Chiral Separation Of Phenylalanine Derivatives

Chiral high performance liquid chromatography is one of the most effective methods forthe enantioseparation. Both chiral stationary phase and chiral mobile phase method are used forchiral liquid chromatography. In this paper, ligand exchanged chiral mobile phase, crown chiralstationary phase and polysaccharide based chiral stationary phases are used for phenylaminederivatives enantioseparation. The main contents are described as follows:1.6of phenylalanine derivatives with substituent group on bezene ring were enantiomericseparated by ligand exchanged chrial mobile phase.Enantioseparation of six phenylalanine derivatives were investigated on C18column withcopper salts and chiral ligand in mobile phase. The results showed that all the six enantiomersare separated with this ligand exchanged method with good resolutions. The influences of thechromatographic conditions such as the type and concentration of copper（Ⅱ） salt and chiralligand, pH of the mobile phase, flow rate, column temperature and so on were investigated. Itwas shown that Dopa,3,5-diiodo-tyrosine, phenylalanine and4-F-phenylalanine enantiomerscould be separated using N,N-dimethyl-L-phenylalanine as chiral ligand, while4-Cl-phenylalanine and4-trifluoromethyl-phenylalanine using L-proine as chiral ligand. Thecomplexation equilibrium was affected by the ratio of the Cu²⁺and ligand amino acid. Themore higher concentration of binarycomplex was produced and more effective points wereprovided to form ternary complexes when the ratio of CuSO₄:N,N-DM-L-Phe got to1:3.5orCuSO₄:L-Pro got to1:4.0. The mobile phae pH needed to be controlled at3.5³.8as complexdissociation occured if pH was less than3.5and poor peak tailing and precipitate were observedif pH was more than3.8. Thermodynamic studies showed that the separation of theseenantiomers on the stationary phase were enthalpy controlled process. The lower columntemperature, the better enantiomer separation.3,5-diiodo-tyrosine method validation resultsindicated that the method was suitable to quantitative control of D enantiomer inL-3,5-diiodo-tyrosine as the results of resolution, linearity, sensitivity, precison, accuracy,robustness and solution stability meet the accept criteria.2.6of phenylalanine derivatives with substituent group on bezene ring were enantiomericseparated by crown ether chiral stationary phase.The chiral separation of six phenylalanine derivatives was also investigated on Crownpak CR（+） column with HClO₄solution with good resolutions. The influences of thechromatographic conditions such as the pH of the mobile phase, methanol content, flow rate,column temperature and so on were investigated. The polarity of mobile phase was decreasedand hydrophobic was enhanced when methanol content increased, which was good to R-NH₃⁺entering into18-crown-6-ring space, Then hydrogen bonding between R-NH₃⁺and Oxygen wasenhanced, to get better resolution. The higher pH solution, the more difficult to generate ofR-NH₃⁺, the less hrgrogen bonding and worse resolution. Thermodynamic studies showed thatthe separation of these enantiomers in the stationary phase were enthalpy controlled process.Better resolution and longer retention time observed when column temperature decreases.Method validation on3,5-diiodo-tyrosine showed that the method was suitable to quantitativecontrol the D-enantiomer in L-3,5-diiodo-tyrosine with good accuracy, precision, robustnessand solution stability.3.5of protected phenylalanine derivatives were enantiomeric separated by polysaccharidebased chiral stationary phaseThe enantiomeric separation of five protected phenylalanines was studied on ChiralPakAD-H. The results showed that all the five enantiomers were separated with good resolutionswith the following conditions: mobile phase was hexane,isopropanol and trifluoroacetic acid atthe ratio of90:10:0.2; column temperature is30℃; flow rate is0.8mL/min; detection wavelengthwas set at210nm and injection volume was10μL. The influence of the chromatographicconditions such as acidic additive, the type and concentration of the polar additive, flow rate,column temperature were investigated. Hydrogen bonding was the dominant element of theinteractions between enantiomers and CSP. Trifluoroacetic acid in mobile phase could enhancethe hydrogen bonding and enhance the chiral selectivity. Isopropanol as a polar additive inmobile phase was better than ethanol to enantiomeric separation. The separation of five kinds ofenantiomers in the stationary phase was an enthalpy controlled process. Lower columntemperature was good to enantiomeric separation. Method validation on Cbz-Phenylanineshowed that the method was suitable to quantitative control the D-enantiomer inL-Cbz-Phenylanine with good accuracy, precision, robustness and solution stability.