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RBNavarro Thin Layer Chromatography Chem263Lab

RBNavarro Thin Layer Chromatography Chem263Lab

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Published by: Rafaél Berroya Navárro on Oct 10, 2012
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 Thin-Layer Chromatography of Lipidsin Selected Food Products
Rafael B. Navarro
University of the Philippines Los BañosLos Baños, Laguna, Philippines 4024rafael.berroya.navarro@gmail.com
Lipids are diverse biomolecules which serve as energy sources, signaling molecules, and main components of the cellmembrane. Lipids in a mixture or biological sample can be identified and quantified using various methods such asspectroscopy 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 (2:1v/v) and analyzed with the classical method of thin-layer chromatography. The stationary phase used was silica and the mobilephase 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, R
, of each band wascalculated 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, Iodine1. INTRODUCTION1.1. Basic Information about Lipids
Lipids are biomolecules that are usually hydrophobic oramphiphilic in nature. Additionally, what makes thesemolecules different from other biomolecules like proteinsand nucleic acids is not about the structure, but rather, thesolubility in aqueous systems, which is attributed to theirpronounced hydrophobicity. Biological organismssynthesize and secrete diverse lipids and use them as energysources, as signalling molecules, and most importantly, asthe primary components of the cell membrane.Lipids can be divided into fatty acid derivatives andisoprenoids. Moreover, the fatty acid derivatives include freefatty acids, oxidated fatty acids, glycerolipids, waxes andsphingolipids which constitute the lipid membranes; whileisoprenoids include sterols (cholesterol), steroids, and lipid-soluble vitamins which function as hormones or signallingmolecules. Furthermore, the sources of lipids for humans areeither from plants and animals and these lipids beingconsumed 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 materialusually carried out through solvent extraction usually usingorganic solvents, detergents, or supercritical carbon dioxide.The lipid content can be determined through spectroscopyincluding UV-visible light spectrometry and infraredspectroscopy whereas to separate the different classes of lipids in a sample can be achieved through chromatographicmethods such as high performance liquid chromatography(HPLC), gas chromatography (GC) for volatile lipidcomponents, and thin-layer chromatography (TLC). Tofurther characterize the individual composition of lipids andtheir structure, the chromatographic techniques mentionedearlier can be coupled with spectroscopic instruments suchas mass spectrometry (MS), infrared (IR) spectroscopy, ornuclear magnetic resonance (NMR) spectroscopy.
1.3. Thin-Layer Chromatography
Thin-layer chromatography is probably the most popularmethod to analyze for lipids for the reason that it is easy tobe carried out and it only requires a minimum amount of inexpensive materials and chemicals. This method can beused to check the purity of given substances, to separatecomponents in a mixture, and to obtain quantitative analysisof 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 indifferent biological samples, detection of poisons andpesticides in food samples, comparison of lipid componentsin different food products, detection of drugs in blood, etcetera.This technique involves a stationary phase and a mobilephase. The stationary phase is usually an adsorbent materiallike silica, cellulose, or aluminium oxide, and magnesiumsilicate on a sheet of glass or aluminum plate. The strengthwith which an organic compound will bind to the adsorbent
material will depend on the strength of the interaction likedipole-dipole, ion-dipole, hydrogen bonding, dipole-induceddipole and van der Waals forces. After the sample has beenapplied at the bottom of the adsorbent material, the mobilephase which is usually a solvent system is drawn up the platethrough capillary action. In a stationary phase which is polarin the case of silica and a mobile phase which is composedof a mixture of nonpolar and slightly polar solvents, theseparation of the components of a sample will depend on thepolarity of the individual components. As a result, polarsubstances will be held by the stationary phase while therelatively nonpolar substances will move forward in themobile phase and will be held less by the stationary phase. Inother words, the order of the components of a sample fromthe top to the bottom of the stationary phase after each runwill be in increasing polarity. The order can be describedquantitatively through the retention factor or R
which is theratio of the distance migrated by the individual component tothe distance migrated by the solvent front.The detection of each component after the separation inthin-layer chromatography will depend on the nature of molecules of each component. If the component can absorbultraviolet light, the plate can be exposed under ultravioletradiation to visualize the bands on the plate while in somecases, each band is already visible to the naked eye so thereis no need for applying radiation or derivatization.Sometimes it is more convenient to subject thechromatographic plate to derivatization to improve thesensitivity of detection, and this technique involves a widerange of methods. It can be done thermochemically or byirradiation with light of high energy waves such asultraviolet light. In some cases, visualization may requiretreatment of reagents which cause reactions such asoxidation, reduction, hydrolysis, decomposition, nitration,halogenations, esterification, and diazotization. Othervisualization methods also include incorporation of radionuclides to be detected by scintillation counters orGeiger counters.In the recent decades, thin-layer chromatography has beenmodified and improved. There is now such improvedmethod as high performance thin-layer chromatography(HPTLC) which involves automation and two dimensionalanalysis using two different solvent systems to increase theresolution of component separation. Other modificationsinclude chiral separations, elution gradients, reversed-phasetechnique, et cetera.In this experiment, the most popular and probably theeasiest method of thin-layer chromatography is carried outparticularly through the use of silica as stationary phase, amixture of petroleum ether, diethyl ether and glacial aceticacid as mobile phase, and iodine crystals as the reagent usedfor visualization. The objective of the experiment is todetermine the lipid components of various food productsincluding milk, margarine, egg yolk, fresh chicken skin, andfried pork rind using the classic form of thin-layerchromatography.
2. MATERIALS AND METHODS2.1. Food Products and Preparation of Samples
The evaporated milk, margarine, egg yolk, fresh chickenskin, and fried pork rind were obtained from the localsupermarket. The fresh chicken skin was finely choppedwhile the fried pork rind was manually pulverized usingmortar and pestle. The egg yolk was manually separatedfrom 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 (2:1v/v) in a centrifuge tube. The samples were centrifuged forthree minutes. Meanwhile, ten milliliters of milk sample wascentrifuged for fifteen minutes. The fat layer was separatedand mixed with five milliliters of ethanol-ether (2:1 v/v).Each sample with the extraction solvent were transferred tofifty milliliter-beakers and left under the hood for an hour toconcentrate.
2.3. Thin-layer Chromatography of Samples
Samples were applied to the plates using fine glass capillarytubes. The reference samples used were pure cholesterol anda mixture of triglycerides. The chromatogram was developedin a solvent system containing petroleum ether, diethyl ether,and glacial acetic acid (70:23:3 v/v). After the solvent hadreached the top of the plate, the plate was allowed to be air-dried.After drying, the plate was developed inside a chamberwith iodine crystals for about 10-15 minutes. As spots hadappeared, the photograph of the plate was taken and theretention factor of each spot was determined.
3. RESULTS AND DISCUSSION3.1. Detection of Lipids in Food Samples
The staining method used in this experiment washalogenation using iodine vapor. The brownish spotobserved was due to iodine vapor that probably reacted withthe double bonds found in the lipid components.In theory, phospholipids such asphosphatidylethanolamine and phosphatidylcholine are morepolar than fatty acid derivatives such as triglycerides,diglycerides and free fatty acids; while the fatty acidderivatives are more polar than cholesterol and cholesterolesters. It will be expected that in a mixture of the said lipidclasses under a nonpolar mobile phase such as chloroform,phospholipids will stay at the bottom of the plate, followedby fatty acid derivatives, and then followed by cholesterolester then cholesterol at the topmost area of the plate;however in this experiment, fatty acid derivatives wereexpected to go higher than cholesterol since the acidic chainof the fatty acids has relatively high affinity to the solventsystem containing 3 % acetic acid.

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