to be 20 m M sodium citrate (pH 3), 0.5% (w/v) PVP, 9% 2-propanol and 1% tert.-butylmethyl ether.