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Rafael B. Navarro University of the Philippines Los Baños Los Baños, Laguna, Philippines 4024 firstname.lastname@example.org
ABSTRACT Lipids are diverse biomolecules which serve as energy sources, signaling molecules, and main components of the cell membrane. Lipids in a mixture or biological sample can be identified and quantified using various methods such as spectroscopy and chromatographic techniques such as thin-layer chromatography. In this experiment, lipid components of different food samples such as milk, margarine, egg yolk, chicken skin and pork rind were extracted with ethanol-ether (2:1 v/v) and analyzed with the classical method of thin-layer chromatography. The stationary phase used was silica and the mobile phase was a solvent system containing petroleum ether, diethyl ether, and glacial acetic acid (70:23:3 v/v). The separation of lipids on the plate was visualized through halogenation using iodine vapor. The retention factor, Rf, of each band was calculated and results had shown that samples contain phospholipids (chicken skin and egg yolk), fatty acids (milk, pork rind, chicken skin, margarine), triglycerides, and cholesterol (except margarine). Keywords: Thin-layer Chromatography, Triglyceride, Cholesterol, Silica, Iodine 1. INTRODUCTION 1.1. Basic Information about Lipids Lipids are biomolecules that are usually hydrophobic or amphiphilic in nature. Additionally, what makes these molecules different from other biomolecules like proteins and nucleic acids is not about the structure, but rather, the solubility in aqueous systems, which is attributed to their pronounced hydrophobicity. Biological organisms synthesize and secrete diverse lipids and use them as energy sources, as signalling molecules, and most importantly, as the primary components of the cell membrane. Lipids can be divided into fatty acid derivatives and isoprenoids. Moreover, the fatty acid derivatives include free fatty acids, oxidated fatty acids, glycerolipids, waxes and sphingolipids which constitute the lipid membranes; while isoprenoids include sterols (cholesterol), steroids, and lipidsoluble vitamins which function as hormones or signalling molecules. Furthermore, the sources of lipids for humans are either from plants and animals and these lipids being consumed exhibit their role as sources of fuel molecules, essential fatty acids, and vitamins. 1.2. Analysis of Lipids There are many ways to analyze for lipid content in samples. Lipids are initially extracted from the biological material usually carried out through solvent extraction usually using organic solvents, detergents, or supercritical carbon dioxide. The lipid content can be determined through spectroscopy including UV-visible light spectrometry and infrared spectroscopy whereas to separate the different classes of lipids in a sample can be achieved through chromatographic methods such as high performance liquid chromatography (HPLC), gas chromatography (GC) for volatile lipid components, and thin-layer chromatography (TLC). To further characterize the individual composition of lipids and their structure, the chromatographic techniques mentioned earlier can be coupled with spectroscopic instruments such as mass spectrometry (MS), infrared (IR) spectroscopy, or nuclear magnetic resonance (NMR) spectroscopy. 1.3. Thin-Layer Chromatography Thin-layer chromatography is probably the most popular method to analyze for lipids for the reason that it is easy to be carried out and it only requires a minimum amount of inexpensive materials and chemicals. This method can be used to check the purity of given substances, to separate components in a mixture, and to obtain quantitative analysis of the individual components present in a given sample. Throughout the decades, it has been used to a wide range of analyses ranging from quantification of amino acids in different biological samples, detection of poisons and pesticides in food samples, comparison of lipid components in different food products, detection of drugs in blood, et cetera. This technique involves a stationary phase and a mobile phase. The stationary phase is usually an adsorbent material like silica, cellulose, or aluminium oxide, and magnesium silicate on a sheet of glass or aluminum plate. The strength with which an organic compound will bind to the adsorbent
material will depend on the strength of the interaction like dipole-dipole, ion-dipole, hydrogen bonding, dipole-induced dipole and van der Waals forces. After the sample has been applied at the bottom of the adsorbent material, the mobile phase which is usually a solvent system is drawn up the plate through capillary action. In a stationary phase which is polar in the case of silica and a mobile phase which is composed of a mixture of nonpolar and slightly polar solvents, the separation of the components of a sample will depend on the polarity of the individual components. As a result, polar substances will be held by the stationary phase while the relatively nonpolar substances will move forward in the mobile phase and will be held less by the stationary phase. In other words, the order of the components of a sample from the top to the bottom of the stationary phase after each run will be in increasing polarity. The order can be described quantitatively through the retention factor or Rf which is the ratio of the distance migrated by the individual component to the distance migrated by the solvent front. The detection of each component after the separation in thin-layer chromatography will depend on the nature of molecules of each component. If the component can absorb ultraviolet light, the plate can be exposed under ultraviolet radiation to visualize the bands on the plate while in some cases, each band is already visible to the naked eye so there is no need for applying radiation or derivatization. Sometimes it is more convenient to subject the chromatographic plate to derivatization to improve the sensitivity of detection, and this technique involves a wide range of methods. It can be done thermochemically or by irradiation with light of high energy waves such as ultraviolet light. In some cases, visualization may require treatment of reagents which cause reactions such as oxidation, reduction, hydrolysis, decomposition, nitration, halogenations, esterification, and diazotization. Other visualization methods also include incorporation of radionuclides to be detected by scintillation counters or Geiger counters. In the recent decades, thin-layer chromatography has been modified and improved. There is now such improved method as high performance thin-layer chromatography (HPTLC) which involves automation and two dimensional analysis using two different solvent systems to increase the resolution of component separation. Other modifications include chiral separations, elution gradients, reversed-phase technique, et cetera. In this experiment, the most popular and probably the easiest method of thin-layer chromatography is carried out particularly through the use of silica as stationary phase, a mixture of petroleum ether, diethyl ether and glacial acetic acid as mobile phase, and iodine crystals as the reagent used for visualization. The objective of the experiment is to determine the lipid components of various food products including milk, margarine, egg yolk, fresh chicken skin, and fried pork rind using the classic form of thin-layer chromatography.
2. MATERIALS AND METHODS 2.1. Food Products and Preparation of Samples The evaporated milk, margarine, egg yolk, fresh chicken skin, and fried pork rind were obtained from the local supermarket. The fresh chicken skin was finely chopped while the fried pork rind was manually pulverized using mortar and pestle. The egg yolk was manually separated from the egg white. 2.2. Extraction of Lipids from Food Samples Each samples except milk were weighed and one gram of each was mixed with five milliliters of ethanol-ether (2:1 v/v) in a centrifuge tube. The samples were centrifuged for three minutes. Meanwhile, ten milliliters of milk sample was centrifuged for fifteen minutes. The fat layer was separated and mixed with five milliliters of ethanol-ether (2:1 v/v). Each sample with the extraction solvent were transferred to fifty milliliter-beakers and left under the hood for an hour to concentrate. 2.3. Thin-layer Chromatography of Samples Samples were applied to the plates using fine glass capillary tubes. The reference samples used were pure cholesterol and a mixture of triglycerides. The chromatogram was developed in a solvent system containing petroleum ether, diethyl ether, and glacial acetic acid (70:23:3 v/v). After the solvent had reached the top of the plate, the plate was allowed to be airdried. After drying, the plate was developed inside a chamber with iodine crystals for about 10-15 minutes. As spots had appeared, the photograph of the plate was taken and the retention factor of each spot was determined.
3. RESULTS AND DISCUSSION 3.1. Detection of Lipids in Food Samples The staining method used in this experiment was halogenation using iodine vapor. The brownish spot observed was due to iodine vapor that probably reacted with the double bonds found in the lipid components. In theory, phospholipids such as phosphatidylethanolamine and phosphatidylcholine are more polar than fatty acid derivatives such as triglycerides, diglycerides and free fatty acids; while the fatty acid derivatives are more polar than cholesterol and cholesterol esters. It will be expected that in a mixture of the said lipid classes under a nonpolar mobile phase such as chloroform, phospholipids will stay at the bottom of the plate, followed by fatty acid derivatives, and then followed by cholesterol ester then cholesterol at the topmost area of the plate; however in this experiment, fatty acid derivatives were expected to go higher than cholesterol since the acidic chain of the fatty acids has relatively high affinity to the solvent system containing 3 % acetic acid.
it does not possess cholesterol since cholesterol is only present in animal fat. 3.4. Lipid Composition of Egg Yolk The yolk is a component of the chicken egg which is actually a fat-in-water emulsion which consists of one-third proteins and two-thirds lipids. In the total lipid content, the yolk is composed of 66 % triglycerides, 28 % phospholipids, and 6 % cholesterol. The two bands with Rf values of 0.2 and 0.6 probably represent phospholipids while Rf of 0.93 probably represents cholesterol. 3.5. Lipid Composition of Chicken Skin and Pork Rind Chicken skin is composed of 75 % lipids and the total lipid consists of 30 % saturated fatty acids, 62 % unsaturated fatty acids, and the rest are cholesterol, phospholipids and fatty acid derivatives. Pork rind or skin, on the other hand, is composed of 58 % lipids and the total lipid consists of 28 % saturated fatty acids and 66 % unsaturated fatty acids, and the rest are cholesterol, phospholipids and fatty acid derivatives. The bands at the bottom with the lowest R f values were probably phospholipids and the bands with Rf values within 0.63 to 0.85 were most likely fatty acids. The bands with Rf values above 0.90 were most likely triglycerides, cholesterol, and cholesterol ester. 3.6. Possible Sources of Errors 3.2. Lipid Composition of Milk Cow’s milk is the secreted fluid of the mammary glands of the female cow. It is supposed to be the primary source of nutrition for the calf but humans in the earliest times learned to utilize milk as food. The color of milk is white and opaque due to the scattering and absorption of light by milk fat globules and protein micelles. Milk fat is mainly composed of 95-96 % triglycerides in total lipid content and less than two percent of other fatty acid derivatives. Low molecular weight fatty acids in milk include saturated and unsaturated, straight chain and branched chain fatty acids. Sterols such as cholesterol also constitute 0.2-0.4 % of the total lipid content of milk. As result has shown, the band with an R f value of 0.92 probably represents cholesterol while the band with an Rf value of 0.89 represents low molecular weight fatty acids. 3.3. Lipid Composition of Margarine Margarine is a semi-solid water-in-fat emulsion which was manufactured to imitate dairy butter. The difference between butter and margarine is that butter is derived from milk fat while margarine is derived from a combination of plant oils, animal fat, and some milk constituents. Margarine is 80 % fat and the rest is water and some milk proteins. Margarine typically has 10-20 % of saturated fatty acids, and relatively high amounts of unsaturated fatty acids which came from oilseeds. Margarine which is plant-based only has no cholesterol but has plant sterols which help lower cholesterol in blood. The band with an Rf value of 0.91 perhaps represents fatty acids. Among all the food samples,
Figure 1. Separation of lipids of different food samples by thin-layer chromatography in silica; development with solvent system containing petroleum ether, diethyl ether, and glacial acetic acid (70:23:3 v/v); Rf values were indicated near each band.
4. CONCLUSION AND RECOMMENDATIONS
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