Chlorophyll color analysis

Analysis of Synthetic Chlorophyll-Based Food Colorants.442... 

The development and reports of methods for colorless chlorophyll derivative (RCCs, FCCs, and NCCs) analysis are relatively recent and the structures of the compounds are being elucidated by deduction from their chromatographic behaviors, spectral characteristics (UV-Vis absorbance spectra), mass spectrometry, and nuclear magnetic resonance analysis. The main obstacle is that these compounds do not accumulate in appreciable quantities in situ and, moreover, there are no standards for them. The determination of the enzymatic activities of red chlorophyll catabolite reductase (RCCR) and pheophorbide a monoxygenase (PAO) also helps to monitor the appearance of colorless derivatives since they are the key enzymes responsible for the loss of green color. ... 

ANALYSIS OF SYNTHETIC CHLOROPHYLL-BASED FOOD COLORANTS... 

In contrast to the well-established methods for identifying and quantifying naturally occurring chlorophylls, very few reports concern quantitative analysis of chlorophyllin copper complexes in color additives and in foodstuffs. Analytical methods proposed are based on spectral properties, elemental analysis, chromatographic separation, and molecular structure elucidation or a combination of these procedures. 

Usually, HPLC analysis resolves four peaks identified by co-chromatography with authentic standards as copper pheophorbide a, Cn(II) chlorin e6, Cn(II) chlorin e4, Cu rhodin g7, and their degradation products, but a sum of other colored components can also be found, for example, native chlorophylls, pheophytins, pheophor-bides, and rodochlorins (free carboxyl forms of pheophorbides) besides epimers, allomers, and degradation products that have been only tentatively identified. 

Immediately cork the tube filled with nitrogen and then add a drop or two of dichloromethane to dissolve the pigments for TLC analysis. Carry out the analysis without delay by spotting the mixture on a TLC plate about 1 cm from the bottom and 8 mm from the edge. Make one spot concentrated by repeatedly touching the plate, but ensure that the spot is as small as possible—less than 1.0 mm in diameter. The other spot can be of lower concentration. Develop the plate with 70 30 hexane acetone. With other plates try cyclohexane and toluene as eluents and also hexane/ethanol mixtures of various compositions. The container in which the chromatography is carried out should be lined with filter paper that is wet with the solvent so the atmosphere in the container will be saturated with solvent vapor. On completion of elution, mark the solvent front with a pencil and outline the colored spots. Examine the plate under the uv light. Are any new spots seen Report colors and i /values for all of your spots, and identify each as lycopene, carotene, chlorophyll, or xanthophyll. 

Based on TLC analysis, the procedure used allows the extraction of at least five different pigments from the spinach leaves. Judging from colors, one of these is a carotene, three are xanthophylls, and the last is chlorophyll b. 

Analysis A good separation will reveal as many as eight colored spots. These are the carotenes (orange), chlorophyll a (blue-green), the xanthophylls (yellow), and chlorophyll b (green). Calculate the // -values of all spots on the plate(s) you developed. Compile these data, together with those of others if you worked as a team, and determine which solvent mixture provided the best and which the... 

Chlorophylls and their derivatives are foimd, for example, in many vegetables, and they are also used as food colorants. They have a strong hydrophobic character, so they are usually analyzed by RPLC with a C18 column and an organic mobile phase, except when chlorophyll c (polar molecule) is present. In this case, the addition of a small amount of water to the organic solvent is recommended. Spectrophotometric and MS detectors are the most commonly employed detection modes for the analysis of chlorophylls. 

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