Separation of 9-Fluorenylmethyloxycarbonyl Amino Acid Derivatives in Micellar Systems of High-Performance Thin-Layer Chromatography and Pressurized Planar Electrochromatography

Abstract

The problems with separation of amino acid mixtures in reversed-phase mode are the result of their hydrophilic nature. The derivatisation of the amino group of mentioned above solutes leads to their solution. For this purpose, 9-fluorenylmethoxycarbonyl chloroformate (f-moc-Cl) as the derivatisation reagent is often used. In our study, the separation of some f-moc- amino acid derivatives (alanine, phenylalanine, leucine, methionine, proline and tryptophan) with the use of micellar systems of reversed-phase high-performance thin-layer chromatography (HPTLC) and pressurized planar electrochromatography (PPEC) is investigated. The effect of surfactant concentration, its type (anionic, cationic and non-ionic) and mobile phase buffer pH on the discussed above solute migration distances are presented. Our work reveals that the increase of sodium dodecylsulphate concentration in the mobile phase has a different effect on solute retention in HPTLC and PPEC. Moreover, it also affects the order of solutes in both techniques. In PPEC, in contrast to the HPTLC technique, the mobile phase pH affects solute retention. The type of surfactant in the mobile phase also impacts solute retention and migration distances. A mobile phase containing SDS improves system efficiency in both techniques. Herein, such an effect is presented for the first time.

Figure 1

The effect of concentration of sodium dodecylsulphate in the mobile phase on the retention (HPTLC technique, (a)) and migration distances of f-moc amino acid derivatives (PPEC technique (b)). The mobile phase: acetonitrile (45%) and an aqueous acetic buffer of pH 4.25 (acetic acid and sodium acetate 7 and 3 mM, respectively). The stationary phase: HPTLC RP-18W. TLC experiment time − 15 min; PPEC polarization voltage 800 V, experiment time − 8 min.

Figure 2

Separation of f-moc Trp with the use of HPTLC (eluent without SDS – (a); eluent with SDS – (c)) and PPEC (eluent without SDS – (c); eluent with SDS – (d)). The mobile phase: 40% acetonitrile, aqueous universal buffer of pH 2.5 (5% v/v), system with surfactant 15 mM SDS. Polarisation voltage in PPEC: 1.5 kV, experiment time 15 min.

Figure 3

Separation of test mixtures with the use of HPTLC (mixture 1 – (a); mixture 2 - (b)) and PPEC (mixture 1 – (c); mixture 2 – (d)). The mobile phase: 40% acetonitrile, aqueous universal buffer of pH 2.5 (5% v/v), 15 mM SDS. Polarisation voltage in PPEC: 1.5 kV, experiment time 15 min. The order for mixture 1 in HPTLC: 1 - f-moc Trp + f- moc – Met; 2 - f-moc Pro; 3- f-moc Ala; 4- f-moc Cit; in PPEC: 1- f-moc Trp; 2 - f-moc Met; 3- f-moc Pro; 4 – f-moc Ala; 5- f-moc Cit. The order for mixture 2 in HPTLC: 1 - f-moc Met; 2- f- moc –Phe + f-moc Pro; 3- f-moc Ala; 4- f-moc Cit; in PPEC: 1- f-moc Phe; 2- f-moc Pro; 3- f-moc Leu; 4 – f-moc Ala; 5- f-moc Cit.