Chromophores identification

Chromophore identification Identification of species and functional groups via group absorbance peaks Determination of molecular structure of crystalline compounds Shows structure of metal complexes, environment around central element Structure determination, shows bonding conditions... 

Luminescence lifetimes are measured by analyzing the rate of emission decay after pulsed excitation or by analyzing the phase shift and demodulation of emission from chromophores excited by an amplitude-modulated light source. Improvements in this type of instrumentation now allow luminescence lifetimes to be routinely measured accurately to nanosecond resolution, and there are increasing reports of picosecond resolution. In addition, several individual lifetimes can be resolved from a mixture of chromophores, allowing identification of different components that might have almost identical absorption and emission features. 

To identify a colorant, its excitation and emission spectra must be measured. This can be done under standard conditions if the colorant has been extracted from a foodstuff. Usually the spectral patterns taken from real conditions will not deviate too much from standard conditions. One must be aware that the main spectral patterns are determined by the chromophore of the colorant and that further molecular identification needs to recognize special fine structures of the spectra or employ additional analytical tools. 

The identification and quantification of potentially cytotoxic carbonyl compounds (e.g. aldehydes such as pentanal, hexanal, traw-2-octenal and 4-hydroxy-/mAW-2-nonenal, and ketones such as propan- and hexan-2-ones) also serves as a useful marker of the oxidative deterioration of PUFAs in isolated biological samples and chemical model systems. One method developed utilizes HPLC coupled with spectrophotometric detection and involves precolumn derivatization of peroxidized PUFA-derived aldehydes and alternative carbonyl compounds with 2,4-DNPH followed by separation of the resulting chromophoric 2,4-dinitrophenylhydrazones on a reversed-phase column and spectrophotometric detection at a wavelength of378 nm. This method has a relatively high level of sensitivity, and has been successfully applied to the analysis of such products in rat hepatocytes and rat liver microsomal suspensions stimulated with carbon tetrachloride or ADP-iron complexes (Poli etui., 1985). 

Polar or thermally labile compounds - many of the more modern pesticides fall into one or other of these categories - are not amenable to GC and therefore LC becomes the separation technique of choice. HPLC columns may be linked to a diode-array detector (DAD) or fluorescence detector if the target analyte(s) contain chromophores or fluorophores. When using a DAD, identification of the analyte(s) is based on the relative retention time and absorption wavelengths. Similarly, with fluorescence detection, retention time and emission and absorption wavelengths are used for identification purposes. Both can be subject to interference caused by co-extractives present in the sample extract(s) and therefore unequivocal confirmation of identity is seldom possible. 

UV/VIS spectrophotometry can be used to determine many physico-chemical characteristics of compounds and thus can provide information as to the identity of a particular compound. Although UV/VIS spectra do not enable absolute identification of an unknown, they are frequently used to confirm the identity of a substance through comparison of the measured spectrum with a reference spectrum. However, UV spectrophotometry is not highly specific, and can obviously only be applied to polymer additives which are absorbers of UV radiation, i.e. contain chromophoric groups. Both UV and IR monitor functional entities rather than the entire molecular structure. A functional group s proximity to other electropositive or electronegative structures in a molecule affects the absorbance spectrum, allowing one to infer some details of molecular structure. 

A method suitable for quantification of the functional class of bis(ethanol)amine antistatics, which lack UV chromophores, consists of reaction with methyl orange [53]. Atmer 163 (alkyl-diethanol amine) has been determined as a yellow complex at 415 nm after interaction with a bromophenol/cresole mixture [64]. Hilton [65] coupled extracted phenolic antioxidants with diazotised p-nitroaniline in strongly acidic medium and carried out identification on the basis of the visible absorption spectrum in alkaline solution. The antioxidant Nonox Cl in... 

Both the identification of a species and the determination of the kinetics of its formation or decay can be achieved with longer pathlength cells, such as that depicted in Figure 2.103. In kinetic experiments, however, there is the proviso that the experiment can be performed before natural convection currents interfere with the measurements i.e. the operator must be certain that the removal of a chromophore from the optical path is due to reaction and not due to convection currents. It should be noted that the strength of UV-visible spectroscopy does not lie primarily in the identification of unknown species as the information it provides is not of a molecularly specific nature. 

The utility of mass selective detection is greatest when analyzing compounds that do not contain chromophores or when structural information is needed for chemical identification. Triterpene saponins contain very weak chromophores and have long been associated with a variety of biological activities including... 

Pongor S, Ulrich PC, Bencsath FA and Cerami A (1984) Aging of proteins isolation and identification of a fluorescent chromophore from the reaction of polypeptides with glucose. Proc Natl Acad Sci USA 81, 2684-2688. 

This method suffered from sensitivity problems initially as the bile-acid molecules lack a chromophore, but did offer the distinct advantage that conjugated bile acids could be determined without hydrolysis. The sensitivity issue was addressed by use of fluorescent derivatives such as dimethoxycoumarin esters with a C18 reverse phase column and were able to resolve endogenous mixtures of bile acids. The combination of hplc and mass-spectroscopy detection has further improved the sensitivity along with providing specific identification, important as the resolution of bile acids by hplc is not as good as capillary column glc. ... 

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