Phospholipids separation techniques

Preparative thin-layer chromatography, therefore, has much merit as a separation technique and should be considered seriously as an excellent tool in isolation of sufficient quatities of particular phospholipids for further chemical or biochemical study. 

Lipids are made up of many classes of very different molecules that all show solubility properties in organic solvents. Mass spectrometry plays a key role in the biochemistry of lipids. Indeed, mass spectrometry allows not only the detection and determination of the structure of these molecules but also their quantification. For practical reasons, only the fatty acids, acylglycerols and bile acids are discussed here, although other types of lipids such as phospholipids, [253-256] steroids, [257-259] prostaglandins, [260] ceramides, [261,262] sphingolipids [263,264] and leukotrienes [265,266] have been analysed successfully by mass spectrometry. Moreover, the described methods will be limited to those that are based only on mass spectrometry, even if the majority of these methods generally are coupled directly or indirectly with separation techniques such as GC or HPLC. A book on the mass spectrometry of lipids was published in 1993. [267]... 

Lipids may be quantified by charring with either dichromate or H2SO4 and then analyzed by photodensitometry (112). The charring technique may be combined with liquid scintillation, a procedure often used to quantify radiolabeled lipids (113). Phospholipids separated by TLC and then eluted from the plates may be quantified by classical wet chemical determination techniques for phosphorus (114). Similar techniques, although infrequently used, are also available for the hydrolysis products of various neutral lipids (46). Gravimetric methods can also be used to quantify lipids separated by TLC, but such methods may be unreliable since small amounts of sorbent, binder, or other... 

The properties of membranes commonly studied by fluorescence techniques include motional, structural, and organizational aspects. Motional aspects include the rate of motion of fatty acyl chains, the head-group region of the phospholipids, and other lipid components and membrane proteins. The structural aspects of membranes would cover the orientational aspects of the lipid components. Organizational aspects include the distribution of lipids both laterally, in the plane of the membrane (e.g., phase separations), and across the membrane bilayer (phospholipid asymmetry) and distances from the surface or depth in the bilayer. Finally, there are properties of membranes pertaining to the surface such as the surface charge and dielectric properties. Fluorescence techniques have been widely used in the studies of membranes mainly since the time scale of the fluorescence lifetime coincides with the time scale of interest for lipid motion and since there are a wide number of fluorescence probes available which can be used to yield very specific information on membrane properties. 

Based on the 96-well format, OCT-PAMPA was proposed and has proved its ability to determine (indirectly) log Poet [87]. PAM PA is a method, first developed for permeability measurements, where a filter supports an artificial membrane (an organic solvent or phospholipids) [88, 89]. With this method, the apparent permeability coefficient (log P ) of the neutral form of tested compounds is derived from the measurement of the diffusion between two aqueous phases separated by 1-octanol layer (immobilized on a filter). A bilinear correlation was found between log Pa and log Poct> therefore log Poet of unknown compounds can be determined from log Pa using a calibration curve. Depending on the detection method used a range oflog P within —2 to +5 (with UV detection) and within —2 to +8 (with LC-MS detection) was successfully explored. This method requires low compound amounts (300 pi of 0.04 mM test compound) and, as for the previous method, samples can be prepared in DM SO stock solutions. For these experiments, an incubation time of 4h was determined as the best compromise in term of discrimination. The limitation of the technique lies in the lower accuracy values... 

Measurements of the quantities of glycolipids inserted into the membrane have also been reported by a technique based on the use of C-labeled lipid anchors. In this method, the carbohydrate (a-o-Man) was covalently coupled to the anchor at the surface of a pre-formed vesicle. Indeed, the liposome structure was shown to remain intact in the treatment. Nevertheless, the measurement of the incorporated mannose was performed after separation of bound and unbound material by centrifugation. The yields of coupling were shown to increase with the increase of the initial mannose/ C-anchor ratio, but non covalent insertions were displayed at high initial mannose concentrations. Therefore, the aforementioned method was not as accurate as could have been expected for the use of radioactive materials [142]. Radiolabeled phospholipids were also used for such determinations thus the amounts of glycosphingolipids incorporated into liposomes were quantified by the use of H-phospholipids whereas the amounts of glycolipids were determined by a sphingosine assay [143]. 

The separation of phospholipids by micellar electrokinetic capillary electrophoresis (MEKC) has been described (17-19). In this technique, solutes are separated based on their distribution between a mobile (usually aqueous) and a pseudostationary (micellar) phase. Szucs et al. found that the major soybean phospholipids were fully resolved in only 7 minutes using deox ycholic acid for micelle formation in combination with 30% n-propanol at 50°C (18). However, quantification of the separated compounds remains troublesome. This is due first of all to the fact that only UV detection can be used, thus making the response highly dependent on the degree of unsaturation of the phospholipids. Besides, the comparison of peak areas in MEKC is more complicated than in HPLC, because all compounds are moving with different velocities. 

Actually, solid-phase extraction is used not only as a rough preliminary fractionation procedure. Prieto et al. described the complete fractionation of the total lipids from wheat into eight neutral lipid, two glycolipid, and four phospholipid classes in addition to PC and LPC, TV-acyl PE and A-acyl LPE were detected (37). However, two separate stationary phases (silica and aminopropyl) as well as seven different mobile phases were needed. Moreover, 14% crosscontamination of PC and LPC was observed, and the recovery of the phospholipids was limited to about 85%. Hence, SPE is a rapid and efficient technique for preliminary fractionation, but loses its advantages if more complex separations are tried. 

Chemical analysis of lipid composition has been described previously In detail. (Ill The technique employs der1 vat 1zat1 on by methylatlon of phospholipid acyl chains followed by separation and analysis with capillary gas chromatography. 

The major challenge in any study in which phospholipids are central components in a biological reaction of interest or in which there is evidence for a new type of phospholipid is their identification. While there are many excellent techniques available to accomplish this goal, there is absolutely no magic route to a satisfactory identification of specific phospholipids in a crude lipid extract except through the use of separation procedures. Basically it is next to impossible to identify or detect a specific phospholipid species in such a complex mixture. Thus, one must bite the bullet and resort to several analytical techniques for definitive proof. Perhaps the most important facet of this approach is that the compounds must be extracted from a tissue and then subjected to chromatographic separation. 

In the usual instance it is necessary to use more than one analytical tool for identification (and separation) of the phospholipids in a biological extract. Other chromatographic techniques of value will be discussed later. However, now it is worthwhile to describe a methodology by which a nearly quantitative... 

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