Chiroptical detection analysis

CD detection for colored derivatives might just as easily be used for structural information about the carbohydrates as well as for their analysis, but so far little attention has been given to either application. Cyclic oligomers of p-D-glucose have important supporting roles to play in analytical applications that are discussed in a later section. The union of chirality in the carbohydrate moiety of a glycoside metabolite with the unsaturation in the base in such compounds as nucleosides and nucleotides, saponins and flavones, etc., is another area that will ultimately be developed for applications of chiroptical detection methods. New and imaginative ideas are needed for the analysis of carbohydrates, and CD should be one of the favored methods. 

Cellulose, starch, and their derivatives are commonly used as chromatographic stationary phases. They are, in principle, potential hosts for inducing CD activity in small molecules and could be used with effect for analysis in homogeneous media with chiroptical detection. An example might be the starch (amylose)-iodide complex [86]. Low aqueous solubility however is an obstacle to their general use in homogeneous solutions. Linear oligomers of maltose are more soluble than starch and could theoretically be used as alternatives to Cy, however they do not really compete in terms of the stability of the association complexes. 

The seminal work on steroid analyses using chiroptical detection was done by Djerrasi by the determination of hecogenin acetate in the presence of tigonenin acetate.Every steroid is chiral and therefore amenable to polarimetric detection after chromatographic separation. Chromophores are fairly uncommon, and analysis by ORD or CD is therefore less suitable. The only unsaturation in the cholesterol molecule, for example, is the isolated A -double bond, which has an absorbance maximum at 205 nm. Unsaturation coupled with chirality provides some selectivity, as ably demonstrated by the work of Potapov for analogs of progesterone Even simpler than that is the direct discrimination between the ketosteroids testosterone and dihydrotestosterone, which have opposite signs in methylene chloride solution (Fig. 6). 

The special problems for vaUdation presented by chiral separations can be even more burdensome for gc because most methods of detection (eg, flame ionization detection or electron capture detection) in gc destroy the sample. Even when nondestmctive detection (eg, thermal conductivity) is used, individual peak collection is generally more difficult than in Ic or tic. Thus, off-line chiroptical analysis is not usually an option. Eortunately, gc can be readily coupled to a mass spectrometer and is routinely used to vaUdate a chiral separation. 

Other more conventional detectors that might ostensibly outperform CD in selectivity are nmr and mass spectrometry, and in fact they do for the analysis of diastereomers, although quantitation is a much more difficult task. They cannot compete with chiroptical methods for the distinction between enantiomers. In nmr detection, derivatization to diastereomers is a prerequisite to enantiomer analysis, and chiral forms of lanthanide reagents can been used with good effect [16,17]. For the analysis of mixtures by either nmr or mass spectrometry, total chromatographic separation is a necessity, so the completeness of the baseline separation is the limiting step not the detector. In contrast CD can be applied to the analysis of enantiomers in mixtures in methods that require no prior separation. 

Derivatization is the preferred option for aminoacids (making use of the anomalous ORD or CD effects introduced with the chromophore) to move the Cotton bands to longer wavelengths and to lower the limits of detection. Derivatizations might include N-acetylation as in the case of aminoglycosides [67], dansylation [72], or binding to metal complexes [73,74], see later. On the other hand aminoacids have an auxiliary role to play in the analysis of other substances, where advantage is taken of their chiroptical properties. 

In its broadest terms the discussion of HPLC detection for chiral species must include the analysis of mixtures with achiral substances as well as the quality testing of, for example, the enantiomeric purity of a chemically pure drug form. The distinction between the definitions of chemical purity versus optical purity can not be overemphasized. In an efficient chiral HPLC system the latter problem is trivial, and if retention times are significantly different then any conventional detector such as RI, electrochemical, absorption, etc., could be used. Co-elutions are a major experimental concern in separations of mixtures and at this juncture it is not only prudent but absolutely necessary to involve a chiroptical detector to preferentially identify the chiral analyte. 

These advantages inherent to chiroptical detectors are particularly useful in the analysis of physiological fluids. The unique selectivity of chirality detection for LC is often due to the fact that optical activity is frequently associated with biological activity, another form of a derivatization reaction. 

See also Atomic Absorption Spectrometry Principles and Instrumentation. Chiroptical Analysis. Chromatography Overview Principles. Clinical Analysis Glucose. Enzymes Enzyme-Based Electrodes. Food and Nutritional Analysis Overview. Infrared Spectroscopy Overview. Mass Spectrometry Overview. Nuclear Magnetic Resonance Spectroscopy Overview. Nuclear Magnetic Resonance Spectroscopy Applications Food. Optical Spectroscopy Detection Devices. Sampling Theory. Spectrophotometry Overview. Sweeteners. X-Ray Absorption and Diffraction Overview. 

UV detection is used in most chiral analysis by HPLC and other liquid chromatographic modalities. However, some other detectors, such as conductivity, fluorescent and refractive index types, are also used. The choice of detector depends on the properties of the racemic compound to be resolved [41, 144]. Chiroptical detectors, which are based on the principle of polarimetry [145] or circular dichroism [146, 147], are also available. The enantiomer (+)- or (—)-notation is determined by these detectors. Some organochlorine pesticides are not UV-sensitive, and hence they are difficult to detect in liquid chromatography. The detection of these types of pollutant can be achieved by using a mass spectrometry (MS) detector, and therefore LC-MS instruments are now being put on the market for routine use [148, 149]. 

The chiroptical properties of the monoterpenoids found in the Hepaticae have not been clarified, since most of the monoterpenoids listed in Table I have been detected by GC-MS analysis. 

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