Chlorophyll extraction techniques

Traditionally, dried or powdered plant material is used and extracts can be obtained by mixing the material with food-grade solvents like dichloromethane or acetone followed by washing, concentration, and solvent removal. The result is an oily product that may contain variable amounts of pheophytins and other chlorophyll degradation compounds usually accompanied by lipid-soluble substances like carotenoids (mainly lutein), carotenes, fats, waxes, and phospholipids, depending on the raw material and extraction techniques employed. This product is usually marketed as pheophytin after standardization with vegetable oils. 

Natural colours include annatto, anthocyanins, beetroot red (betalaines), caramel, carotenoids, cochineal and lac pigments, flavanoids, chlorophylls and tumeric. There is a trend towards encapsulating natural colours for food use, but this is not yet reflected in the extraction techniques described in the published analytical methods. Lancaster and Lawrence (1996) described the extraction and... 

Selection of a suitable extraction procedure can increase the concentration of phenolic diterpenes relative to the plant material. In addition, undesirable components can be removed prior to adding extracts to foods. For example, chlorophylls present in plant material may reduce the light stability of food during storage. Several extraction techniques have been patented (Nakatani et al., 1984 Aeschbach and Philippossian, 1989) using solvents with different polarity, such as petrol ether, hexane, toluene, acetone, methanol and ethanol. To obtain tailored extracts, the successive use of different solvents has been applied. 

In general, procedures used for these studies involve the treatment of soil plots and the removal of samples from treated and control plots over a specified time frame after treatment. The algal population in the sample is then estimated, most frequently by a soil dilution technique (Fields and Hemphill 1968, Cullimore and McCann 1977, Calle 1970, Huge 1970). Pantera, however, used chlorophyll extraction as an index of population size in soil samples pre-enriched with a nutrient medium (1970). 

High performance liquid chromatography (HPLC) has been by far the most important method for separating chlorophylls. Open column chromatography and thin layer chromatography are still used for clean-up procedures to isolate and separate carotenoids and other lipids from chlorophylls and for preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography. The whole analysis should be as brief as possible, since each additional step is a potential source of epimers and allomers. 

Post-column detection of chlorophyll derivatives is often accomplished by ultraviolet and visible spectroscopic techniques, which take advantage of the strong electronic absorption spectra of these pigments (units F4.3 F4.4). While these methods have enjoyed wide application (Schwartz et al., 1981 Khachik et al., 1986), a major advance was made with the introduction of photodiode array (PDA) detection. Multichannel photodiode array detection allows for simultaneous monitoring of multiple wavelengths, resulting in the generation of online electronic absorption spectra of a compound as it elutes from the HPLC column. Because of the uniqueness of electronic absorption spectra of individual chlorophyll derivatives, these techniques have enjoyed extensive application for tentative identification of components from complex mixtures and extracts... 

Sensidoni, A., Bortolussi, G., Orlando, C. and Fantozzi, P. (1996) Borage oil (Bortago officinalis L.) an important source of gamma linolenic acid. II. Tocopherols and chlorophyll content and sensorial analysis of borage oils extracted by different techniques and blended with extra virgin olive oil. lnd. Aliment., 35, 664-669. 

Figure 16 shows an example of the excitation spectra measurement in the same bay. The continuous dot plot shows the fluorescent emission intensity obtained every 6 s. The upper half shows the results of excitation at 499 nm, and the lower half shows the results at 578 nm. The horizontal axis shows the distance in meters. The three filled circles show the chlorophyll a concentration of the sampled water (chlorophyll a was obtained by extraction in acetone). The results show a decrease where the vessel crossed the estuarine region. Thus, continuous measurement by this technique can determine the distribution of phytoplankton, including the patch size, and the small-scale variability. 

Carotene was first extracted from the carrot by Wackenroder in 1831 and later from green leaves, although the identical nature of the material from the two sources was not established until 1907 by Willstatter. The former source comprises in fact a mixture of a- and p-carotenes which was later separated by Kuhn in the early thirties by the then new technique of column chromatography although this procedure was first described in 1906 by Tswett for the separation of carotene and chlorophyll. 

Hydrodiffusion is a steam distillation method, whereby steam is fed to the plant material from the top. Steam penetrating the plant matrix condenses and forces the essential oil to diffuse to the surface. Because of gravity, oil leaves the vessel together with the condensate water from the bottom of the still. Powdering the material results in better oil yields. This technique is suitable both for superficial and subcutaneous oils. The disadvantage of this technique is the undesired extraction of coumarins, psoralens, and chlorophyll into the oil. Therefore, hydrodiffusion has not become a widely used distillation technique. 

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