Carotenoids fine structure

In this protocol, commercially purchased carotenoid standards are dissolved in a suitable solvent and the absorbance measured at its maximum wavelength (A.max). Using published extinction coefficients and taking into consideration the dilution factor, the concentration of the standard carotenoid is calculated. The spectrum is also scanned in order to evaluate the fine structure (see Spectral Fine Structure in Background Information). The carotenoid solution should ideally be assayed by HPLC as described in unit F2.3 to establish chromatographic purity and thus correct the calculated concentration. 

The same principle as described above can be used for the estimation of the carotenoid content of extracts of food colorants, pharmaceuticals, foods, biological samples, or chromatographic fractions. This procedure employs calculations used for individual carotenoids of high purity and thus will estimate the total carotenoids present in a food or biological extract, where a mixture of carotenoids would be expected. Greater accuracy can be obtained as extracts are purified to contain single components (see Commentary). A spectrum scan is not employed in this procedure as the fine structure of a mix of carotenoids can only be identified after HPLC separation (see Commentary). 

In addition to the absorption maxima of the carotenoids, the shape of the spectra provides important information for identification of purified carotenoid extracts or pure standard (while the identity of the standard is generally not in question, it is a good idea to check the purity by fine structure analysis). Fine structure... 

The spectral characteristics of a standard can be monitored during HPLC using a diode-array detector (unitfu). A directory of standard spectra can be stored, enabling additional identification of sample peaks. The actual absorption maxima and fine structure will be dependent on the composition of the mobile phase (see Fig. F2.2.4). Peak I may only occur as a shoulder with civ-carotenoids. while an additional peak is observed at around 340 nm (see Fig. 2.2.1). 

These changes are illustrated in Fig. 2, where the visible absorption spectra of cells from exponentially-growing and carbon-starved cultures are shown for comparison. The shift of the three-peaked carotenoid band is clearly indicated by the marked variation in the depth of the trough to the left of the Qx(0, 0) bacteriochlorophyll band at 588 nm. There is also a change of the band s fine structure, which is lower in the spectrum of exponential cultures. This additional difference is enhanced by the variation of the extent of overlapping between carotenoid peak III and the Qx(0, 1) band of bacteriochlorophyll near 555 nm. 

This section includes identification and measurement of carotenoids by UV/visible spectrophotometry and column chromatography. The UV/visible absorption and the chromatographic behavior spectrum provide the first clues for the identification of carotenoids. Both the wavelengths of maximum absorption (Xroax) the shape of the spectrum (spectral fine structures) are characteristics of the conjugated unsaturated part of the carotenoid molecule cmitaining delocalized 7t-electrons called the chromophore. The values of the carotenoids commonly found in natural products in various solvents are listed in Table 111.2. 

This method is based in the light-absorbing chromospheres and the visible absorption spectrum. The wavelength of maximum absorption (A,max) and the shape of the spectrum (spectral fine structure) are specific characteristics of each chromophore structure. In the past, most data on the amount of carotenoids found in foods were based on the total absorbance at a specified wavelength (450 nm). In some cases, a separation in an OCC was previously made followed by a spectroscopy. 

The chromatographic smdy must be complemented spectroscopically, with UV-visible spectroscopy being the most commonly used because of its availability and simplicity. The UV-visible absorption spectrum of a pure pigment is usually recorded in various solvents, and the values obtained for the absorption maxima and fine structure are compared with those listed in the bibliography, and if possible, with the spectmm of a pure standard recorded under the same conditions. In practice, it is normal to find differences of a few nanometers with respect to the Aj values in the literature, because basically of instmment-related factors. Table 6.12 details the absorption maxima in different solvents for the carotenoid pigments commonly found in foods of plant and animal origin. 

Proteobacteria (Imhoff, 1995). The functions of carotenoids in photosynthetic bacteria have been investigated in most detail in the Rhodospirillaceae (other chapters in this book). Their RC resembles that of PS 11 of green plants. Their major BChl is BChl a or b. The RC was firstly crystallized from Bla. (previously, Rhodopseudomonas) viridis, and the localization of one carotenoid, 1,2-dihydroneuro-sporene, four BChl b and two bacteriopheophytin b molecules was determined (Deisenhofer et al., 1995). A similar localization of spheroidene in the RC of Rba. sphaeroides has also been described (Yeates et al., 1988 Ermler et al., 1994). The fine crystal structure of the LH II antenna complex from Rps. acidophila strain 10050 has shown the localization of one rhodopin glucoside and three BChl a molecules per ap monomer (McDermott et al., 1995). A similar localization of lycopene in the LH II complex from Rsp. molischiamm has also described (Koepke et al,... 

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