In analytical chemistry, A chiral derivatizing agent (CDA), also known as a chiral resolving reagent, is a derivatization reagent that is a chiral auxiliary used to convert a mixture of enantiomers into diastereomers in order to analyze the quantities of each enantiomer present and determine the optical purity of a sample. Analysis can be conducted by spectroscopy or by chromatography. Some analytical techniques such as HPLC and NMR, in their most commons forms, cannot distinguish enantiomers within a sample, but can distinguish diastereomers. Therefore, converting a mixture of enantiomers to a corresponding mixture of diastereomers can allow analysis. The use of chiral derivatizing agents has declined with the popularization of chiral HPLC. Besides analysis, chiral derivatization is also used for chiral resolution, the actual physical separation of the enantiomers.
History
Since NMR spectroscopy has been available to chemists, there have been numerous studies on the applications of this technique. One of these noted the difference in the chemical shift (i.e. the distance between the peaks) of two diastereomers. Conversely, two compounds that are enantiomers have the same NMR spectral properties. It was reasoned that if a mix of enantiomers could be converted into a mix of diastereomers by bonding them to another chemical that was itself chiral, it would be possible to distinguish this new mixture using NMR, and therefore learn about the original enantiomeric mixture. The first popular example of this technique was published in 1969 by Harry S. Mosher. The chiral agent used was a single enantiomer of MTPA (α-methoxy-α-(trifluoromethyl)phenylacetic acid), also known as Mosher's acid. The corresponding acid chloride is also known as Mosher's acid chloride, and the resultant diastereomeric esters are known as Mosher's esters. Another system is Pirkle's Alcohol developed in 1977.
Requirements
The general use and design of CDAs obey the following rules so that the CDA can effectively determine the stereochemistry of an analyte: The CDA must be enantiomerically pure, or (less satisfactorily) its enantiomeric purity must be accurately known. The reaction of the CDA with both enantiomers should go to completion under reaction conditions. This acts to avoid enrichment or depletion of one enantiomer of the analyte by kinetic resolution. CDA must not racemize under derivatization or analysis conditions. Its attachment should be mild enough so that the substrate does not racemize either. If analysis is completed by HPLC, the CDA must contain a chromophore to enhance detectability. If analysis is completed by NMR, the CDA should have a functional group that gives a singlet in the resultant NMR spectrum, where the singlet must be remote from other peaks.
Mosher's method
Mosher's acid, via its acid chloride derivative, reacts readily with alcohols and amines to give esters and amides, respectively. The lack of an alpha-proton on the acid prevents loss of stereochemical fidelity under the reaction conditions. Thus, using an enantiomerically pure Mosher's acid allows for determination of the configuration of simple chiral amines and alcohols. For example, the (R)- and (S)-enantiomers of 1-phenylethanol react with (S)-Mosher acid chloride to yield (R,S)- and (S,S)-diastereomers, respectively, that are distinguishable in NMR.
CFNA (alternative to Mosher's acid) A newer chiral derivatizing agent (CDA), α-cyano-α-fluoro (2-naphthyl)-acetic acid (2-CFNA) was prepared in optically pure form by the chiral HPLC separation of a racemic 2-CFNA methyl ester. This ester was obtained by fluorination of methyl α-cyano (2-naphthyl) acetate with FClO3. 2-CFNA has been shown to be a superior CDA than Mosher's agent to determine the enantiomeric excess of a primary alcohol.
Chromatography using CDAs
Upon reaction of a CDA with the target analyte, chromatography can be used to separate the resulting products. In general, chromatography can be used to separate chiral compounds to bypass difficult crystallizations and/or to collect all diastereomer pairs in solution. Chromatography also has many variations (e.g. HPLC, Gas Chromatography, flash chromatography) with a wide array of applicability to diverse categories of molecules. The ability for CDAs to separate chiral molecules is dependent on two major mechanisms of chromatography: Differential solvation in the mobile phase Differential adsorption to the stationary phase