Lipid silica HPLC

Figure 2. Silica HPLC of myelin lipids. NC, nonhydroxycerebroside HC, hydroxycerebroside NS, nonhydroxysulfatide HS, hydroxy sulfatide and GD, monogalactosyl diglyceride. See text for details of TLC conditions.

Although sample cleanup on open silica columns or Sep-Pak silica cartridges (and so on) removes parts of the lipids that show different polarity in comparison to vitamin K, many equipolar lipids are collected together with the vitamin fraction. Using silica as stationary phase, only little variability is possible in the choice of solvent, due to the lipophilic character of the vitamin K molecules. Hexane with a content of 3% to 5% diethylether or minimal amounts of acetonitrile, ethyl acetate, or diisopropylether (up to 1%) can be used in these systems, with the consequence of a relative poor separation from interfering lipids. That is why in the last few years adsorption chromatography (inclusive semipreparative silica HPLC) is used only as a cleanup step in sample preparation for re-versed-phase HPLC (see Table 1). 

Plattner RDK, ayne-Wahl K. Separation of triglycerides by chain length and degree of unsaturation on silica HPLC columns. Lipids 1979 14 152—3. 

An excellent example of PLC applications in the indirect coupling version is provided by the works of Miwa et al. [12]. These researchers separated eight phospholipid standards and platelet phospholipids from the other lipids on a silica gel plate. The mobile phase was composed of methylacetate-propanol-chloro-form-methanol-0.2% (w/v) potassium chloride (25 30 20 10 10, v/v). After detection with iodine vapor (Figure 9.2), each phospholipid class was scraped off and extracted with 5 ml of methanol. The solvent was removed under a stream of nitrogen, and the fatty acids of each phospholipid class were analyzed (as their hydrazides) by HPLC. The aim of this study was to establish a standardized... 

Chloroform-methanol extracts of Borrelia burgdorferi were used for the identification of lipids and other related components that could help in the diagnosis of Lyme disease [58]. The provitamin D fraction of skin lipids of rats was purified by PTLC and further analyzed by UV, HPLC, GLC, and GC-MS. MS results indicated that this fraction contained a small amount of cholesterol, lathosterol, and two other unknown sterols in addition to 7-dehydrocholesterol [12]. Two fluorescent lipids extracted from bovine brain white matter were isolated by two-step PTLC using silica gel G plates [59]. PTLC has been used for the separation of sterols, free fatty acids, triacylglycerols, and sterol esters in lipids extracted from the pathogenic fungus Fusarium culmorum [60]. 

Takafuji, M., Rahman, A.M., Ansarian, H.R., Derakhshan, M., Sakurai, T., and Ihara, H., Dioctadecyl L-glntamide-derived lipid-grafted silica as a novel organic stationary-phase for RP-HPLC, J. Chrvmatogr. A, 1074, 223, 2005. 

The major difficulty in analyzing OPPs in fatty samples has to do with the wide polarity range for both pesticides and lipids present in the matrix. Normal-phase HPLC is an adequate technique for cleaning up this type of sample using silica gel and modifiers with different polarity. In fact, an automated sample-cleanup system based on normal-phase HPLC using a silica gel column has been reported efficiently to clean up and fractionate chlorpyriphos, chlorpyriphos methyl, and their metabolites in molluscs. The system presents several advantages The procedure is fully automated, from the injection of the extract to the collection of fractions, which are injected directly into the GC system, and a diode array detector (DAD) allows online monitoring of the elution of lipids (68). 

Mussels NP-HPLC, online 3.9-mm-ID X 15-cm Silica Nova-pack, 4 fim w-Hexane to elute lipids hexane-ethyl acetate to remove pesticide... 

Myelin galactolipid analysis by HPLC. Fig. 2 and 3 show HPLC chromatograms obtained from myelin lipids on Silica column and reverse phase column, respectively. Reverse phase HPLC of mono-galactosyl diglyceride and its 1-0-alkyl ether homolog was not examined but typical chromatograms of these lipids obtained from calf brain stem were presented previously (11 ). Myelin was obtained from 25 day-old rat brains. 

Such new RP-HPLC stationary-phase materials have been available for some years (Regis Chemical Company, Morton Grove, IL, USA). These so-called immobilized artificial membrane (IAM) columns consist of lipid molecules covalently bound to propylamine-silica. The unreacted propylamine moieties are end-capped with methylglycolate. The membrane lipid, phosphatidylcholine, possesses polar head groups and two non-polar hydrocarbon chains (C18). One of the alkyl chains is linked to the propylamine-silica surface. 

A method using HPLC has also been proposed (Schulte, 1994) whereby an oil solution is first eluted through a silica gel column to separate the steradienes from the more polar lipids. The eluate is then concentrated and analysed on a reverse phase HPLC column using UV detection at 235 nm. This method involves a little more sample preparation than the LC-GC method proposed by Grob et al. (1992) but the equipment is more widely available. 

As regards the lipids, two types of adsorbents are available, one of which is a form of silica gel and is utilized in normal-phase HPLC, and the other of which can be a silica gel bonded to a hydrophobic chain and is employed in reverse-phase HPLC. In normal-phase HPLC the phospholipids appear to be separated based on the molecular classes present (PE, PC, Sph, etc.), whereas in reverse-phase HPLC the separation is closely related to the lipophilic character of the acyl (fatty acyl, hydrocarbon chain) of the particular phospholipids. High-quality adsorbents suitable for HPLC are easily available from commercial companies. 

In an interesting experimental protocol, Silvestro et al. (1993) utilized HPLC-mass spectrometry with an ion spray (electrospray) interface for determination of PAF and lysoPAF in human PMN (neutrophils). Both unstimulated and stimulated (with complement-activated zymosan) cells were used as starting material. The total lipids were isolated in the usual way, and the PAF was isolated and purified by a combination of thin-layer chromatography, HPLC, and silica chromatography. This final PAF preparation was subjected to a bioassay with the inclusion of 3H 16 0 PAF to monitor recoveries. 

Determination of free 4-hydroxy-2,3-trans-alkenals by HPLC Esterbauer (1982) has developed a procedure for the qualitative detection and quantitative measurement of steady-state concentrations of free hydroxyalkenals (specifically HNE) in tissues, tissue extracts and lipid containing foodstuffs. Their method utilizes UV-detection of the free aldehyde at its 220 nm UV-absorption maximum and peak identification was confirmed by mass spectrometry. An effective purification and concentration step is employed using dichloromethane to extract hydroxyalkenals from samples trapped on Extrelut columns. The samples are subsequently purified by solid-phase extraction on octadecyl-bonded silica (ODS) disposable cartridges and then analysed by HPLC. 

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). 

Five inositolphosphosphingolipids (compounds II, m, V, VI and VIII) have been isolated, purified and analyzed from the yeast phase of H. capsulation [61], a dimorphic fungus that causes histoplasmosis. Cells uniformly labeled with [3H] inositol and [32P] were treated with 5% TCA at 0°C and extracted according to Hanson and Lester s method [62]. Isolated lipids were purified by preparative liquid chromatography (HPLC) on silica gel columns and utilized for structural analysis. 

---