Chiral polymeric reagents for off-line and on-line derivatizations of enantiomers in high-performance liquid chromatography with ultraviolet and fluorescence detection: an enantiomer recognition approach

Abstract

Optically active and detector-sensitive polymeric reagents have been synthesized, loadings determined, derivatizations/separations/detection optimized, and applications to simple amines and amino alcohols described. Such reagents have been designed to contain different chiral centers, usually amino acids, leashed via an activated ester attachment to an insoluble, structurally rigid, organic polymer backbone. 9-Fluorenylmethyl (FMOC) moieties chemically bonded to the amino acids were used as ultraviolet (UV) and fluorescence (FL) sensitive detector probes to the final diastereomers of enantiomer substrates. Such diastereomers can be readily separated by isocratic or gradient elution normal-phase methods. The kinetics for diastereomer formation have been determined, and final UV/FL responses for known mixtures of enantiomers have been compared to demonstrate overall validity of the method. Minimum detection limits, linearity of calibration plots, dual detector responses, and linear diode array spectra and absorbance ratios have also been demonstrated. In some cases, authentic standards have been prepared to calculate absolute percent derivatizations for specific enantiomer pairs. The overall approach permits, for the very first time, off-line or on-line precolumn derivatization for the formation of diastereomers having unique detector properties. It has been proven that the rates and rate constants for such formations are identical for at least those pairs of enantiomers studied. Separations are base-line or near-base-line, permitting accurate and precise quantitative determinations, by both UV and FL, of enantiomer/optical purity and chemical purity.

Size-selective Derivatizations with Polymer Immobilized Reagents

“…9-FAbutylamide, 9-FA-adamantanamide, and 9-FA-nonylamide were synthesized and characterized in a previous publication (Zhou et al, 1993b Preparation of polymeric reagents. The polymeric intermediates were prepared according to a reaction scheme previously described (Gao et al, 1989). Polymeric intermediates were prepared on both microporous and macroporous supports.…”

“…Because only the amount of reagent involved in the derivatization reaction was actually released from the polymer surface, the reactor was stable for multiple derivatizations. These reagents have been used in HPLC for the on-line determination of chiral (Chou et al, 1989) and achiral amine drugs (Gao et al, 1989), amino acids (Bourque and Krull, 1991), and peptides (Zhou et al, 1993a). Other applications include combining several reagents in a reactor to form a multiple derivatives precolumn, so that each of these derivatives may be used to quantitate and confirm the presence of an analyte (Szulc and Krull, 1992).…”

Chiral derivatization reagents for drug enantioseparation by high‐performance liquid chromatography based upon pre‐column derivatization and formation of diastereomers: enantioselectivity and related structure

“…The 9-fluorenylmethyl moiety in some compounds is used as a ultraviolet/fluorescence sensitive probe. Reagents such as FMOC-L-Pro (37) and FMOC-L-Phe (38), in which fluorene moiety is chemically bonded to a chiral amino acid, have been used as CDRs for the resolution of enantiomeric amines (Herraez-Hernandez et al, 1998) or polymeric derivatization reagents for the separation of enantiomeric amines and aminoalcoholes (Chou et al, 1989). It was pointed out that the FMOC-L-Pro method combines the high sensitivity provided by chloroformates with the possibility of resolving the isomers of secondary amphetamines, and that the reagent FMOC-L-Pro should be superior to the reagent (À)-MCF because its bulky and rigid fluorenyl group should produce better enantioselectivity and detectability (Herraez-Hernandez et al, 1998).…”

“…Accordingly, FMOC-L-Pro diastereomers exist in very different, more rigid conformations and exhibit very different interactions with the stationary phase (Chou et al, 1989).…”

A mixed‐bed, multi‐derivatization approach using polymeric reagents for derivatizations of amines in high performance liquid chromatographic detection

“…Reagents IV, V, and VI have been derived from the corresponding FMOC-CI, pnitrobenzoyl chloride, and o-Ac chloride, all commercially available or readily synthesized from the precursor acids. Each of these reagents was characterized in terms of loading using two basic approaches, previously described (Gao et al, 1989b(Gao et al, , 1990Chou et al, 1989). One approach involved base hydrolysis, after organic solvent washing of the polymeric reagent to remove adsorbed reagents/solvents.…”