Lipid sample

A method which uses supercritical fluid/solid phase extraction/supercritical fluid chromatography (SE/SPE/SEC) has been developed for the analysis of trace constituents in complex matrices (67). By using this technique, extraction and clean-up are accomplished in one step using unmodified SC CO2. This step is monitored by a photodiode-array detector which allows fractionation. Eigure 10.14 shows a schematic representation of the SE/SPE/SEC set-up. This system allowed selective retention of the sample matrices while eluting and depositing the analytes of interest in the cryogenic trap. Application to the analysis of pesticides from lipid sample matrices have been reported. In this case, the lipids were completely separated from the pesticides. 

The basic technology for the preparation of sample material is similar in all TLC preparations, irrespective of the origin of the hpid and specific preparation method for a variety of biological samples [43]. The most important factor is the solubihty of the sample. The lipid sample must be completely soluble in the dissolving solvent prior to the application and must be free from water. Either toluene or chloroform is commonly used as the solvent to dissolve hpid materials. The dissolving solvent should be nonpolar in namre and volatile at such a concentration that the hpid components in the sample are completely adsorbed throughout the entire thickness of the layer as quickly as possible. Although sample sizes as small as 1 to 10 pi can... 

The amoimt of lipid applied to the plate varies depending on the ease of separation of individual lipid components in the sample. Usually 25 to 50 mg of the neutral lipid sample can be applied to a 20 X 20 cm preparative plate with the silica gel G layer thickness of 0.5 mm, whereas only about 4 mg of phospholipids can be applied on these plates. 

This is the most polar group of lipids in natural lipid samples. When developed in a nonpolar solvent system, phospholipids remain at the origin and more polar solvent system should be used to elute and separate individual phospholipids. The most popular system is the Wagner system, which consists of chloroform metha-nohwater (65 25 4) [51] for the separation of common phospholipid species in natural tissue samples. 

Rainey JK, Sykes BD (2005) Optimizing oriented planar-supported lipid samples for solid-state protein NMR. Biophys J 89 2792-2805... 

Avoid contamination by modern high lipid samples, e.g., keep ancient samples separate from modern samples (store and post separately) and avoid contact with other sources of oil (lunch, car engines, cigars, etc. ) whilst sampling. 

Only a very small proportion of the fatty acids are present in the free, unester-ified form and the vast majority are components of other lipids. Nevertheless it is important to be able to measure and identify the free fatty acids present in either form and for this they must be first extracted into an organic solvent and then usually converted to their methyl ester. The simplest method of methyla-tion, which is applicable to both esterified and non-esterified fatty acids, is to heat the lipid sample for 2 h under a current of nitrogen at 80-90°C with 4% sulphuric acid in methanol. After cooling and the addition of water, the resulting methyl esters are extracted several times into hexane and the combined extracts are dried over sodium carbonate and anhydrous sodium sulphate. The solvent fraction is then reduced in volume by a stream of nitrogen. 

Figure 10 shows the NMR spectrum of sonicated POPC-TTC vesicles at room temperature and selected pressures. Already the one-dimensional NMR spectra exhibit some interesting features. With increasing pressure, the signal intensity of the acyl-chain protons at 0.85 and 1.24 ppm decrease due to the pressure-induced rigidization of the acyl-chains, as it is also observed for pure phospholipid samples. At pressures above the fluid-gel main transition, which is detected at a pressure of about 1200 bar at 20 °C in pure POPC dispersions, the acyl-chain signals of pure lipid samples disappear completely, whereas in the spectra of the POPC-TTC system considerable signal intensities remain even up to pressures of 2800 bar. Furthermore, we observe for the... 

Determination of CholesteroL For meat extraction, the procedures for determining the cholesterol of extracted lipid samples were described Chao et al. (2i). For edible beef tallow extraction, the preparation of samples for cholesterol content was based on the AOAC (22) method Section 28.110. The prepared sample was then injected into a Supelco SPB-1 fused silica capillary column of 30 meters x 032 mm i.d. in a Varian Model 3700 gas chromatograph equipped with dual flame ionization detectors. The initial holdup time was 4 min at 270°C and then programmed to a temperature of 300°C at a ramp rate of 10°C/min. Helium flow rate and split ratio were 13 ml and 50 1, respectively, while the injector/detector temperature was 310°C. 

To remove lipids, sample extracts are frequently also partitioned with n-hexane (25, 33-35, 37, 40, 43, 45, 47, 49, 50, 53-57, 62), petroleum ether (31, 38, 63), isooctane (36, 41, 48), or toluene (26, 58, 59, 61). Use of n-toluene is not recommended, however, in chloramphenicol and florfenicol analysis, because these drugs have the tendency to transfer into toluene to some extent during the partitioning process. As an alternative to the classic liquid-liquid partitioning cleanup, some workers in the field (24, 26, 34, 58, 59) have suggested use of diatomaceous earth columns as another option of a liquid-liquid partitioning process that offers substantial reduction in emulsification problems and, thus, allows a high recovery increase. 

Liquid-liquid partitioning is intended either to extract the drugs from an organic solvent into an aqueous solution or to wash out interfering substances from organic or aqueous solutions. In general, quinolones are extracted from chloroform or ethyl acetate sample extracts into alkaline buffers, to then be back-extracted into chloroform or ethyl acetate at acidic conditions (191, 195, 196, 200). Occasionally, sodium chloride may be added to the sample extracts in order to increase the extraction efficiency of ethyl acetate or chloroform (193-196, 200). To remove lipids, sample extracts are often also partitioned with u-hexane (183-186, 193-196, 202, 204), or diethyl ether (189, 190, 201). 

Polyether antibiotics are hydrophobic compounds that are characterized chemically by their low polarities and their instability under acidic conditions. These antibiotics can be quantitatively extracted from the primary organic extract into carbon tetrachloride (393-395). When partitioning from a sodium chloride solution into an organic solvent, high yields have been achieved using dichloromethane (396, 397), carbon tetrachloride (391, 399), and chloroform (14, 398) as extraction solvents. In a different approach, water extracts containing lasalocid residues have been purified by partitioning into the mobile phase, which was a complex mixture of tetrahydrofuran, methanol, n-hexane, and ammonia (387, 389, 390, 392). To remove lipids, sample extracts have often been partitioned with n-hexane. 

For the lipid sample that you analyzed, report the fatty acid composition in mole %. Compare your results with those of other students. 

BASIC PROTOCOL I PREPARATION OF FATTY ACID METHYL ESTERS FROM LIPID SAMPLES CATALYZED WITH BORON TRIFLUORIDE IN METHANOL In this method, lipid samples are first saponified with an excess of NaOH in methanol. Liberated fatty acids are then methylated in the presence of BF3 in methanol. The resulting fatty acid methyl esters (FAMEs) are extracted with an organic solvent (isooctane or hexane), and then sealed in GC sample vials for analysis. Because of the acidic condition and high temperature (100°C) used in the process, isomerization will occur to those fatty acids containing conjugated dienes, such as in dairy and ruminant meat products, that contain conjugated linoleic acids (CLA). If CLA isomers are of interest in the analysis, Basic Protocol 2 or the Alternate Protocol should be used instead. Based on experience, this method underestimates the amount of the naturally occurring cis-9, trans-11 CLA isomer by -10%. The formulas for the chemical reactions involved in this protocol are outlined in Equation D1.2.1 Saponification RCOO-R + NaOH, RCOO-Na + R -OH v 100°C DC Esterification RCOO-Na + CH,OH r 3 v RCOO-CH, + NaOH ioo°c ... 

Extracted lipid sample in organic solvent (see Support Protocol)... 

Lipid samples are generally dissolved in 2 1 (v/v) chloroform/methanol if the lipid is extracted using a protocol similar to the Support Protocol, as both polar and neutral lipids are soluble in this mixture. 

PREPARATION OF FATTY ACID METHYL ESTERS FROM LIPID SAMPLES CATALYZED WITH SODIUM METHOXIDE IN METHANOL... 

In this method, lipid samples are first dissolved in dry toluene, then treated with sodium methoxide, an alkaline catalyst. Fatty acids in complex lipids are then transesterified to form their corresponding methyl esters. This is a relatively mild reaction and does not produce isomerization of conjugated dienes. The limitation of this method is that, unlike the BF3 method (see Basic Protocol 1), free fatty acids are not methylated and the procedure requires more time and glassware. 

It is important to keep the solvent ratio as close as possible to 8 4 3 (v/v/v) chloro-form/methanol/water, to prevent selective loss of lipid classes during this step (Christie, 1989a). Therefore, it is necessary to estimate the water content of the lipid sample before extraction and take this into consideration when determining the amount of 0.88% KCl needed. In addition, during filtration, a portion of the organic solution is lost due to evaporation directly from the funnel, increasing with filtration time. One should try to estimate the loss and adjust the volume of the 0.88% KCl based on empirical observation for a specific type of sample. 

Sample preparation is probably the most important step in any analytical procedure. Poor preparation of lipid samples will only yield inferior or questionable results. Some commonly performed sample-preparation procedures for gas-liquid chromatographic (GC) analysis of fatty acids in food samples are introduced in this unit. Since the introduction of gas chromatography in the 1950s, significant progress has been made in fatty acid analysis of lipids however, fatty acid methyl esters (FAMEs) are still the most commonly used fatty acid derivative for routine analysis of food fatty acid composition. 

Precautions should be taken to prevent oxidation during lipid analysis. Polyunsaturated fatty acids in lipid samples are easily attacked by active oxygen species (e.g., free radicals), exacerbated by the presence of strong light and metal ions. Therefore, it is arule of thumb while working with lipids that samples should be handled in a way that minimizes contact with air, light, and metals. To accomplish this, handle samples in glass vessels, use Teflon-lined or coated materials, and maintain the samples... 

The quality of FAME prepared by the methods described in this unit must be examined by GC analysis. Generally, impurities in the extracted lipid samples are not removed before methylation. If the GC results are not satisfactory due to sample contamination, additional steps may be necessary to clean the sample either before or after methylation. Commonly used techniques for purifying lipid samples are thin-layer chromatography (TLC), solid phase extraction (SPE), and column chromatography. 

Add 20 ml cyclohexane and swirl to dissolve the sample. Add 25 ml of Wijs solution using a pipet (not a graduated cylinder) for best control of data. Seal the flask with a glass stopper and place in a cool (25° to 30°C), dark place for 1 or 2 hr depending on expected IV (time must be consistent across samples). Also prepare a blank using all ingredients except the lipid sample. 

Prepare a lipid sample stock solution ( 20 mg/ml) by accurately weighing -200 mg total lipid into a 10-ml volumetric flask. Dilute to the mark with methylene chloride and mix well. 

Prepare lipid sample solution and calibration standard solutions as described (see Basic Protocol, steps 2 and 3) but include dihexadecanoyl cephalin in the standard solution. 

Weigh 100 mg lipid sample into a 125-ml flat-bottom flask with standard taper neck containing a Teflon-coated magnetic stir bar. Add 70 ml chloroform and 5 mg analytical-grade platinum oxide (catalyst). 

Lipid samples from natural sources generally contain various classes of lipid compounds in which concentration of individual lipid class varies substantially. Lipids from fat-rich tissues of biological samples are usually dominated by triglycerides. On the other hand, those from low-fat tissues tend to have even distribution of compounds among lipid classes. The range of calibration standard solutions described in this unit is the same for all lipid classes. To improve the accuracy in lipid-class quantification, it would always be a good approach to adjust the range of individual calibration standards based on the lipid class profile of lipids from particular sources. 

To provide constant humidity, Chromarods that have been spotted with a lipid sample are incubated in a constant humidity chamber for a fixed period of time (10 to 15 min) to reach equilibrium before being transferred to a developing tank. A saturated sodium chloride solution is widely used to provide constant humidity, as saturated solutions of inorganic salts give... 

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