Lipid traditional methods

Other traditional methods available for monitoring the extent of lipid oxidation include the Anisidine value, the Kreis test (Mehlenbacher, 1960), methods based on the carbonyl content of oxidized fats (Henick et al., 1954 Lillard and Day, 1961), and measurement of oxygen uptake either by manometry or polarography (Tappel, 1955 Hamilton and Tappel, 1963). 

Today, HPLC is the dominant analytical technique used for the analysis of most classes of compounds. The analyses can be carried out at room temperature and the collection of fractions for reanalysis or further manipulation is straightforward. The main reason for the slow acceptance of the HPLC technique for Upid analysis has been the detection system. Traditionally, HPLC used ultraviolet/visible (UV/vis) detection, which requires the presence of a chromophore in the analyte. Most lipid molecules do not contain chromo-phores and therefore would not be detected by UV/vis. Modern HPLC detection techniques, such as the use of a mass spectrometer as the detector, derivatization techniques to introduce chromophores, and the availability of pure solvents to reduce interference, have allowed HPLC to compete with and/or complement GC and other traditional methods of lipid analysis. In addition to analytical HPLC, preparative HPLC has been used extensively to collect pure samples of the lipids for the derivatization or synthesis of new compounds. 

Matrix solid-phase dispersion (MSPD) was developed by researchers at Louisiana State University s School of Veterinary Medicine in order to isolate, identify, and quantify veterinary drug residues in livestock (Barker and Hawley, 1992). Compared to traditional methods, MSPD reduces solvent use by 98% and turnaround time by as much as 90%. The method involves the mechanical blending of a sample matrix with bulk C-18 sorbent. The C-18 hydrophobic phase has the ability to incorporate the lipids in meat and other food products into its matrix. Mechanical shearing forces initially disrupt the sample structure and disperse the sample over the surface of the C-18 bonded silica. The process causes the sample and polymer phase to become semidry, which then allow the material to be packed into a column (see Fig. 9.4). 

Of the many analytical techniques now available to the lipid chemist, mass spectrometry (MS), is probably the one that has experienced the fastest growth in the last two decades. This is due both to the development of new techniques (gas and liquid chromatography combined with MS, soft-ionization MS, field desorption MS, atmospheric pressure MS etc.) and to the refinement of more traditional methods and their successful application to very complex problems, e.g. the elucidation of glycolipid structure, or the study of structures in lipid mixtures. Much progress has been made since the pioneering work of Ryhage and Stenhagen (1963) on fatty acid methyl esters. 

The concentration of phospholipids in a lipid mixture can be estimated as phosphate after ashing the sample. The ash can be treated with nitric acid, dissolved in hydrochloric acid, and the phosphorus determined by inductively coupled plasma-atomic emission spectrometry at 214.9 nm. Alternatively, the phosphorus can be determined by atomic absorption spectrometry at 213.547nm with a graphite furnace. Traditional methods for phosphorus determination include the precipitation of phosphate as quinoli-nium molybdophosphate and gravimetric determination, or colorimetric methods such as the reaction with ammonium molybdate and acidic ammonium vanadate solution followed by determination of the absorbance at 460 nm. 

See also Carbohydrates Overview. Elemental Speciation Overview. Food and Nutritional Analysis Overview. Gas Chromatography Mass Spectrometry. Lipids Overview. Liquid Chromatography Liquid Chromatography-Mass Spectrometry Food Applications. Mass Spectrometry Overview Principles Ionization Methods Overview Atmospheric Pressure Ionization Techniques Eiectrospray Matrix-Assisted Laser Desorption/lonization Pyrolysis. Proteins Traditional Methods of Sequence Determination. Vitamins Overview. 

The technique of SCIR analysis has not previously been applied to the analysis of oils and fats, and it will therefore be strange to those familiar with the more traditional methods of lipid analysis. It is therefore appropriate to explain the background in a little more depth. 

As suggested above, the presence of salt and fat on the solid matrixes may influence the extraction of PAHs from solid matrixes. We applied the SPME-DED method for analysing the effect of the presence of salt and fat in the solid matrix on the analysis of PAHs. It is known that the presence of lipids in the matrixes hinders the release of some compounds during the extraction by SPME (Keszler and Heberger, 1999) or by other traditional methods such as saponification or liquid-liquid partition (Phillips, 1999 Moret and Conte, 2000) since lipophilic compounds, as PAHs, interact with the non-polar groups of lipids. 

Supercritical fluid extraction (SFE) is a technique in which a supercritical fluid [formed when the critical temperature Tf) and critical pressure Pf) for the fluid are exceeded simultaneously] is used as an extraction solvent instead of an organic solvent. By far the most common choice of a supercritical fluid is carbon dioxide (CO2) because CO2 has a low critical temperature (re = 31.1 °C), is inexpensive, and is safe." SFE has the advantage of lower viscosity and improved diffusion coefficients relative to traditional organic solvents. Also, if supercritical CO2 is used as the extraction solvent, the solvent (CO2) can easily be removed by bringing the extract to atmospheric pressure. Supercritical CO2 itself is a very nonpolar solvent that may not have broad applicability as an extraction solvent. To overcome this problem, modifiers such as methanol can be used to increase the polarity of the SFE extraction solvent. Another problem associated with SFE using CO2 is the co-extraction of lipids and other nonpolar interferents. To overcome this problem, a combination of SFE with SPE can be used. Stolker et al." provided a review of several SFE/SPE methods described in the literature. 

However, during the past three decades, an analytical method has been developed that currently rivals and may soon surpass the traditional liquid chromatographic techniques in importance for analytical separations. This technique, high-performance liquid chromatography (HPLC), is ideally suited for the separation and identification of amino acids, carbohydrates, lipids, nucleic acids, proteins, pigments, steroids, pharmaceuticals, and many other biologically active molecules. 

Prior to phospholipid analysis, it is imperative to extract the lipids from their matrix and free them of any nonlipid contaminants. Phospholipids are generally contained within the lipid fraction, which may be recovered by the traditional Bligh and Dyer or Folch extraction procedure (9,22). In any phospholipid extraction method it is recommended to include a rather polar solvent in addition to a solvent with high solubility for lipids. The former is needed to break down lipid-protein complexes that prevent the extraction of the lipids in the organic phase. Traditionally, mixtures of chloroform and methanol (especially 2 1, v/v) have been recommended. These are washed with water or aqueous saline to remove nonlipid contaminants. Comparing the recovery of phospholipids, Shaikh found that the neutral phospholipids PC, PE, SPH as well as DPG were nearly quantitatively extracted by all solvent systems studied (Table 1), although Bligh and Dyer, in which the lower phase was removed only once, was somewhat worse (23). 

In order to prevent the formation of a stable emulsion at any stage of the extraction procedure, the water content of the hydrated WPC has to be controlled so as not to obtain a biphasic solvent system during extraction with mixtures of chloroform and methanol. Besides, nonlipid contaminants are removed from the extract by gel filtration on nonlipophilic Sephadex G-25 instead of traditional aqueous washing total lipids were eluted with a 19/1 (v/v) mixture of chlo-roform/methanol, saturated with water, whereas a 1/1 (v/v) mixture of water and methanol eluted nonlipid contaminants. The method yields a similar total lipid content to the Folch method, but it is about four times faster (24). 

Ions and small molecules may be transported across cell membranes or lipid bilayers by artificial methods that employ either a carrier or channel mechanism. The former mechanism is worthy of brief investigation as it has several ramifications in the design of selectivity filters in artificial transmembrane channels. To date there are few examples where transmembrane studies have been carried out on artificial transporters. The channel mechanism is much more amenable to analysis by traditional biological techniques, such as planar bilayer and patch clamp methods, so perhaps it is not surprising that more work has been done to model transmembrane channels. 

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