The purpose of this research was to explore a convenient method for bulk separations of volatile fatty acid (VFA) mixtures in dilute aqueous solution, and to develop new chiral stationary phases (CSPs) for enantiomeric separations by high-performance liquid chromatography (HPLC).
Direct separations of acetic, propionic, and butyric acids in dilute water solution by azeotropic distillation were demonstrated. Despite of its highest boiling point among these three acids, the butyric acid is the first acid to distill, followed by propionic acid. Over half of acetic acid still remains in the pot even when the other two acids are gone. Quantitative carbon-13 nuclear magnetic resonance (NMR) was employed as an effective and accurate detecting method for the acid mixtures. A comparison of short (1 second) vs. long (30 seconds) delay (D1) between successive scans was made. The effects of a paramagnetic reagent, disodium (diethylenetriaminepentaacetato)iron(III) (Na2[Fe(DTPA)]), and its concentration on the quantitative carbon-13 analysis was also discussed.
The basis for most chiral high performance liquid chromatographic separations is the chiral stationary phases. The design, synthesis and evaluation of new chiral stationary phases are the other important part of this research.
We first evaluated the chromatographic performance of the polymeric ( R,R) and (S,S) poly (trans-1,2-cyclohexanediyl-bis acrylamide) based CSPs, which are known as (R,R) and ( S,S) poly-cyclic amine polymer (P-CAP) HPLC columns. These two columns are effective in separating enantiomers under normal phase and polar organic conditions, and with mobile phases containing halogenated solvents. The retention behavior, effects of mobile phase additives, normal-phase organic modifier, flow rate, column efficiency under different mobile phase conditions, sample loading capacity, and reversal of analyte elution order are discussed in detail. Hydrogen bonding interactions between these CSPs and analytes are the dominant interactions for chiral recognition by P-CAP columns. Dipole-dipole interactions and steric repulsion may also contribute to enantiomeric separations.
For the first time, we prepared stable β-cyclodextrin (β-CD) derivatives which contain π-electron deficient substituents (i.e., π-acidic moieties) for enantiomeric separations by HPLC. A variety of different dinitro-substituted aryl groups are investigated and compared in terms of their enantioselectivity. These CSPs are highly stable and robust under three mobile phase modes, including the reversed-phase, normal phase, and polar organic modes and exhibit very broad selectivity for a wide variety of compounds, including heterocyclic compounds, chiral acids, chiral amines, chiral alcohols, chiral sulfoxides and sulfilimines, amino acid derivatives, and other chiral compounds. The effects of the mobile phase pH, buffer composition, number and position of the dinitro groups on the phenyl ring substituent, degree of substitution, and the bonding strategy on the chromatographic performance of the CSPs are investigated. The 2,6-dinitro-4-trifluoromethylphenyl (DNP-TFM) substituted β-cyclodextrin-based CSPs have the best column performance overall. For each mobile phase mode, no degradation in column performance was observed even after more than 1000 injections.
Finally, we optimized the 2,6-dinitro-4-trifluoromethylphenyl derivatized β-cyclodextrin-based CSPs for enhanced enantiomeric separations. Five different CSPs, which differ from each other in the linkage chain, the position of DNP-TFM groups on β-CD ring, or the sequence of synthetic procedure, were prepared and evaluated with 14 pairs of enantiomeric analytes in the reversed-phase mode. The spacer effect is much more pronounced for the β-cyclodextrin derivatives with the DNP-TFM substituted only on the secondary hydroxyl groups. The synthetic sequence of derivatization and bonding chemistry also affects the chiral recognition capability of the CSPs. The optimized CSP based on DNP-TFM derivatized β-CD exhibits broad enantioselectivity and high separation efficiency which makes it one of the most useful derivatized cyclodextrin CSPs.