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preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography.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

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... 

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 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]. 

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

Carotenoid isolation is carried out in open-column chromatography (OCC). OCC should be carried out under a fume hood to protect the analyst from inhaling solvent vapor. Breathing hexane, for example, should be avoided due to neurotoxicity of some of its oxidative metabolites [87]. The isolation procedure is as follows. 

Although thin-layer chromatography (TLC) and open-column chromatography have played important roles historically in analysis of retinoids and carotenoids, both methods have been almost completely supplanted by HPLC. Paper chromatography has never been employed extensively for retinoid or carotenoid analysis. 

The removal of sterols, vitamin E vitamers, carotenoids, and other interfering material from the unsaponifiable fraction of food samples has been achieved using one or more of the following techniques coprecipitation of sterols with digitonin (91), precipitation of sterols from a methano-lic solution (195,209), adsorption chromatography on open columns of alumina (70,91,96), thin-layer chromatography on silica plates (209), and solid-phase extraction on silica (68,100) and reversed-phase (210) cartridges. 

Chen BH, Yang SH, Han LH (1991) Characterization of major carotenoids in water convolvulus (Ipomoea aquatica) by open-column, thin-layer and high-performance liquid chromatography. J Chromatogr 543 147-155... 

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