Reference standards chiral purity

Chiral or achiral assay and purity determinations are done according to an external calibration calculation procedure, either with or without internal standardization. The calibration is performed against a 10% w/w (compared to the nominal concentration of the sample solution at 100% w/w) reference standard solution. The sample solution for the purity determination remains at the 100% w/w level, while that of the assay determination is diluted 10 times. The reason for the difference in concentration levels is similar to the purity method. A suggested sample injection sequence can be... 

The ultimate stereochemical identity test is, of course, the direct resolution of the enantiomers using chiral liquid or gas chromatography (9). When compared to a reference standard of the racemate, and under experimental conditions that will resolve the peaks of both enantiomers, the occurrence of two equal peaks will identify the racemate, and one peak will signify an enantiopure material. A proof of the stereochemical identity of the analyte can be provided, based on a match of retention times with a reference standard of known stereochemistry. Inequality between the peaks is a measure of enantiomeric enrichment. Therefore, it is conceivable that both stereochemical identity and purity can be established from a single experiment. 

Documentation of preparation of stock and sub-stock solutions must start with receipt of the reference standard, its Certificate of Analysis, assigned purity (and possibly chiral purity) and its history of storage and use after receipt (Section 9.4.4). Procedures for preparation and subsequent dilution of stock solutions are described in Section 9.5.4 and stability testing for these solutions in Section 10.4.Ih. Some of the relevant documentation might be included in general laboratory SOPs but full documentation of all study-specific procedures and data regarding preparation, storage and validation of stock solutions is required. 

Online CD detectors are now commercially available for use with HPLC that are inherently more sensitive than corresponding OR detectors and not affected by solvent changes to the same extent and are thus more gradient compatible [121]. Provided Ae and the concentration of an analyte are known with good precision/accuracy, the measurement of CD will allow the determination of enantiomeric purity. In addition, with CD-based detection systems, both chiroptical and ordinary absorbance can be determined simultaneously allowing the measurement of the g-factor (or dissymmetry factor), which is defined as the ratio of the CD to the absorbance (AA/A) [122]. The g-factor is concentration independent and its measurement allows a more reliable determination of enantiomeric purity (without using a CSP) with reference to standards of known enantiomeric composition irrespective of their concentration [123]. A small number of recent literature examples have suggested the potential use of achiral HPLC with online CD detection for the determination of extreme enantiomeric ratios [121, 124-126] however, chiral separation techniques currently provide a more reliable measurement of enantiomeric purity. 

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