Lipid analysis purification

As plant extracts mainly comprise large amonnts of ballast substances (e.g., lipids and chlorophylls), their purification is often a priority in the analysis. Such purification can be expensive in terms of both time and solvent consumed and can lead to losses of sample components. Online purification and separation of extracts contaminated with plant oil, can be readily performed by TLC in equilibrium chambers [1] that enable the use of continuous elution. 

By means of gel electrophoresis on cross-linked, hydrolyzed starch,99 with simultaneous checking for proteins, lipids, and pectinesterase activity, it was found, however, that the product isolated after the separation on CM-Sephadex C-50 constitutes but one of five multiple forms of tomato pectinesterase, and is the one present in preponderant proportion98 (see Fig. 4). The accompanying lipid and sugar components were separated from this pectinesterase form in the course of the purification procedure. After analysis of the hydro-lyzate of the final product for fatty acids, as well as for carbohydrate components, it was possible to exclude the possibility of a lipoprotein,30 as well as glycoprotein,100 character of this form of tomato pectinesterase. 

Takayama and coworkers (60) introduced the h.p.l.c. separation technique for such amphiphilic molecules as lipid A, and in earlier experiments they applied paired-ion reverse-phase h.p.l.c. for the preparation of homogeneous fractions deriving from 4,-monophosphated lipid A of S. typhimur-ium. The purified preparations obtained were suitable for f.a.b. - m.s. analysis. However, monophosphated lipid A isolated in this way expressed a considerable heterogeneity with respect to the number and location of 0-acyl residues (60). In order to further improve the purification procedure, as well as to obtain lipid A derivatives suitable for n.m.r. spectroscopy, Qureshi et al. (174) prepared the dimethyl phosphate derivative of S. minnesota (R595) lipid A, which, after purification by reverse-phase h.p.l.c. (C18), could be analyzed by1 H-n.m.r. The n.m.r. spectrum of, for example, the heptaacyl lipid A dimethyl monophosphate fraction, unequivocally revealed 0-acyl substitution [14 0(3-OH)J at position 3 and a free hydroxyl group at position 4 of GlcN(I). 

Most existing methods are based on instrumental analysis involving exhaustive sample pretreatment and preconcentration steps, followed by purification and fractionation before final chromatographic separation and detection. For fat and oil samples, dissolving the lipids in an appropriate solvent is usually the first treatment. This has been achieved by melting the fat at 90°C followed by LLE or direct solid liquid extraction (SEE) with an apolar solvent [37], column extraction with a mixture of apolar solvents after drying of the sample with anhydrous Na2S04, Soxhlet extraction and/or sonication with apolar solvents. Typically, sample intake is between 0.5 g and 1 g and quantitative recoveries >60% have been reported. 

Structure-Function Dissection and sequence analysis of phospholipases A2, 197, 201 cloning, expression, and purification of porcine pancreatic phospholipase A2 and mutants, 197, 214 preparation of antibodies to phospholipases A2, 197, 223 thermodynamics of phospholipase A2-ligand interactions, 197, 234 activation of phospholipase A2 on lipid bilayers, 197, 249 ... 

Retinoids The challenge in fat-soluble vitamins analysis is to separate them from the lipid fraction that contains interferents. Alkaline hydrolysis, followed by LLE, is widely applied to remove triglycerides. This technique converts the vitamin A ester to all-trani-retinol. A milder process, which does not hydrolyze vitamin A ester, is alcoholysis carried out with metha-nolic KOH solution under specific conditions that favor alcoholysis rather than saponification. A more accurate explanation of this technique is reported in the book Food Analysis by FIPLC [409]. For some kind of matrices a simple liquid extraction can be sufficient with [421-423] or without [424,425] the purification... 

This experiment provides students with the opportunity to isolate a biomolecule from its natural source, followed by its purification and identification. In addition, students will follow a procedure that is typical of the general extraction and characterization of lipids. However, unlike most lipids, the plant pigments are highly colored and may be characterized and quantified by visible spectrophotometry. Several types of plant tissue may be used. Some recommendations are fresh leaves (tree, plant, grass, spinach), green algae, or mosses. For variety, students may be asked to bring their own samples for analysis. 

Nelson, G.J. 1991. Isolation and purification of lipids from biological matrices. In Analysis of Fats, Oils and Lipoproteins (E.G. Perkins, ed.) pp. 20-59, American Oil Chemists Society, Champaign, 111. 

Anthocyanins are generally more stable at an acidic pH. Therefore, anthocyanins are commonly extracted under cold conditions using either acidic methanol or ethanol to avoid degradation1 5169 (Table 3.4). In comparison, acetone allows more reproducible extraction and avoids problems with pectins. However, it is limited by the coextraction of proanthocyanins.39 In general, ethanol is preferable as an extraction solvent, although it can require an additional step for the removal of lipid-soluble substances. SPE using Ci8, polyamide, HLB (hydrophilic lipophilic balanced stationary phases), or Amberlite has been employed for the purification of anthocyanidins prior to HPLC analysis.39-51 66 69... 

Abstract Lipopolysaccharides are the major components on the surface of most Gram-negative bacteria, and recognized by immune cells as a pathogen-associated molecule. They can cause severe diseases like sepsis and therefore known as endotoxins. Lipopolysaccharide consists of lipid A, core oligosaccharide and O-antigen repeats. Lipid A is responsible for the major bioactivity of endotoxin. Because of their specific structure and amphipathic property, purification and analysis of lipopolysaccharides are difficult. In this chapter, we summarize the available approaches for extraction, purification and analysis of lipopolysaccharides. 

Keywords Lipopolysaccharide LPS Lipid A Extraction Purification Analysis... 

As an important membrane anchored molecule in Gram-negative bacteria that can activate the immune response, LPS and lipid A are largely needed for research to understand infection mechanism of bacterial pathogens. Methods for extraction and purification of LPS and lipid A still need to be modified to increase the yield and purity, so do the methods for analysis of LPS. 

Kane, C. D.,Coe, N. R., Vanlandingham, B., Krieg, P., Bernlohr, D. A. (1996). Expression purification, and ligandbinding analysis of recombinant keratinocyte lipid-binding protein (MAL-1), an intracellular lipid-binding found overexpressed in neoplastic skin cells. Biochemistry 35, 2894-2900. 

NP-HPLC Normal-phase liquid chromatographic methods applying Diol-columns or common silica columns are well suited for the analysis of the total steryl ferulate content. They require very little sample preparation, as total lipid extracts can frequently be directly injected into the column without purification or fractionation. Run times for SFs are also relatively short, and a good separation from other lipid components can be obtained in less than 10 min in traditional HPLC systems. Depending on the column type and the sample, SFs elute as one or two peaks. Two peaks are obtained from the separation of SFs, which have ferulic acid both in cis- and trans- configuration (Nystrom et ah, 2008). The relative retention time (obtained with a silica column and hexane/ethyl acetate 97 3 as eluent) of the cis- form is about 0.5 smaller than that of steryl irans -ferulates (Akihisa et al., 2000). 

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