Tocopherols, separation

The commercial membrane separation processes are offered in the areas of nitrogen production and waste treatment applications (1). Developing membrane applications in oil milling and edible oil processing are (1) solvent recovery, (2) degumming, (3) free fatty acid removal, (4) catalyst recovery, (5) recovery of wash water from second centrifuge, (6) coohng tower water recovery, (7) protein purification, and (8) tocopherol separation. 

Figure 3. Both a-tocopherol and y-tocopherol have similar abilities to quench in vitro lipid peroxidation. The addition of 16 iM i ,i ,i -a-tocopherol ( ) or 16 pM / ,/ ,/ -Y-tocopherol (+) separately, or a mixture of both 8 pM i ,i ,i -a-tocopherol and 8 pM R,R,R-y-tocophero (A), equally inhibited the formation of fluorescent lipid peroxidation products (assayed as described by Shimasaki, 1994) compared with levels seen in control samples ( ) of large unilamellar liposomes oxidized in the presence of 7.4 mM 2,2 -azobis(2-amidinopropane) dihydrochloride.

An example of the efficacy of the resin phases used as an alternative to a conventional silica based reverse phase is shown in figure 12 where the separation of the three tocopherols are shown separated on the Cl 8 Polymer Column and The ODA-A 120A silica gel based columns. The columns were 15 cm long, 4.6 mm i.d., operated at a flow rate of 0.5 ml/min at 30°C with a mobile phase of 98% methanol/2% water. 

Comparison of the Separation of Some Tocopherols on a Polymer C18 Column and a Conventional Silica Based ODS... 

The reaction of eq. 16.9 will regenerate the antioxidant Arj-OH at the expense of the antioxidant At2-OH. Despite the fact that such regeneration reactions are not simple electron transfer reactions, the rate of reactions like that of eq. 16.9 has been correlated with the E values for the respective Ar-0. Thermodynamic and kinetic effects have not been clearly separated for such hierarchies, but for a number of flavonoids the following pecking order was established in dimethyl formamid (DMF) by a combination of electrolysis for generating the a-tocopherol and the flavonoid phenoxyl radicals and electron spin resonance (ESR) spectroscopy for detection of these radicals (Jorgensen et al, 1999) ... 

Basically, the Rf value of a solute is determined by its distribution ratio which in turn is dependent on relative solubilities for partition systems or relative polarities for adsorption systems. For example, if adsorption TLC is used to separate a mixture of squalene (a hydrocarbon), methyl oleate, cholesterol and a-tocopherol (vitamin E), then squalene, being the least polar, will move furthest and the cholesterol, being the most polar, will remain close to the origin. Methyl oleate is less polar than a-tocopherol and will therefore be found between it and the squalene. The role of polarity is discussed more fully on p. 82. 

Phases of extended length (C30) have been utilized for the separation of larger-size constrained solutes, such as carotenoids and steroids [27-29,93,106,107]. Apractical limit of alkyl chain length of C34 to C36 is imposed by the commonly employed silan-ization chemistry techniques [106]. Immobilization of longer alkyl stationary phases has been achieved through the use of poly(ethylene-co-acrylic acid) materials for use in carotenoid separations [27,28,93]. Rimmer et al. [28] have recently compared the selectivity of both alkyl and poly(ethylene-co-acrylic acid) stationary phases on the basis of separations of carotenoids in food matrices (Figure 5.12), in addition to mixtures of tocopherols and PAHs. 

Strohschein, S., Pursch, M., Lubda, D., and Albert, K., Shape selectivity of C-30 phases for RP-HPLC separation of tocopherol isomers and correlation with MAS NMR data from suspended stationary phases. Anal. Chem., 70, 13, 1998. 

Although the majority of analytes do not possess natural fluorescence, the fluorescence detector has gained popularity due to its high sensitivity. The development of derivatization procedures used to label the separated analytes with a fluorescent compound has facilitated the broad application of fluorescence detection. These labeling reactions can be performed either pre- or post-separation, and a variety of these derivatization techniques have been recently reviewed by Fukushima et al. [18]. The usefulness of fluorescence detectors has recently been further demonstrated by the Wainer group, who developed a simple HPLC technique for the determination of all-trani-retinol and tocopherols in human plasma using variable wavelength fluorescence detection [19]. 

In spite of the obvious advantages of elevated temperature, there are examples of cases where better separation is achieved at a reduced temperature, even for simple solutes. Craft et al. [20] recently demonstrated an improved separation of P and y tocopherol at -20°C in THF/acetonitrile when compared to the ambient temperature separation of the compounds in Acetonitrile water. Bohm [21] reported the temperature dependence of the separation of a mixture of five xanthophylls and six carotenes on a C-30 column. The optimum temperature in this case was 23°C with a coelution of some peaks at temperatures below 20°C and others above 35°C. In a study using a 300 A pore C-18 column, Bohm [22] reported dramatic changes in the elution order over the temperature range -7°C to 35°C. On this column, the optimal separation was achieved at low temperatures... 

Brunner et al [1, 2] investigated separations of fatty acids according to chain length, using methyl esters of different carbon chain length from C14 to Cl8, separation of tocopherols from a by-product of the edible oil production and separation of fish oil esters [3], Stahl et al [4] proposed the supercritical fractionation of orange peel oil and Reverchon et al [5,6] of an orange flower concrete. Different authors treated citrus peel oil [7,8] and citrus oil [9-12]. 

Currently, high-performance liquid chromatography (HPLC) methods have been widely used in the analysis of tocopherols and tocotrienols in food and nutrition areas. Each form of tocopherol and tocotrienol can be separated and quantified individually using HPLC with either a UV or fluorescence detector. The interferences are largely reduced after separation by HPLC. Therefore, the sensitivity and specificity of HPLC methods are much higher than those obtained with the colorimetric, polarimetric, and GC methods. Also, sample preparation in the HPLC methods is simpler and more efficiently duplicated than in the older methods. Many HPLC methods for the quantification of tocopherols and tocotrienols in various foods and biological samples have been reported. Method number 992.03 of the AOAC International Official Methods of Analysis provides an HPLC method to determine vitamin E in milk-based infant formula. It could probably be said that HPLC methods have become dominant in the analysis of tocopherols and tocotrienols. Therefore, the analytical protocols for tocopherols and tocotrienols in this unit are focused on HPLC methods. Normal and reversed-phase HPLC methods are discussed in the separation and quantification of tocopherols and tocotrienols (see Basic Protocol). Sample... 

There are numerous papers that refer to the quantification of tocopherols and tocotrienols using HPLC methods. Normal-phase HPLC methods with a silica column as well as reversed-phase HPLC methods with a C18 column are commonly used. A silica normal-phase column is able to separate all eight tocopherols and tocotrienols in a typical chromatographic procedure. Because plant tissues possess most forms of tocopherol and tocotrienol, it is recommended that the normal-phase HPLC method be applied to food samples from plants. In the reversed-phase HPLC method, [3- and y-tocopherol and (3-and y-tocotrienol are not usually completely separated. This method can be used in animal tissues, which either lack or have reduced levels of [3- and y-tocopherol and (3- and y-tocotrienol. The resolution of the normal-phase HPLC method is higher than that of reversed-phase HPLC method however, the reversed-phase HPLC column is more long-lasting than the normal-phase HPLC column (see Critical Parameters and Troubleshooting). 

Compared to refined vegetable oils, the compositions of crude vegetable oils and oil and fat products are more complicated. These samples contain proteins, carbohydrates, and minerals that interfere with HPLC separation and reduce the lifetime of the HPLC column. These compounds need to be largely eliminated from the extract before HPLC analysis. Saponification and heating are used to weaken sample matrices to allow the solvent to fully access all tocopherols and tocotrienols of the sample. Liquid/liquid extraction is used to remove these polar compounds from the organic solvent layer that contains tocopherols and tocotrienols. The normal-phase HPLC method is usually used for crude vegetable oils and vegetable oil products reversed-phase HPLC can be used for animal fat products. 

In normal-phase HPLC on a silica column, separation is based on the number and position of methyl substituents on the chromanol ring. In reversed-phase HPLC on a Cl8 column, separation is based on the structure of the side chain and the number of methyl substituents. It is therefore difficult to completely separate [3-and y-tocopherol and (3- and y-tocotrienols by reversed-phase HPLC, because both have the same side-chain structure and number of methyl substituents on the chromanol ring. Only six peaks are usually found in the reversed-phase HPLC method. Thus, reversed-phase HPLC is recommended for samples from animal tissues, which contain little or no P and y vitamers. 

Column. Although the advantage of the normal-phase column is the high resolution in the separation of all tocopherols and tocotrienols, the silica packing material in a normal-phase column is very reactive to strong polar chemicals. Any high-polarity compounds in the sample extract and mobile phase will diminish the column performance and shorten the column lifetime. It is very important to ensure that the sample extract is free of water and metal ions, and it is also advisable to regenerate the column routinely. 

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