Phenolic lipids separation

This chapter focuses on the use of different methods for isolation of alkylresorcinols. Alkylresorcinols are members of a lipid group called non-isoprenoid phenolic lipids. Different aspects of the extraction by classical methods and supercritical CO2 are discussed. Supercritical CO2 extraction of alkylresorcinols from rye bran is discussed for the first time. As compared to the classical extraction methods, supercritical CO2 gives higher yields and it allows the separation of the crude extrac into long- and short-chain alkylresorcinol homologues. 

For resorcinolic lipids, particularly those with long saturated side-chains, the use of polar solvents is important due to their amphiphilicity. The crude extracts in many cases are subjected to preliminary fractionation/purification either by solvent fractionation/partition or by application of chromatography. For prepurification of the material and its separation from polymerized phenolics, gel filtration on hydrophobic Sephadex or TSK gel is sometimes used. Silica gel is most frequently employed for the separation and/or purification of resorcinolic lipids, notably in some studies with Ononis species (12-14). The array of compounds reported appears partly attributable to methylation or acetylation reactions occurring during column chromatographic separation. An interesting approach for I the pre-purification and selective separation of resorcinolic lipid from phenolic lipids or resorcinolic lipids from impurities has recently been reported. A selective partitioning of different non-isoprenoid phenolic lipids... 

Supercritical CO2 extraction of AR or other phenolic lipids have been only sparcely reported (49,50). The solubility of a compound in a solvent depends on its physico-chemical properties. The ARs present in a crude extract, especially those with consecutive number of carbon atoms in the acyl chain (e.g. Cis and Ci7 homologues in rye alkyiresorcinols) have very similar physico-chemical properties. Therefore, when supercritical CO2 is used, the separation of individual homologues from crude extracts still remains a challenge. Illustrative studies on the extraction of phenolic lipids by supercritical CO2 are given in the following sections. 

Prior to structural elucidation and possible eventual synthesis, the isolation of component phenolic lipids in a pure state is essential. The cold methods of thin layer chromatography (TLC), column chromatography (CC), flash chromatography, high performance liquid chromatography (HPLC) and hot methods (GC), often after derivatisation, are well established. Argentation versions of these separatory methods are less common but are desirable for the rapid separation of unsaturated constituents. 

In general phenolic lipids have been separated from natural sources for compositional studies and structural determination by solvent extraction and chromatographic techniques. The individual component phenols of the major phenolic lipid (CNSL) from Anacardium occidentale have assumed some significance in certain chemical applications and detailed purification processes... 

CNSL used in polymerisation with formaldehyde as for example in friction dusts may not require elaborate analysis. Nevertheless interest in the industrial chemical uses of phenolic lipids has led to a study of quantitative methods of analysis by a variety of chromatographic methods. For cashew phenols these were first based on GLC. Thus the (15 3), (15 2), (15 1) and (15 0) constituents of methyl anacardate, cardol and cardanol have been separated by GLC on PEGA columns (ref.206), the free phenols (anacardic acid as methyl anacardate) by GLC on SE30 (ref207) and the hydrogenated anf fully methylated phenols on Dexsil and PEGA columns (ref.208). A further number of stationary phases have been investigated... 

A number of applications of commercial lacs and of separated urushiol have been referred to (ref. 2). As with the phenolic lipids of Anacardium occidentale a great deal of work has been carried out particularly in Japan and China to diversify the uses of lacs from Rhus vemicifera. It is widely employed in artistic decoration, building materials, textile equipment and furniture. The industrial utilisation of polyketide natural products including the phenolic lipid urushiol has been reviewed (ref. 314). The great number of uses largely comprise polymerisation reactions and some non-polymeric processes, some of both of which are described in the next sections. 

Typically lipids, chlorophyll, and phenolic acids can be separated by liquid-liquid partition. Lipids and chlorophyll can be removed from acetone-water extracts by chloroform while phenolic acids have higher affinities for ethyl acetate at a pH close to nentral and water. °°... 

Plant metabolism can be separated into primary pathways that are found in all cells and deal with manipulating a uniform group of basic compounds, and secondary pathways that occur in specialized cells and produce a wide variety of unique compounds. The primary pathways deal with the metabolism of carbohydrates, lipids, proteins, and nucleic acids and act through the many-step reactions of glycolysis, the tricarboxylic acid cycle, the pentose phosphate shunt, and lipid, protein, and nucleic acid biosynthesis. In contrast, the secondary metabolites (e.g., terpenes, alkaloids, phenylpropanoids, lignin, flavonoids, coumarins, and related compounds) are produced by the shikimic, malonic, and mevalonic acid pathways, and the methylerythritol phosphate pathway (Fig. 3.1). This chapter concentrates on the synthesis and metabolism of phenolic compounds and on how the activities of these pathways and the compounds produced affect product quality. 

Jansson et al. [189] used the conventional approach of blending the solid particles with solvent after which an aliquot was taken to determine the volatile compounds (e.g., phenols and chlorobenzenes). A second fraction was taken after the lipid removal for determination of compounds sensitive to concentrated sulfuric acid. The bulk lipids were removed by oxidative dehydration with Si02 /H2S04 and further cleaned-up with GPC. The chloroparaffins were isolated at this stage. Separation on silica isolated the OCPs, and the organochlorines and organobromines were finally fractionated on active charcoal. 

In the last years, the use of comprehensive liquid chromatography has been greatly increased and it has been widely used to separate and characterize various complex samples, such as biomolecules [10-15], polymers [16,17], lipids [18-21], essential oils [22], acidic and phenolic compounds [23-28], pharmaceuticals and traditional medicines [29-31], etc. Comprehensive LC has been reviewed by several authors [32-37]. 

The first edition of Food Analysis by HPLC fulfilled a need because no other book was available on all major topics of food compounds for the food analyst or engineer. In this second edition, completely revised chapters on amino acids, peptides, proteins, lipids, carbohydrates, vitamins, organic acids, organic bases, toxins, additives, antibacterials, pesticide residues, brewery products, nitrosamines, and anions and cations contain the most recent information on sample cleanup, derivatization, separation, and detection. New chapters have been added on alcohols, phenolic compounds, pigments, and residues of growth promoters. 

Further purification of the total lipid extract from neutral lipids like triacylglycerols is desirable to increase the separation performance and lifetime of reverse-phase HPLC columns. Several authors have utilized a procedure of base-acid wash for this purpose (Evershed et al., 1988 Seitz, 1989 Hakala et al., 2002), where one takes advantage of the phenolic nature of the steryl femlates. Total lipid extract in acetone or... 

Silica gel is the most extensively used adsorbent in thin layer chromatography because it leads to excellent, uncomplicated separations. It can be successfully employed for both qualitative and quantitative thin layer chromatographic analyses. It is usually used as a stationary phase in separations and analysis of alkaloids, various organic acids (especially amino acids and their derivatives), steroids, lipids, vitamins, plant pigments, pesticides, drugs, carbohydrates, phenolic substances, etc. 

Direct Measurements—UVNIS Techniques. The conjugated diene (CD) formed among the polyene hydroperoxide products that are formed as a result of oxidation of polyunsaturated fatty acids (PUFAs) have a UV absorbance that can be monitored to follow the progress of the oxidation. The effect of antioxidants on the suppressed rate of product formation can be followed with time. For example, conjugated dienes from oxidation of linoleate lipid molecules absorb at 234 nm and can be monitored directly , or else after HPLC separation (via normal phase or reverse phase j of the individual isomers. In order to use these findings to calculate the antioxidant activity of phenols and relate it to oxygen uptake studies (equations 7 and 14), one also has to make a correction to account for loss of absorbance due to loss (from decomposition) of hydroperoxides (equation 22) . 

Peat materials are often richer in nonhumified plant residues than in humus. Exhaustive alkaline extraction and estimation of humic substances are therefore hampered by the presence of hydrophobic lipids, and by coextraction of phenolic compounds from undecomposed plant residues, particularly from lignihed tissues. Artifacts may occur also during the solvation, separation, or concentration steps of the procedure, as discussed by Hayes in Chapter 13. Some of the problems can be mitigated by prior extraction of the lipids, soluble phenols, and sugars, as in the various schemes of proximate analysis discussed by Walmsley (1973). 

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