Carotenoids column chromatography

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preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography.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. 

Although saponification was found to be unnecessary for the separation and quantification of carotenoids from leafy vegetables by high performance liquid chromatography (HPLC) or open column chromatography (OCC), saponification is usually employed to clean the extract when subsequent purification steps are required such as for nuclear magnetic resonance (NMR) spectroscopy and production of standards from natural sources. 

Carotenoids were discovered during the nineteenth century. Wachen in 1831 proposed the term carotene for the hydrocarbon pigment crystallized from carrot roots Berzelius called the more polar yellow pigments extracted from autumn leaves xanthophylls and Tswett separated many pigments by column chromatography and called the whole group carotenoids. ... 

Traditional column chromatography has also been employed for the extraction of carotenoids from palm oil. Separations were carried out on silica columns, carotenoids were eluted with n-hexane while the free fatty acids of the oil were removed from the stationary phase with ethyl acetate. The recovery of the method was 45 per cent and the purity of the cartotenoid fraction about 20 per cent w/w [23],... 

This protocol begins with the extraction of a dehydrated sample. It continues with a saponification scheme to initiate the isolation of the carotenoid mixture. During saponification, the esters are hydrolyzed and the free pigments released. Then, to continue the isolation, column chromatography is suggested as a simple and fast means of separating the three main groups of carotenoids based on their different polarities. 

Once the carotenoids have been isolated as described in Basic Protocol 1, they can generally be crystallized as an initial step to purification. Actually, what is most likely to happen is a co-crystallization. When working with a nonpolar fraction, a- and (3-carotene may co-crystallize. In the same way, a polar fraction may yield lutein-zeaxanthin crystals. A pure carotenoid product may be obtained by crystallization of a fraction derived from a preparatory chromatographic procedure, which can be done using TLC, HPLC unit F2.3), or in some cases column chromatography. 

It must be stressed that the value obtained in this way for mixtures of carotenoids is only an approximation. For more definitive analysis column chromatography (unit F2.3) should be used. 

Accurately quantifying the amount of provitamin A carotenoids in a food product is essential for determining nutritional value of foods. The AO AC method for determination of vitamin A (974.29) and carotenoids (941.15 and 970.64) in foods utilizes open column chromatography combined with a colorimeter (vitamin A) or spectrophotometer (carotenoids) (Horwitz, 2006). It is recommended to extract with acetone-hexane followed by filtration, then remove the acetone by rinsing with water. The extracts in hexane are applied to an activated Mg02 diatomaceous earth column and eluted using acetone and hexane... 

The Interaction of the carotenoid and the fatty acid fractions on Cellte were both necessary for the odor development to occur. Studies designed to confirm an Interaction of the carotenoid and fatty acid fractions In the development of salmon flavors showed that when carotenoid fractions from salmon oils were separated from the acylglycerol fraction by column chromatography, neither yielded a salmon-llke aroma during oxidation (Table III). 

The ionised sulphate group makes carotenoid sulphates strongly polar. This results in significant solubility in water with values up to 0.4 mg/ml. The high polarity and solubility properties determine the choice of isolation procedure. Special methods are employed and detailed isolation procedures available [68]. The isolation methods include column chromatography on Sephadex or silica, TLC and reversed phase HPLC. Sulphur analysis is also desirable. Further characterisation is carried out by spectroscopic methods. 

Isolation and purification. Carotenoids were extracted with CHCl /MeOH from the wet cells of aerobic or semi-aerobic ciilture, or from those of aerobic culture inhibited by DPA or nicotine. The polar carotenoid group was purified by silica gel column chromatography, SEP-PAK NH2 and reversed phase TLC. The others were purified with silica gel and DEAE-Sepharose CL-6B column chromatography. The purity was analyzed with the reversed phase HPLC system using water (20-0%, v/v) in methanol as eluent [3>4] ... 

Carotenoids are commonly extracted from liquid samples (plasma/serum) into lipophilic solvents such as hexane, hexane-ethyl acetate, or diethyl ether, mostly after deproteinization with ethanol or methanol, which also helps to liberate the lipidic substances from protein binding. Extracts should be protected from light and acids and antioxidants may usefully be added. The extract is either used as such or is concentrated under oxygen-free nitrogen. Solid samples, e.g., foods, are either extracted with a solvent miscible with water (acetone, methanol) or, after dehydration of the sample, with a water immiscible solvent. Cleanup of the extract and fractionation of the pigments may involve saponification and/or open-column chromatography. 

In pure samples such as pharmaceutical products tocopherols can sometimes be directly determined by UV spectrophotometry. Improved selectivity is derived from the Emmerie-Engel reaction, based on the reduction by tocopherols of iron(III) ions to iron(n) ions. The latter form a red complex with 2,2 -bipyridyl or bathophenanthroline, which can be measured colorimetrically (2max = 520nm). However, as many reducing substances, including carotenoids and sterols, interfere with the reaction, their removal by column chromatography or TEC is imperative. Such prefractionation should also be carried out in the spectrofluorimetric assays of vitamin E, although these are more specific than the colorimetric ones. 

Isolation and purification of individual carotenoids has been accomplished with countercurrent distribution, but the most important technique is separation by column chromatography. Two types of separation have been commonly employed. In zonal chromatography, carotenoids are applied to a column of a suitable adsorbent and then separated through development of the chromatogram with an appropriate solvent. The packing material of the column is then extruded, and the bands are scraped separately into beakers... 

In the field of carotenoids, most TLC-MS utilizations to date have been made offline and have used TLC merely for the purpose of purification or isolation of these pigments. Mass spectrometry was introduced into carotenoid analysis in 1965 [16]. In the 1970s, separations by open column chromatography on aluminum oxide were often combined with TLC separations on silica gel and MgO/Kieselguhr to achieve sufficient purification degree of carotenoids from tomato. These were analyzed afterward by direct-insertion electron impact-MS (EI-MS) [17-19]. Such isolation procedures, applied reactions (acetylation, saponification, and reduction), Rf values, absorption, and MS spectra enabled identification of phytoene 1,2-oxide, and related compounds as the first naturally occurring epoxides of acyclic carotenoids [17]. 

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