Carotenoids quantitative determination

Because of their versatility and simplicity, TLC methods have been frequently applied to the separation and semi-quantitative determination of carotenoid pigments in synthetic mixtures and various biological matrices. The retention of pure carotenoid standards has been measured in different TLC systems. Separations have been carried out on silica plates using three mobile phases (1) petroleum ether-acetone, 6 4 v/v (2) petroleum ether-tert-butanol 8 2 v/v, and (3) methanol-benzene-ethyl acetate 5 75 20 v/v. Carotenoids were dissolved in benzene and applied to the plates. Developments were performed in presaturated normal chambers. The chemical structure and the Rv values of the analytes measured in the three mobile phases are listed in Table 2.1. It was concluded from the retention data that mobile phase 3 is the most suitable for the separation of this set of carotenoids [13],... 

Because of their considerable role in human wellfare, carotenoids have been measured not only in plants as primary sources and in human and animal tissues but also in a wide variety of other matrices to find new and economical sources for carotenoids. Thus, the carotenoid accumulation capacity of algae and microalgae have been vigorously investigated. A validated liquid chromatography-electrospray mass spectrometry method have been developed and employed for the separation and quantitative determination of... 

Chlorophylls occur very frequently in the plant kingdom, they are responsible for the colour of vegetables and some fruits. They also occur in algae and several bacteria. Chlorophylls in plants are photoreceptors and in photosynthesis the presence of a closed circuit of conjugated double bonds allows them to absorb light. Because of their predominant importance as photoreceptors a considerable number of analytical methods have been developed for the separation and quantitative determination. The analytical methods applied for the measurement of chlorophylls and carotenoids in food products have been reviewed previously [273],... 

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. 

M Kimura, DB Rodriguez-Amaya, HT Godoy. Assessment of the saponification step in the quantitative determination of carotenoids and provitamins A. Food Chem 35 187-195, 1990. 

Glaser, T. Lienau, A. Zeeb, D. Krucker, M. Dachtler, M. Albert, K. 2003. Qualitative and quantitative determination of carotenoid stereoisomers in a variety... 

Larsen, E. Christensen, L.P. 2005. Simple saponification method for the quantitative determination of carotenoids in green vegetables. J. Agric. Food Chem. 53 6598-6602. 

Turning now to chemical methods used for stereochemical studies of carotenoids the modified Horeau method (32) has been successfully used to confirm the configuration of (2/ )- 3, 3-caroten-2-ol [(33), R=H] (38) and to establish the configuration of aleuriaxanthin (36) (34). However, no conclusive results could be obtained for plectaniaxanthin [(35), R = R =H] (28). In principle the Horeau method is based on partial resolution of racemic and meso a-phenylbutyric anhydride by means of an optically active secondary alcohol, which readts preferentially with one diastereomer. The unreacted anhydride is quantitatively determined by an appropriate procedure (32, 68, 94). 

Carotenoids and chlorophylls can he quantitatively determined either on the layer by visual comparison with a series of standards chromatographed simultaneously or spectrophotometrically after elution [127]. The separated pigments can be eluted from impregnated layers using dimethylformamide and freed from interfering oil by washing with petrol ether [36]. 

Reversed phase partition TLC may be applied to fractionate alcohols [78, 86], aldehydes and ketones [10, 125], fatty acids [4, 80, 84, 124] and their methyl esters [114, 213], keto-acids, hydroxy-acids and lactones [82]. More complete separations of these compounds or of suitable derivatives, are, however, obtained with gas chromatography and it is appreciably more sensitive than TLC it is moreover more suitable for quantitative determinations. Reversed phase partition TLC offers substantial advantages only in the analysis of lipids of relatively high molecular weight, like wax esters [86], steryl esters [79] (see also Chapter L), carotenoid esters (see Chapter K) and triglycerides [4, 81, 89, 124] some derivatives of naturally occurring lipids are also best fractionated on hydrophobic layers [21, 130, 131, 165]. 

Elucidation of the structure of carotenoids requires, in addition to VIS/UV spectrophotometry, supplemental data from mass spectrometry and IR spectroscopy. Carotenoids are determined photometrically with high sensitivity based on their high molar absorbancy coefficients. This is often used for simultaneous qualitative and quantitative analysis. New separation methods based on high performance liquid chromatography have also proved advantageous for the qualitative and quantitative analysis of carotenoids present as a highly complex mixture in food. 

Thin-Layer Chromatography (TLC) Methods for Quantitative Determination of Carotenoids in Foods... 

The vitamin A activity of plant foods is usually based on the HPLC determination of the three most ubiquitous provitamins, namely, a- and /3-carotene and /3-cryptoxanthin. It is necessary to separate the provitamins from other carotenoids and to quantify them individually. An obvious prerequisite to accurate quantitation is the conclusive identification of the provitamins. 

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