The use of countercurrent chromatography in the separation of nonpolar lipid compounds.

Isolation of lipophilic compounds by countercurrent chromatography (CCC) is a challenge because biphasic solvent systems in which these compounds distribute evenly are difficult to obtain. In this article we present novel applications of lipid compound isolation from natural sources. Conjugated linolenic acids (CLnAs, log KOW ∼7) were isolated from pomegranate oil using a solvent system consisting of n-heptane/methanol/water 100:91:9 (v/v/v). The CLnA fraction was free of other fatty acids but consisted of different isomers which were not resolved from each other. In the less polar range (log KOW ∼12), three tocotrienols (α-, γ- and δ-tocotrienol) were isolated from a vitamin E capsule produced from palm oil by using the solvent system n-hexane/acetonitrile/benzotrifluoride (BTF) at a ratio of 10:6.5:3.5 (v/v/v). Between 36 and 65mg of each of the three tocotrienols were obtained in one injection with purities >97%. Advantages and disadvantages of the "BTF system" are discussed by comparing the phase composition with the simple n-hexane/acetonitrile system and by the fractionation of phytosterols (log KOW ∼9.5) from rapeseed oil.

Development of solvent systems with room temperature ionic liquids for the countercurrent chromatographic separation of very nonpolar lipid compounds.

Solvent systems are not readily available for the separation of very nonpolar compounds by countercurrent chromatography (CCC). In this study we therefore evaluated the suitability of room temperature ionic liquids (IL) in organic solvents for the CCC separation of the extremely nonpolar lipid compounds tripalmitin (PPP) and cholesteryl stearate (CS). The four IL tested were [C10mim][OTf], [C2mim][NTf2], [P66614][NTf2], and [P66614][Cl]. Search for a CCC-suited solvent system started with solubility studies with fourteen organic solvents. Following this, combinations were made with one organic solvent miscible and one organic solvent immiscible with IL (147 combinations). Twenty-four initially monophasic mixtures of two organic solvents became biphasic by adding IL. Several unexpected results could be observed. For instance, n-hexane and n-heptane became biphasic with [P66614][Cl]. Further nine systems became biphasic although the IL was not miscible in any of the two components. These 33 solvent systems were investigated with regard to phase ratio, settling time, share of IL in the upper phase and last not least the KU/L values of PPP and CS, which were 8.1 and 7.7 respectively. The most promising system, n-heptane/chloroform/[C10mim][OTf] (3:3:1, v/v/v) allowed a partial separation of PPP and CS by CCC which was not achieved beforehand.

Isolation of a furan fatty acid from Hevea brasiliensis latex employing the combined use of pH-zone-refining and conventional countercurrent chromatography.

Furan fatty acids are valuable and bioactive minor fatty acids that usually contribute <0.1% to the fatty acid content of food samples. Their biological role still remains unclear as authentic furan fatty acid standards are not readily available and thorough experimental studies verifying the relevance of furan fatty acids are thus virtually impossible. An efficient protocol for the isolation of the furan fatty acid 9-(3-methyl-5-pentylfuran-2-yl)-nonanoic acid from hydrolyzed and centrifuged latex of Hevea brasiliensis was developed using countercurrent chromatography. A first run using pH-zone-refining countercurrent chromatography provided 48.4 mg of 9-(3-methyl-5-pentylfuran-2-yl)-nonanoic acid from 210 mg latex extract in a purity of 95%. The purity was increased to 99% by means of one second run in conventional countercurrent chromatography mode. The Structure and purity of 9-(3-methyl-5-pentylfuran-2-yl)-nonanoic acid were determined by gas chromatography coupled to mass spectrometry and (1)H and (13)C NMR spectroscopy.

Accelerated separation of GC-amenable lipid classes in plant oils by countercurrent chromatography in the co-current mode.

Triacylglycerols represent the major part (>90%) in most plant oils and have to be eliminated, when the minor compounds such as phytosterols or tocopherols should be analyzed. Here, we used an all liquid-liquid chromatographic technique, countercurrent chromatography (CCC), to fractionate the minor lipids before gas chromatography (GC) analysis. To cover the wide range of polarity of the minor compounds, we used the co-current mode, in which both mobile and stationary phase are pumped through the system. This allowed to elute substances which partitioned almost exclusively in the stationary phase within 90 min. After testing with standard compounds, the method was applied to the separation of sesame oil and sunflower oil samples. The abundant triacylglycerols could be effectively separated from tocopherols, phytosterols, diacylglycerols, and free fatty acids in the samples, and these compounds could be analyzed (after trimethylsilylation) by GC coupled with mass spectrometry. After the enrichment caused by the CCC fractionation, we were also able to identify the tocopherol derivative α-tocomonoenol, which had not been described in sunflower oil before. Also, separation of sesame oil yielded a mixture of the polar compounds sesamin and sesamolin without further impurities.

Heart-Cut Two-Dimensional Countercurrent Chromatography with a Single Instrument.

Countercurrent chromatography (CCC) is a separation technique based on the liquid-liquid partitioning of compounds between the two phases of a biphasic solvent system. Limitations in the separation efficiency and peak capacity of one-dimensional (1D) CCC often result in insufficiently resolved peaks. Therefore, partially resolved peak fractions have to be rechromatographed in order to improve the yield and purity. Additional solvent evaporation steps can be circumvented by the application of two-dimensional CCC (2D CCC). Existing 2D CCC configurations are based on the linking of two CCC instruments which are not readily available in every laboratory. In this study, we introduce a technical improvement which allows performing multiple heart-cuts and 2D CCC separations with one instrument which has up to four independent coils, one pump, and one detector. For this purpose, we modified a commercially available CCC system by the addition of three six-port selection valves and a T-piece. The applicability of the 2D CCC system was shown under overloading conditions with eight alkyl hydroxybenzoates with nonideal conditions. We used two solvent systems which were hydrodynamically compatible and which showed different selectively characteristics with regard to the compounds. Four unresolved compounds in the first dimension were baseline resolved by means of the heart-cut technique, in which between 4 and 9 mL of the flow from coil 1 was transferred to coil 2. Three successive heart-cuts were performed that led to baseline resolution of unresolved compounds in the first dimension. The obtained recovery rates were 94-100%, and the purities of the compounds as determined by GC/MS were 90-100%.

Tubing modifications for countercurrent chromatography (CCC): Stationary phase retention and separation efficiency.

Countercurrent chromatography (CCC) is a separation technique in which two immiscible liquid phases are used for the preparative purification of synthetic and natural products. In CCC the number of repetitive mixing and de-mixing processes, the retention of the stationary phase and the mass transfer between the liquid phases are significant parameters that influence the resolution and separation efficiency. Limited mass transfer is the main reason for peak broadening and a low number of theoretical plates along with impaired peak resolution in CCC. Hence, technical improvements with regard to column design and tubing modifications is an important aspect to enhance mixing and mass transfer. In this study we constructed a crimping tool which allowed us to make reproducible, semi-automated modifications of conventional round-shaped tubing. Six crimped tubing modifications were prepared, mounted onto multilayer coils which were subsequently installed in the CCC system. The stationary phase retention of the tubing modifications were compared to the conventional system with unmodified tubing in a hydrophobic, an intermediate and a hydrophilic two-phase solvent system. Generally, the tubing modifications provided higher capabilities to retain the stationary phase depending on the solvent system and flow rates. In the intermediate solvent system the separation efficiency was evaluated with a mixture of six alkyl p-hydroxybenzoates. The peak resolution could be increased up to 50% with one of the tubing modifications compared to the unmodified tubing. Using the most convincing tubing modification at fixed values for the stationary phase retention, a reasonable comparison to the unmodified tubing was achieved. The peak width could be reduced up to 49% and a strong positive impact at increased flow rates regarding peak resolution and theoretical plate number was observed compared to unmodified tubing. It could be concluded that the tubing modification enhanced the interphase mixing and mass transfer of the two phases by additional and more vigorous agitation.

Overcoming the equivalent-chain-length rule with pH-zone-refining countercurrent chromatography for the preparative separation of fatty acids.

Purification of individual fatty acids from vegetable oils by preparative liquid chromatography techniques such as countercurrent chromatography (CCC) is a challenging task due to the equivalent-chain-length (ECL) rule. It implies that one double bond equals two carbon atoms in the alkyl chain of a fatty acid and therefore causes co-elutions of saturated and unsaturated fatty acids. Accordingly, existing methods for the purification of individual fatty acids are cumbersome and time-consuming as two or more steps with different conditions are required. To avoid additional purification steps, we report a method utilizing pH-zone-refining CCC which enabled the purification of all major fatty acids from sunflower oil (purities >95 %) in one step by circumventing co-elutions caused by the ECL rule. This method is based on the involvement of acid strength and hydrophobicity of fatty acids during the separation process. By exploiting the preparative character of the pH-zone-refining mode, a tenfold sample amount of free fatty acids from sunflower oil could be separated in comparison to regular CCC.

Isolation of ß-carotene, α-carotene and lutein from carrots by countercurrent chromatography with the solvent system modifier benzotrifluoride.

A carotenoid purification method with dual-mode countercurrent chromatography (CCC) for ß-carotene, α-carotene and lutein from a fresh carrot extract was developed. The fluorinated liquid benzotrifluoride (IUPAC name: (trifluoromethyl)benzene) was used as a novel modifier in the non-aqueous ternary solvent system n-hexane/benzotrifluoride/acetonitrile. The ternary phase diagram of the type I solvent system was used to select two-phase solvent mixtures which enabled an efficient preparative separation of α-carotene, ß-carotene and lutein from concomitant pigments in crude carrot extract. By means of the modifier, high separation factors (α ≥ 1.2) were obtained, allowing baseline resolution between α-carotene and ß-carotene due to specific chemical interactions such as π-π molecular interactions. After optimizing the injection step with a pseudo-ternary phase diagram, 51 mg of ß-carotene, 32 mg of α-carotene and 4 mg of lutein could be isolated from 100.2mg crude carrot extract in a short time and with high purities of 95% and 99% by using dual-mode CCC, respectively. Temperatures > 22°C had a negative impact on the separation of α-carotene and ß-carotene.

Solvent systems with n-hexane and/or cyclohexane in countercurrent chromatography--Physico-chemical parameters and their impact on the separation of alkyl hydroxybenzoates.

Countercurrent chromatography (CCC) is an efficient preparative separation technique based on the liquid-liquid distribution of compounds between two phases of a biphasic liquid system. The crucial parameter for the successful application is the selection of the solvent system. Especially for nonpolar analytes the selection options are limited. On the search for a suitable solvent system for the separation of an alkyl hydroxybenzoate homologous series, we noted that the substitution of cyclohexane with n-hexane was accompanied with unexpected differences in partitioning coefficients of the individual analytes. In this study, we investigated the influence of the subsequent substitution of n-hexane with cyclohexane in the n-hexane/cyclohexane/tert-butylmethylether/methanol/water solvent system family. Exact phase compositions and polarity, viscosity and density differences were determined to characterize the different mixtures containing n-hexane and/or cyclohexane. Findings were confirmed by performing CCC separations with different mixtures, which led to baseline resolution for positional isomers when increasing the amount of cyclohexane while the resolution between two pairs of structural isomers decreased. With the new methodology described, structurally similar compounds could be resolved by choosing a certain ratio of n-hexane to cyclohexane.