Chemo- and enantio-selective method for the analysis of amino acids by capillary electrophoresis with in-capillary derivatization

A novel dual chiral CE method was developed for the separation of l- and d-amino acids (AAs), using in-capillary derivatization with 9-fluoroenylmethyl chloroformate (FMOC). Firstly, using pre-column derivatization, the enantioseparation of FMOC–AAs was optimized according to the nature of cyclodextrins (CD). A background electrolyte (BGE) composed of 30 mM β-CD, 30 mM octakis(2,3-dihydroxy-6-O-sulfo)-γ-CD (OS-γ-CD), 40 mM tetraborate and 15% isopropanol (IPA) was selected and led to 17 baseline resolved pairs (Rs = 1.7–5.8) and two partially resolved pairs (Lys, Rs = 0.5 and Arg, Rs = 1.2). Experimental conditions for in-capillary derivatization were then optimized. Several parameters, such as mixing voltage and time, concentration of labeling solution and the length of the spacer plug were studied. The optimal conditions for in-capillary derivatization procedure were obtained using successive hydrodynamic injections (30 mbar) of AAs for 2 s, borate buffer for 4 s and 10 mM FMOC solution for 6 s, followed by a mixing at 3 kV for 72 s and wait time of 1 min. Moreover, a particular attention was paid to improve separation chemoselectivity. The effect on stereoselectivity and chemoselectivity of different factors, such as decrease of pH and tetraborate concentration and the addition of sodium dodecyl sulfate (SDS), was investigated using the in-capillary derivatization procedure. The best separation of a standard mixture of ten AA racemates was observed using a BGE containing 30 mM β-CD, 30 mM OS-γ-CD, 25 mM SDS, 40 mM sodium tetraborate and 17% IPA.

► Careful optimization leading to fast in-capillary derivatization of amino acids.
► Baseline enantioseparation of 17 amino acid pairs.
► MEKC conditions for the simultaneous separation of 10 amino acid pairs.