Biochem 34 FR Expt #7 & 9

8/17/2019 Biochem 34 FR Expt #7 & 9

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Biochemistry 34.1 Isolation of Lipids from Egg Yolk & Determination of Acid Value of Fats of 10

Experiment No. 7 & 9 – ISOLATION OF LIPIDS FROM EGG YOLK & DETERMINATION OF ACID VALUE OF FATS

2013-14728; 2013-68117; 2013-14905Group #5, Biochemistry 34.1 MEJ, Professor Del RosarioNovember 9, 2015

I. AbstractLipids are relatively non-polar macromolecules that contain hydrocarbons, and have various functions

in biological systems. Examples of lipids include glycerol, phospholipids, natural oils and steroids. In thisexperiment, the lipids were extracted from egg yolk sample using liquid —liquid extraction, withchloroform-methanol as the solvent mixture. Using thin layer chromatography and qualitative tests suchas Sudan IV test, Acrolein test, Test for Phosphates, Leibermann-Buchard test and Test for Unsaturation,the different lipid components of the supernate and precipitate were determined. Results of the TLCshowed that the supernatant contained less polar components, with an R f value of 0.3533, compared toprecipitate (Rf = 0.0667). The qualitative tests showed positive results for both the supernate andprecipitate in the Sudan IV test and Test for Phosphates, while both being negative in the Leibermann-Burchard test. In the test for unsaturation, the supernate and precipitate needed 8 and 22 dropsrespectively, while only the precipitate showed positive results in the Acrolein test. Another method ofanalysis for fatty acids is through the calculation of the acid value of the sample. This can be determinedby the amount of KOH needed to neutralize the free acids present. This experiment determined the acidvalue for used commercially available cooking oil, with the results being an average of 0.1683 mg KOHper gram of fat sample, which is relatively a low acid value. The palm oil analysed, therefore contains onlya small amount of free fatty acids. The obtained acid value can be accounted to the deterioration of thefats by environmental factors. Both lipid extraction and acid value determination of fats are essentialprocesses in clinical chemistry, medicine, food technology and various industries.II. Keywords

Lipids, Acid Value, Liquid-liquid extraction, Fatty acids, Thin layer chromatography

III. IntroductionLipids are biomolecules which are relatively

nonpolar as compared to water and otherbiomolecules, thus their solubility in nonpolarsolvents. They vary greatly in structure andfunction, such as cell membrane component andenergy storage (Reusch, 2013).

Since lipids have a wide array of structures, aunified classification system for lipids is hard toachieve. However, lipids can be divided into twogroups: polar and nonpolar lipids, relative to eachother. Nonpolar lipids, such as triglycerides, areless polar than polar lipids because little to nopolar groups are found within the molecule, andthe polar groups available are small as comparedto those of the polar lipids (Hasenhuettl & Hartel,2008). Thus, nonpolar lipids have less interactionwith water and other polar solvents than polarones, allowing them to be easily separated frompolar lipids, and extracted by liquid-liquid

extraction.Most lipids contain fatty acids, an example of a

polar lipid, in their structures. Compared to otherlipids, fatty acids have simple structures, with achain of 10-20 carbon atoms and a carboxylicgroup at the end (Figure 1).

Figure 1. An example of a fatty acid.Source: Classification of lipids (n.d.). Retrieved fromhttp://www.laney.edu/wp/cheli-fossum/files/2012/01/Classification-of-Lipids.pdf.

These fatty acids are also usually products ofhydrolysis of more complex lipids. Lipids in oilhydrolyze spontaneously after prolonged storage

(DPSM, 2013). To determine the amount of freefatty acids present in a sample, the acid value ismeasured.

The acid value of fats measures the amount ofKOH in milligrams required to neutralize the fattyacids present in one gram of a sample (Analyticalmethods to measure the constants of fats and oils,

n.d.). Determination of free fatty acids in a sampleof oil can therefore be achieved through directtitration with a base.

This experiment aims to successfully isolatelipids from egg yolk using liquid-liquid extraction,and confirm the presence of various lipids usingqualitative tests. It also aims to determine the acidvalue of a sample of commercially-available oil bydirect titration.

IV. Experimental

A. Isolation of Lipids from Egg Yolk

Twenty milliliters of a solution containg eggyolk and 1% NaCl solution, in a ratio of 1:3, wasmixed with 60mL of another solution containingCHCl3 and methanol, in a ratio of 1:2. Theresulting mixture was placed in a separatoryfunnel, swirled, and mixed, occasionally releasinggas and pressure through the valve. Theseparatory funnel was then allowed to stand for 30minutes, or until distinct layers are observable.

The organic layer formed at the bottom of thefunnel was collected and added with acetone untilprecipitation stops. The resulting solution wascentrifuged for 10 minutes at 6000rpm.

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The precipitate was collected and dissolved in5mL methanol. The solution was then labelled P,and the supernate was labelled S.

Thin Layer Chromatography A silica gel plate was spotted with the

precipitate from the previous step, P, thesupernate from the last centrifugation, S, and with

suitable standards.

Three milliliters of a methanol-acetone mixturein a 1:2 v/v ratio was placed in thechromatographic chamber. The TLC plate wasthen allowed to stand inside the chamber until thesolvent line was at about 1cm below the top sideof the plate. The plate was then removed from thechamber and allowed to dry.

Several iodine crystals were placed in thechamber, then, after drying, the TLC plate wasreturned to the chamber. At the appearance ofdistinct orange/brown spots, the plate was

removed, and the distance travelled by the spotswas measured and noted. The respective Rf values for each sample and for the standards werethen calculated.

Qualitative Tests

SUDAN IV TESTTen drops of P and S were placed in separate

test tubes, and a tiny crystal of Sudan IV wasadded in each tube. The tubes were shaken andthe results were noted.

ACROLEIN TEST

Ten drops of P and S were placed in separatetest tubes, and a pinch of KHSO4 was added ineach tube. The solutions were then heated gentlyfor a few seconds by flaming, and placed in aboiling water bath for five minutes. The resultingsmells of the solutions were noted.

TEST FOR PHOSPHATES A 0.5mL sample each for P and S was

incinerated in separate porcelain crucibles. Aftercomplete evaporation of the solvents, the residuesobtained were cooled. Three milliliters of distilledwater was added to each of the residues. Aftermixing thoroughly, the solutions were filtered, and

the filtrates were obtained. Each filtrate was mixedwith 0.5mL 10% (NH4)2MoO4 and two drops ofconcentrated HNO3. The solutions were heated fortwo minutes and allowed to stand. The formationof a yellow precipitate, or lack thereof, was noted.

LEIBERMANN-BURCHARD TESTFive drops of P and S were placed in separate

test tubes. Ten drops of acetic anhydride and tendrops of concentrated H2SO4 were mixed witheach of the tubes. The changes in color, or lackthereof, were noted.

TEST FOR UNSATURATION

Five drops of P and S were placed in separatetest tubes. Hubl’s solution was then addeddropwise to each tube, while shaking, until theresulting pink color does not fade. The number of

drops required for decolorization to cease wasrecorded for each sample.

B. Determination of Acids Value of Fats

Twenty milliliters of fat sample was titrateddirectly using KOH after adding two drops ofphenolphthalein as indicator. The volume of titrantrequired to reach the end point was noted.

V. Results and Discussion

Lipids are naturally occurring substances,arbitrarily grouped together on the basis of theirinsolubility in water and solubility in nonpolarsolvents. Due to the fatty acid profile, high oilsoluble vitamin and lecithin content, Egg yolk isconsidered a rich source of a variety ofbiochemically important compounds such asproteins and lipids (Minard & West, 2001),specifically triglycerides (65%), phospholipids (28-

30%) and cholesterol (4-5%).Lipids include a wide variety of differentsubstances, but are commonly subdivided intoseveral classes based on structural similarities.

TRIACYLGLYCEROLSThe triacylglycerols, (fats and oils) are esters

of glycerol and fatty acids generally formed by adehydration reaction as shown below in Figure 2.Triacylglycerols differ in the types of fatty acidsattached to the glycerol backbone. The fatty acidalways contains an even number of carbon atoms,commonly ranging between 10-20 carbon atoms

long. The hydrocarbon chain on the fatty acid canbe either saturated (contains only C-C singlebonds), or unsaturated (containing one or moreC=C bonds. Fats are solids, obtained primarilyfrom animals, and contain a larger proportion ofsaturated fats, while oils are liquids obtainedprimarily from plants and contain a greaterproportion of unsaturated fatty acids (Properties oflipids, n.d.).

Figure 2: Formation Reaction for Triacylglycerols Source:http://science.halleyhosting.com/sci/ibbio/chem/notes/chpt3/triglyceride1.gif

PHOSPHOLIPDSPhospholipids, also known as

glycerophosphatides, are widespread type of lipidwhich occur in all plant and animal cells as majorstructural components of cell membranes. They

play critical roles in the transport of moleculesacross membranes, storage and metabolism offatty acids, and as activators in the blood clottingprocess. Structurally, they are similiar to

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triacylglycerols but phospholipids has one of itsfatty acid group replaced with a very polarphosphate ester group (Minard & West, 2001), asshown in Figure 3, which is produced by theesterification of one of the alcohol groups ofglycerol by phosphoric acid molecule rather thanby carboxylic acid molecule (Campbell & Farrell,2005).

Figure 3. Structure of PhospholipidsSource:http://patentimages.storage.googleapis.com/WO2008019797A2/imgf000005_0002.png

STEROIDSteroids are lipids containing the core

structure of 17 carbons fused in a ring structurecontaining three, six-member rings, and one five-member ring. The different functionality of steroidscomes from the substituent groups attached to thecore structure.

Cholesterol, a typical example of steroid, is amajor component of cell membranes. An egg yolkcontains about 200 milligrams of cholesterol, muchof it bound as complex lipid. Figure 4 below showsthe structure of cholesterol.

Figure 4: Structure of CholesterolSource:http://blogs.dnalc.org/wp-content/uploads/2012/04/cholesterol.png

Isolation of Lipids from Egg YolkThere are several methods for the lipid

isolation. One of which is the Liquid-liquidextraction, which is employed in the experiment to

separate different types of lipids present in eggyolk sample through the difference in miscibility ofthe solution involved.

Liquid egg yolk contains a lot of water andextraction with non-polar solvents is not efficientdue to difference in solvent and egg yolk polarities.Polar solvents, such as lower alcohols, denatureegg yolk proteins destroying hydrogen bonds orelectrostatic interaction in protein structureopening the way to the neutral lipids, what makesextraction with non-polar solvent possible. Withoutprotein denaturation, polar solvents will extractpolar membrane-associated lipids from the eggyolk. However, the combination of polar and non-polar solvents for better lipid extraction from liquidegg yolk can be chosen. The methanol/chloroform

mixture can be a good solvent for extraction oflipids from egg yolk (Ahn et al., 2006).

The egg yolk is composed of polar andnonpolar lipids. Lipids are generally water-insoluble substances that are soluble in non-polarorganic solvents, such as chloroform. Chloroformis relatively the most nonpolar solvent used in theexperiment. It cannot employ dipole-dipole

interaction because chlorine is too bulky and isvery electron rich, thus it prefers van der Waalsinteraction. Chloroform solubilises lipids that areinsoluble in polar solvents such as triglyceride,cholesterol, phospholipids and glycolipids (Berg, etal., 2002).

Figure 5. Structure of ChloroformSource: http://f.tqn.com/y/chemistry/1/S/Q/S/1/Chloroform.jpg

Alcohols, on the other hand, are known to begood solvents for lipids (Lipid Extraction, n.d.).

Methanol and other alcohol components dissociatethe bonds in the lipoprotein complexes, making thelipids readily available for chloroform to bedissolved. Polar compound, such ascarbohydrates, urea, salts and proteins areconsidered as contaminants and are dissolved bymethanol (Murray, et al., 2003).

Figure 6. Structure of MethanolSource:http://d1h8qm6whtl6z3.cloudfront.net/wp-content/uploads/2014/08/Methanol-structure-300x231.png

The process of lipid extraction from tissueswith methanol/chloroform first was mentioned in1957 in Folch et al. (as stated in Axelsson &Gentili, 2014). Folch et al. were the first which

develop the chloroform/methanol/water phasesystem (the so-called ―Folch‖ method) forextraction of lipids from biological material. Themethod is still considered as the classical andmost reliable method for quantitative extraction oflipids. The method relies on a mixture ofchloroform and methanol in 1:2 ratio, forming amonophasic solvent system, to extract anddissolve lipids solution (Abert-VIan et al., 2015).

The addition of chloroform/methanol solvent tothe sample formed three distinguishable layers:the upper aqueous layer, the middle foamy layerand the lower yellowish layer. The upper layer is

mainly composed of water, methanol and otherwater soluble compounds; the middle emulsionlayer composed of aqueous and organic solution(Boyer, 2012). A biphasic system is then produced

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by the addition of water or saline solution (Abert-VIan et al., 2015).

After letting the solution stand for 30 minutes,the organic layer was observed to be separatedfrom the aqueous layer, resulting into 2 layers asshown in Figure 7. The bottom organic layerdisplayed a golden yellow clear liquid while theupper aqueous layer appeared as a pale yellow,

semi-clear liquid.

Figure 7. Liquid-liquid Extraction Set-up

Source:https://upload.wikimedia.org/wikipedia/commons/thumb/d/d1/Liquid_liquid_extraction.png/220px-Liquid_liquid_extraction.png

The addition of NaCl to the egg yolk helpsdesolubilize the proteins in the sample throughsalting out process (Wang, et al., 2000). This leadsto the migration of polar compounds along with themethanol into an upper aqueous phase andleaving the lipids in the lower chloroform phase(Abert-VIan et al., 2015). The separation isemployed by the differences in density, with theaqueous layer having the lowest and the

chloroform-lipid layer having the highest density(Boyer, 2012).In the extraction of lipids of other biological

samples such as the myelin sheath, thechloroform/methanol ratio of 1:2 could bemaintained. Myelin sheaths are cholesterol-enriched nerve cell insulators. The methanol musthave a higher volume than the chloroformcomponent to maximize the amount of lipidsextracted from the tissue without lowering thewater content (Rumsby, et al., 1966).

The solvent-sample ratio greatly influences theper cent yield of the extraction process. A lower

ratio may form two phases which may lower theinteraction between the polar and non-polarsolvent and the membrane lipids. A higher ratiomight not bring any harm to the extraction process,but the extraction of polar lipid might be lower dueto lower water content (Wang, et al. 2003).

A polar acetone solvent, as shown in Figure 8,was added to the organic bottom layer extractedthrough liquid-liquid extraction until precipitationceases. This further facilitates the solubilisationbetween polar and non-polar lipids. Simple lipidsand glycolipids dissolve readily in acetone, whilephospholipids do not due to the presence of the

phosphate groups. The addition of acetoneemploys dipole-dipole interaction and precipitatesthe phospholipids, making them easier to be

separated through centrifugation process (Cristie,2011).

Figure 8. Structure of AcetoneSource:http://www.sigmaaldrich.com/content/dam/sigma-aldrich/structure2/194/mfcd00008765.eps/_jcr_content/renditions/mfcd00008765-medium.png

The precipitate from the extraction process istherefore composed mainly of phospholipids, whilethe supernatant liquid is composed of triglycerides,cholesterol and glycolipids.

Thin Layer ChromatographyThin Layer Chromatography (TLC) is a

technique used to analyse the components of amixture through separation by determining the

number and identity of components in a mixture,as well as the purity of the compound through theprocess of elution. The mobile phase consisting ofa volatile organic solvent or mixture of solvent iseluted through a stationary phase, as shown inFigure 9, via capillary action (Thin LayerChromatography , n.d.).

Figure 9. Thin-Layer Chromatography Process (Boyer, 2012)

The mobile phase moves the sample up thestationary phase at different rates. In theexperiment, the methanol-acetone solvent wasused. Methanol dissolves polar substances, whileacetone can dissolve both polar and non-polarsubstances. The solubility of the compounds in theeluting solvent and the ability of the solvent to beabsorbed on the stationary phase affect how fastthey travel through the TLC plate. Too high solventaffinity to the absorbent may cause the samples tomove up close to the solvent front withoutseparation, while too low solvent affinity may resultto the slow movement of the components withoutenough separation. The stationary phase, on theother hand, is made from silica (SiO2) supportedon a metal foil. It shows maximum selectivity onthe types of compounds being separated so thatthe differences in the elution rates will be largeenough for effective separation (Thin LayerChromatography , n.d.).

The chromatography chamber, as shown inFigure 10, must be saturated with the solventvapour before the start of the elution process. Themethanol-acetone solvent mixture used in TLC arehighly volatile thus, evaporation is spontaneous.

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The saturation of the atmosphere inside thechamber prevents the solvent from the stationaryphase to evaporate even before the elutionprocess is complete (Libal, n.d.).

Figur e 10. A typical chamber for Thin-layer Chromatography(Boyer, 2012)

However, some compounds may not bereadily visible after a chromatography process.Chemical processes are used to make the marksvisible for clear determination of distance travelled.Some of these development processes include theuse of a development chamber with a saturatediodine vapor. The vapour present inside the

chamber complexes with the double bonds inorganic compounds on the plate to form visiblebrown marks. This developing technique isreversible and the iodine marks will disappearupon reheating of the plate (Boyer, 2012).

Upon detection of separated compounds, theRf value (retention factor) of each samples wascalculated using the formula:

Source: Biochemistry 34.1 Laboratory Manual, UP Manila

The supernatant liquid and the precipitate fromthe first part of the experiment, together with astandard palm oil solution, were subjected to thinlayer chromatography. The distance travelled byeach sample was measure and their respective Rf values were computed.

Sample computations of the R f value

Results were obtained for each sample of lipid

isolation and were tabulated in Table 1.

Table 1. Rf values of the different samples subjected to TLC

Samples dtravelled Rf

S 2.65 0.3533

P 0.5 0.0667

Standard 5.7 0.7600

Solvent 7.5 ---

The results of this experiment shows that thestandard palm oil has the greatest Rf value(Rf =0.7600), followed by supernatant liquid sample(Rf =0.3533), and the precipitate having the least

Rf value (Rf =0.0667).The Rf values are always greater than zero

and less than one. Less polar compounds havehigher values than high polar compounds due to

the attraction between the SiO2 molecules of theplate and the polar molecules of the compound(Boyer, 2012). Thus, it can be said that thesupernatant liquid sample, composed oftriglycerides, cholesterol and glycolipids are lesspolar than that of the precipitate which iscomposed mainly of phospholipids.

Qualitative TestsIn order to verify the presence of specific types

of lipids in the isolates, the supernatant liquidsample and precipitate were subjected toqualitative analysis. The tests include Sudan IVTest, Acrolein Test, Test for Phosphates,Leibermann-Burchard Test, and Test forUnsaturation.

SUDAN IV TestSudan IV (C24H2ON4O) is a red nonpolar, lipid-

soluble dye used in the staining process of lipids,triglycerides and lypoproteins. Upon addition to asolution of lipids and water, the dye will mix intothe lipid layer and produce a red colored layer(Testing for Biologically Important Molecules, n.d).

Polar compounds and aqueous solutions, on theother hand, will not interact with the nonpolarSudan IV stain.

Figure 11. Sudan IV Test: Positive result (left) and negativeresult (right)Source:http://emp.byui.edu/wellerg/Molecules%20of%20the%20Cell%20Lab/instruction/Molecules%20of%20the%20Cell%20Instructions.html

The Sudan IV test was done on both thesupernatant liquid and precipitate, and positiveresults were observed in both samples (Table 2)indicating the presence of necessary lipids such astriglycerides and lypoproteins to produce a red

color with the Sudan IV.

Table 2. Sudan IV Test Results

TestCompounds

ExperimentalResults

TheoreticalResults

S +++ +++

P ++ -

However, the precipitate sample musttheoretically not test positive for the test, as itshould no longer contain tryglycerides orlypoproteins, which should have readily dissolvedin the acetone added in the sample. The positive

(++) result of the precipitate sample suggests thatthere are remaining triglycerides in theprecipitates, although in smaller amountscompared to that of the supernatant liquid sample.

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ACROLEIN TestThe Acrolein test is a general test for the

presence of glycerol or fats in a molecule. Theprinciple behind the acrolein test is a specificchemical reaction. When heated strongly in thepresence of a dehydrating agent, potassiumbisulfate (KHSO4), the fat undergoes hydrolysis

forming glycerol which is then dehydrated to formthe simplest unsaturated aldehyde, acrolein(CH2=CHCHO), which has a characteristicirritating odor that can be compared with burntgrease (Ballou et al., 2005).

Figure 12. Acrolein Test ReactionSource:http://image.slidesharecdn.com/activity5abiochemreport-130513015903-phpapp01/95/activity-5-a-biochem-report-14-638.jpg?cb

Experimental data, as shown in Table 3,shows that the precipitate produced burnt greaseodor upon the completion of reaction, indicatingthe presence of glycerol, while the supernatantshowed negative results.

Table 3. Acrolein Test Results

TestCompounds

ExperimentalResults

TheoreticalResults

S - -P ++ +

Test for PHOSPHATESThe oxidation of fatty acids and the formation

of water and carbon dioxide by fatty acidmetabolism yields a reduced coenzyme used forthe production of ATP. The test for phosphatesuses the same principle by incinerating thesamples to destroy organic parts, evaporatingthem as carbon dioxide and water, leaving solidinorganic phosphate residue. The inorganicphosphate residue was separated from

contaminants and other nonpolar lipid componentsby dissolving the residue in water. Componentsnot soluble in water were discarded (Koolman &Roehm, 2005).

When lipids containing phosphate groups intheir structures are added to a strong acid solution,the lipid hydrolyses, producing free phosphate.The presence of free phosphate in a solution wasthen detected by adding ammonium molybdateand nitric acid to the filtrate to produce yellowammonium molybdo-phosphate precipitates,according to the reaction (Koolman & Roehm,2005):

HPO42 –

(aq)+ 12MoO42 –

(aq)+ 3 NH4+

(aq)+ 23 H3O+

(aq) (NH4)3[P(Mo3O10)4] (yellow,s) + 35 H2O(l)

When phospholipids are hydrolyzed in acidicmedium, both the fatty acid ester bonds and thephosphate ester bonds are broken and free fattyacids and inorganic phosphate are releasedreaction (Koolman & Roehm, 2005).

The supernatant of the lipid isolation istheoretically negative for the test for phosphates,because the phosphates must be present only in

the precipitate, since the acetone cannot dissolvethe phosphate groups. However the experimentalresults (Table 4), show positive results for both thesupernatant and precipitate samples. The falsepositive results may be accounted to the probablepresence of phosphate impurities in thesupernatant sample.

Table 4. Test for Phosphates Results

TestCompounds

ExperimentalResults

TheoreticalResults

S + -

P + +

LIEBERMANN-BURCHARD TestThe Lieberman –Burchard is a reagent used in

a colorimetric test to detect the presence ofcholesterol. A positive result for the test isobserved when the solution begins as a purplish,pink colour and finally turns into a bluish-greensolution. The colour is due to the hydroxyl group (-OH) of cholesterol reacting with acetic anhydride(used as solvent and a dehydrating agent), andsulfuric acid (used as dehydrating and oxidizingagent), thereby increasing the conjugation in theadjacent fused ring, as shown in Figure 13.

Cholesterol reacts with acetic anhydride andsulfuric acid to yield pentoenylic cation, a highlyconjugated compound whose maximum lightabsorption is at 620 nm wavelength. This accountsfor the purple or pink color observed initially at theaddition of the reagents. Further reaction withsulfuric acid produces cholestahexone sulfuricacid. The color of the final solution is green due tothe maximum absorption of the compound at 410nm wavelength (Burke et al., 1974).

Figure 13. Liebermann-Burchard ReactionSource:https://upload.wikimedia.org/wikipedia/commons/thumb/8/83/Liebermann-Burchard.svg/600px-Liebermann-Burchard.svg.png

Experimental results, as shown in Table 5,suggest the negative result, therefore the absenceof cholesterol for both the supernatant and

https://en.wikipedia.org/wiki/Reagent

https://en.wikipedia.org/wiki/Colorimetric_analysis

https://en.wikipedia.org/wiki/Colorimetric_analysis

https://en.wikipedia.org/wiki/Reagent

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precipitate sample. However, the supernatantsample from lipid isolation must theoreticallycontain cholesterol, since it can readily dissolve inacetone.

Table 5. Liebermann-Burchard Test Result

TestCompounds

ExperimentalResults

TheoreticalResults

S - +P - -

Test for UNSATURATIONIn order to determine the degree of

unsaturation of the fatty acids in the lipid sample,an alcoholic solution of iodine containing somemercuric chloride called Hubl’s iodine reagent, wasadded to the samples until a change in color wasobserved, The test works in line with the principlethat unsaturated fatty acids become saturated bytaking up iodine and if it contains moreunsaturated fatty acids, it will take up more iodine,and thus producing a color change in the solution.

Figure 14. Reaction with the Hubl’s Reagent Source:http://www.assignmentpoint.com/wp-content/uploads/2013/12/reaction1.jpg

The experimental results (Table 6) show thatthe supernatant sample reacted and employed acolor change in a smaller amount of the Hubl’sreagent, thereby showing that the sample containless double bonds, and thus a smaller degree ofunsaturation. The precipitate sample, on the otherhand, requires a higher amount of the reagentbefore a color change. Hence, it contains a higherdegree of unsaturation.

Table 6. Test or Unsaturation Results

Test

Compounds

Experimental

Results(no. of drops)

S 8

P 22

Determination of Acid Value of FatsFat may become rancid after long storage. It

may be hydrolysed by different microorganisms,thus breaking its ester bonds. This then leads tothe formation of free fatty acids making thesolution acidic or rancid. The amount of free fattyacid present in the oil then indicates the age and

the quality of that oil (Determination of acidnumber of edible oil, n.d.). In order to analyse this,the acid value of the fat sample was determined.

The acid value indicates the proportion of freefatty acid present in oil or fat and may be definedas the number of milligrams of potassiumhydroxide (KOH) required to neutralize the freefatty acid in one gram of oil or fat (Determination ofacid number of edible oil, n.d.). Acid value canhelp determine the purity of the oil or fat. A highacid value thus indicates that the oil is old and

rancid.

Fat SolventThe dissolution of fat/oil samples is done

through the use of a fat solvent, which is generallycomposed of the mixture of an absolute alcohollike ethanol, and a nonpolar compound such asdiethyl ether (Thomas, n.d.).

Figure 15. Structure of Diethyl etherhttp://www.sigmaaldrich.com/content/dam/sigma-aldrich/structure0/009/mfcd00011646.eps/_jcr_content/renditions/mfcd00011646-medium.png

Ethanol, shown in Figure 16, has both polarand nonpolar end, making it able to dissolve bothpolarities. Lipids and fatty acids are arranged withthe hydrophobic parts pointing inwards while thehydrophilic parts are exposed in the aqueousenvironment. The ethanol then dissolves theexposed hydrophilic part so that inner hydrophobicparts will be dissolved by the nonpolar diethylether solvent (Thomas, n.d.).

Figure 15. Structure of EthanolSource:https://upload.wikimedia.org/wikipedia/commons/6/6f/Ethanol_flat_structure.png

The fat sample used for this experiment wasused palm oil. Upon its titration with potassiumhydroxide, the acid value was calculated using theformula:

Source: Biochemistry 34.1 Laboratory Manual, UP Manila

where V KOH is the volume of KOH used in thetitration, M KOH is the concentration of KOH usedand MW KOH is the molecular weight of KOH (56.11g/mol).

Sample computations for Acid Value

mg KOH per g of palm oil

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After the acid values of the samples weredetermined, the results were tabulated. Table 7shows the results of the experiment.

Table 7. Determination of Acid Value of Palm Oil

VKOH at

equivalence point

(mL)

Acid Value

FatSample

Trial 1 0.3 Trial 2 0.3

Trial 3 0.3

Average

From the experimental results, it can be saidthat the used palm oil analysed has a relatively lowacid value. Therefore, the palm oil contains only asmall amount of free fatty acids and may thensuggest that the oil is not yet old and rancid.

Aside from the acid value, the quality of fatsmay be determines using other constants or

values, such as the Saponification Value, EsterValue, Hydroxyl Value and Iodine Value ( Assays,2005).

Saponification ValueThe saponification value (I s ) is the amount in

milligrams of KOH needed to saponify the esterand neutralize the free acids in 1 gram of sample.The procedure in the determination of thesaponification value includes performing a blanktest similar to back titration ( Assays, 2005). Thesaponification value is calculated using theformula:

Source:http://www.ffcr.or.jp/zaidan/ffcrhome.nsf/7bd44c20b0dc 562649256502001b65e9/146fd852cd5e269049256f32001a133e/$file/b11.pdf

where a is the volume (mL) of 0.5 Mhydrochloric acid (HCl) used in the blank test andb is the volume (mL) of 0.5 M HCl consumed in theassay (General Tests, n.d.).

Ester ValueThe ester value (IE) is the amount of KOH in

milligrams needed to saponify the esters presentin 1 g of substance ( Assays, 2005). The I E iscalculated using the saponification value I S andacid value I A as follows:

Ester Value, IE = IS - I A

Source:http://lib.njutcm.edu.cn/yaodian/ep/EP5.0/02_methods_of_analysis/2.5.__assays/2.5.0%20Assays.pdf

Hydroxyl ValueThe Hydroxyl value (I OH ) expresses the

amount of KOH in milligrams required to neutralizethe acid combined by acylation in 1 g of thesubstance ( Assays, 2005), which is calculatedusing the formula:

Source:http://lib.njutcm.edu.cn/yaodian/ep/EP5.0/02_methods_of_analysis/2.5.__assays/2.5.0%20Assays.pdf

where a is the volume in mL of 0.5M

ethanolic KOH consumed in the blank test while bis the volume in mL of 0.5M ethanolic KOHconsumed in the actual test.

Iodine ValueLastly, the Iodine Value (II) is the amount in

grams of halogen, particularly Iodine, that can beconsumed (in certain conditions) by 100 grams ofsample. Iodine values are often tabulated intables, as in Table 8 ( Assays, 2005).

Table 8. Unsaturated Fat Levels in various Foo Ingredients

Source:http://nationalhogfarmer.com/site-files/nationalhogfarmer.com/files/uploads/Keeping%20Iodine%20table1.jpg

VI. ConclusionLipids are major macromolecules that include

relatively non-polar compounds and serve asstorage for energy in biological systems. One wayof extracting water-insoluble lipids from a sample isthrough liquid-liquid extraction, a method thatutilizes the solubility of the lipid in a polar solvent,in this experiment being chloroform-methanolsolvent mixture. The supernate and precipitatefrom the extracted sample then underwent TLCand several qualitative tests.

Results of the TLC showed that supernatanthas a greater Rf value (0.3533) compared to that of

the precipitate (Rf = 0.0667). This means that thatprecipitate is the more polar isolate, since theyhave more interaction with the stationary phase ofthe TLC (silia gel).

The qualitative tests aided the identification oflipids present in the isolates: glycerol for the

Acrolein test, triglycerides for the Sudan IV test,phospholipids for test of Phosphates, cholesterolfor the Leibermann-Burchard test, and unsaturatedfatty acids for test for Unsaturation. The resultssuggest that supernate and precipitate samplescontain triglycerides, phosphates, unsaturated fattybut not cholesterol. The precipitate, in addition,

contains glycerol. Another method of qualitative analysis for fatty

acids is through the calculation of the acid value ofthe sample. In this experiment, this value was

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determined by the amount of KOH required toneutralize the fatty acids present in the process oftitration.

The results gave an acid value of 0.1683 forthe used cooking oil which is a relatively low acidvalue. Therefore, the palm oil contains only a smallamount of free fatty acids and may then suggestthat the oil is not yet old and rancid. Although a low

acid value was calculated, the values still suggestslow purity for the sample and the possibledeterioration of the free fatty acids.

For greater accuracy in the isolation of lipids, itis suggested that the collection of the sample fromthe liquid-liquid extraction be done carefully. In theTLC, it should also be made sure that the samplesspots do not touch one another.

As for the determination of acid values, it isrecommended to have the proper fat solvent ratioto better extract the fats from the sample. It is alsoimportant to use fresh samples to isolate from, inorder to more accurately determine the acid valueof the fats.

VII. References

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Arntz, D. et al. (2012) Acrolein and methacrolein.Ullmann's Encyclopedia of IndustrialChemistry. Wiley-VCH, Weinheim.doi:10.1002/14356007.a01_149.pub2

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Berg J. et al. (2002) Biochemistry . W. H. Freemanand Company ISBN 0-7167-4955-6LipidExtraction, n.d. Retrieved fromhttp://www.cyberlipid.org/extract/extr0001.htm

Boyer, R., 2012. Biochemistry laboratory: Moderntheory and techniques second edition.Pearson Education Inc., New Jersey

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h/VirtTxtJml/lipids.htm.Rumsby, M. & Finean, J. (1966) The action oforganic solvents on the myelin sheath of

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peripheral nerve tissue – 1. University of

Birmingham, Birmingham 15, England.Testing for biologically important molecules (n.d.)

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Alberta, Canada.

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