Absorption spectrophotometry carotenoids

This chapter focuses on the extraction and handling of retinoids and carotenoids, their separation by various chromatographic techniques, and their detection and quantitation, primarily by absorption spectrophotometry, fluorescence, and mass spectrometry. A variety of other methods exist for their identification and characterization, including circular dichroism (333), infrared spectroscopy (334), resonance Raman spectroscopy (335), NMR spectroscopy (336), and x-ray crystallography (337). Although some of these procedures require substantial amounts of a retinoid or a carotenoid in an essentially pure form for study, others, such as resonance Raman spectroscopy, are extremely sensitive and can be used to detect the localization of carotenoids in single cells (338,339). 

The analytical procedures applied in the study were fluoromet-ric determination of chlorophyll pigments / /, absorption spectrophotometry for the analysis of carotenoids /2/. Hydroperoxides were determined by the iodometric titrational or spectro-photometric procedures /3,4/. 

Data taken from Choo et al. (1989). Total carotenoids were estimated using absorption spectrophotometry at 446 nm. ... 

The extraction of chlorophylls and carotenoids from water-containing plant materials requires polar solvents, such as acetone, methanol, or ethanol, that can take up water. These extracts must then be transferred to a solvent such as diethyl ether in order to be stored stably. Samples with very high water content, such as juices and macerated plant material, are usually freeze-dried first, and can then be extracted directly with diethyl ether. After extraction, solutions are clarified and diluted to an appropriate volume to measure chlorophyll content by UV-VIS spectrophotometry. Absorption coefficients and equations needed for quantitative determination are given in unitf4.3. 

Without prior fractionation the total concentration of carotenoids ( total carotenes ) in a sample extract may be estimated by spectrophotometry at 450 nm (using an average molar absorptivity for calculation). As an improvement, in earlier times, individual carotenoids or carotenoid classes were collected after separation on alumina or magnesia-hyflo supercel columns and the absorbance of the eluted fractions measured. This approach once formed the basis of the Association of Official Analytical Chemists (AOAC) method for foods. 

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. 

Detection is normally by absorption or fluorescence spectrophotometry. The high coefficients of extinction of the chlorophyll Soret band enable sensitive detection between 380 and 445 nm. This region of the spectmm also includes the carotenoids, which accompany the chlorophylls in plant pigment extracts and whose analysis and quantification are also usually of interest (see Chapter 6). When these compounds are not of interest, and their possible interference must be excluded, a selective detection of chlorophylls and derivatives can be carried out at 654 [136] or 667 nm [230], where there is no absorption of these pigments. Detection... 

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