Chromatographic separation of tocopherols

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Abstract

α-, β-, γ-, and δ-Tocopherols were separated by reversed-phase high-performance thin-layer chromatography (C18RP-HPTLC), normal-phase high-performance liquid chromatography (NP-HPLC), reversed-phase high-performance liquid chromatography (C18RP-HPLC), and gas chromatography (GC). The selected topological indices based on connectivity (M, 1χν), on distance matrix (W, °B, MTI) and on information theory (IAC, ĪAC) were calculated for these tocopherols. The observed chromatographic separations of investigated tocopherols were compared. This comparison indicated that the C18RP-HPTLC, NP-HPLC, and GC are the best techniques for the separation of these tocopherols. Topological index °B was the most significant. We obtained definite dependence between the numerical values of topological index °B and the chromatographic separation of the investigated tocopherols.

Introduction

Vitamin E has been an enigma in nutrition research for over 60 years. In 1937, Emerson et al. [1] described the existence of various vitamin E homologues that had different abilities to prevent vitamin E deficiency. In nature, vitamin E occurs in eight different forms (α-, β-, γ- and δ-tocopherols and α-, β-, γ- and δ-tocotrienols) with varying biologic activities. Tocopherols are being intensively studied owing to their medical, biological, and physico-chemical significance [2], [3], [4]; the biological properties of α-tocopherol are of particular importance [5], [6]. Of these eight compounds, α-tocopherol is reported to have the highest biological activity [7]. Tocopherol possesses three asymmetric carbon atoms. Because eight stereoisomeric tocopherols can be received. Natural α-tocopherol occurs as the enantiomer about the configuration 2R, 4′R, 8′R. Semi-synthetic α-tocopherol is the mixture of the diasteroisomers about configuration 2R/S, 4′R and 8′R. Although γ-tocopherol is a more effective free radical scavenger than α-tocopherol in vitro [8], the reverse is true in vivo [9]. The biological activity of vitamin E has generally been associated with its well-defined antioxidant property, specifically against lipid peroxidation in biological membranes. The antioxidative effect of the different tocopherols may not be identical. It has been shown repeatedly in antioxidative tests with feedstuffs and fat that the tocopherols have an antioxidative activity of the following order: γ-, δ-, β-, and α-tocopherols [10], [11]. Vitamin E occurs principally in wheat germ, vegetable oil, and vegetables. α-Tocopherol and γ-tocopherol are the most common of these eight naturally occurring vitamin E homologues in the human diet. Tocopherol is nearly insoluble in water, but is soluble in ethanol, ether, chloroform, acetone, and vegetable oils.

The problem of the separation of α-, β-, γ- and δ-tocopherols has been the subject of numerous reviews [12], [13].

The problem of the relationship between the structure of a molecule and its physical, chemical and biological properties is one of the most fundamental in chemistry. Therefore, the aim of these studies was to follow solutes of α-, β-, γ- and δ-tocopherols, which were separated by different chromatographic techniques and were characterized by selected topological indices. Table 1 lists general physicochemical data of investigated tocopherols.

Section snippets

Chemicals

If not otherwise specified, all used chemicals (analytical grade), reference tocopherols and HPTLC and HPLC solvents were from Merck, Darmstadt, Germany. Commercial samples of α-, β-, γ-, and δ-tocopherol (No. 15496, Merck) were used as test solutes.

Separation of α-, β-, γ-, and δ-tocopherols by RP-HPTLC

RP-HPTLC was performed on 10 cm×10 cm C18 reversed-phase UV254 HPTLC plates (No. 1.13724, Merck). Solution of a mixture of tocopherols was spotted on a chromatographic plate in quantities of 10 μg of each tocopherol in 2 μl of chloroform. Mixtures

Results and discussion

The separations of α-, β-, γ-, and δ-tocopherols on RP-C18 plates with HPTLC by use of ethanol and water in different volume ratios as mobile phases are introduced in Table 2. The best separations were achieved with mobile phases S1 and S2 (Table 2).

In preceding investigations the separation of the above-mentioned substances was achieved by partition TLC technique, obtaining similar separation [22].

The following investigations referred to separation of these tocopherols using NP-HPLC. Fig. 1

Conclusion

The chromatographic conditions which were applied in the NP-HPLC, C18 RP-HPTLC and GC can be used to separate α-, β-, γ-, and δ-tocopherols in various biological samples. Additionally, the structures of the particular investigated tocopherols are the best described by the topological index oB.

Further investigations concerning the physicochemical significance of the topological index oB in connection with the chromatographic data are being continued. Future investigations will also be

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