Carotenoids spectral characteristics

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). 

It is important to correctly identify the provitamin A peak(s) of interest in the chromatogram. A tentative identification can be made by a combination of retention time and spectral characteristics, using a photodiode array detector. Identification is aided by comparisons with authentic carotenoid standards in more than one chromatographic mode. Because of the ease of cis-trans isomerization when solutions of carotenoids are exposed to heat, light, oxygen, etc., it is difficult to ascertain whether a cis isomer occurs in nature or whether it is formed during its isolation. 

HPLC is commonly used to separate and quantify carotenoids using C18 and, more efficiently, on C30 stationary phases, which led to superior separations and improved peak shape.32 4046 An isocratic reversed-phase HPLC method for routine analysis of carotenoids was developed using the mobile phase composed of either methanol acetonitrile methylene chloride water (50 30 15 5 v/v/v/v)82 or methanol acetonitrile tetrahydrofuran (75 20 5 v/v/v).45 This method was achieved within 30 minutes, whereas gradient methods for the separation of carotenoids can be more than 60 minutes. Normal-phase HPLC has also been used for carotenoid analyses using P-cyclobond46 and silica stationary phases.94 The reversed-phase methods employing C18 and C30 stationary phases achieved better separation of individual isomers. The di-isomers of lycopene, lutein, and P-carotene are often identified by comparing their spectral characteristic Q ratios and/or the relative retention times of the individual isomers obtained from iodine/heat-isomerized lycopene solutions.16 34 46 70 74 101 However, these methods alone cannot be used for the identification of numerous carotenoids isomers that co-elute (e.g., 13-ds lycopene and 15-cis lycopene). In the case of compounds whose standards are not available, additional techniques such as MS and NMR are required for complete structural elucidation and validation. 

Spectral characteristics and configuration of some stereoisomeric carotenoids including prolycopene and pro-gamma-carotene. J. Am. Chem. Soc. 65 (1943) 1940—1951. (L. Zechmeister, A. L. LeRosen, W. A. Schroeder, A. Polgar, and Linus Pauling). 

The quantitative distribution of the various major provitamin A carotenoids in natural foods and feeds has been studied for the most part using open chromatographic procedures. Quite complete separations of individual carotenoids are possible by these procedures, but they are time-consuming (Davies, 1976). In addition, extended exposure to solvents and adsorbents may alter the carotenoids by oxidation or isomerizations that change their spectral characteristics and thus yield incorrect quantitative results (Rodriguez et al., 1976). Most of the vitamin A values of foods reported in commonly used food-composition tables are based on the Association of Official Analytical Chemists (AOAC) simplified chro-motographic method (AOAC, 1980). This method separates xanthophylls from carotene but does not separate individual carotenes. Separate values, therefore, frequently are not reported for (3- and non-p-carotenoids. 

Zechmeister L, Polgar A (1943) Cis-trans isomerization and spectral characteristics of carotenoids and some related compounds. J Am Chem Soc 65 1522-1528... 

In a previous study, we incubated chromatophores of Rps.caps. with a specific pronase (type III, Sigma) from Streptomyces griseus (5). The effect of pronase digestion on the spectral characteristics of both the field-sensitive and the field-insensitive carotenoid pools was monitored. It was shown that the field-responding carotenoids are more sensitive to pronase treatment than the non-responding pool. In the present study, these experiments were extended to other digestive treatments of chromatophores of Rhodopseudomonas capsulata as well as sphaeroides. 

The fact that the 2 populations remain spectrally different implies that - even at this stage of extensive digestion - solvatochromism as well as specific interactions with the proteins determine the spectral characteristics of the carotenoid pools (see the model of Kakitani et al (6)). 

In the carotenoid radicals, the unpaired electron is highly delocalized over the conjugated polyene chromophore. This has a stabilizing effect and also allows subsequent reactions. The cation and anion radicals can be detected by their characteristic spectral properties, with intense absorption in the near-infrared region. 

The absorption spectrum ofpurified bjhy itself in the presence of 8204 shows a split a-band at ambient temperature, as shown in Fig. 9 (A), reflecting the characteristic absorbances ofcytochrome/at 554 nm and b at 563 nm. The absorption spectra showed no indication of the presence of any chlorophyll, carotenoids, or Cyt b559. At ambient temperature, the Asc reduced-minus-FeCy oxidized difference spectrum revealed cytochrome /at 554 nm, while the 8204 reduced-minus-Asc reduced difference spectrum revealed cytochrome b(, at 563 nm [see Fig. 9 (A), inset]. The amplitudes of the two a-bands indicates a ratio of 2 CyibfJCytf. The same difference spectra measured at 77 K [Fig. 9 (B)] shows the a-band of cytochrome / is split, with a major band at 551.4 nm, while cytochrome b shows an asymmetric a-band at 560.8 nm. The absorption maxima of both cytochromes are shifted to the blue by a few mn at 77 K. Since Cyt bf, contains two hemes, one might expect some spectral difference between... 

Analysis of pigment composition by HPLC revealed that mutant C-6D of Scenedesmjs obliquus forms oily chlor yll a (peak 9 = Chi a, peak 10 = Chi a ) and acyclic precursors of carotenoids (peaks a-f) during heterotroiMc growth in the dark (Fig. 1). The precursors were identified their spectral and chemical characteristics (cf. 1) to be cis-lycopene (a), transMieurosporaije (b), two forms of cis-neurosporene (c+d), S-carotene (e) and -zeacarotene (f). 

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. 

---