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Characterization of Saponifiable lipids3PSY5MembersMercado, Eloisa BellePestano, Julian TerrenceRegino, Paul Andrew R.Tady, Kristine ClaireTee, MargaritaVillanueva, KervinIntroductionREGINO, Paul Andrew R.3PSY5LipidsLipids are naturally organic compounds that are insoluble in water but soluble in organic solvents. Animals: lipids make up most of the fats in the form of adipose tissues which helps in insulation and thermal regulation. Fats are known to store heat.Plants: lipids are seen as the waxy covering known as the cuticle that protects the leaves of the plant and makes the leaves waterproof.

Classification of lipidsOpen-chain compounds with polar heads and long nonpolar tailsFatty acidsTriacyglycerolsSphingolipidsPhospholipidsGlycolipidsFused-ring compoundsSteroids

Classification of lipidsOn the basis of whether they will undergo hydrolysis in alkaline solution:Saponifiable lipidsNonsaponifiable lipids

Fatty AcidsPolar end= the carboxyl groupNonpolar tail= the hydrocarbon chainFatty acids are amphipathicThey usually have even number of carbon atoms & the hydrocarbon chain is unbranched

Two main types: Saturated & unsaturated

TriacylglycerolsThe 3 alcohol groups in glycerol (glycerol contains 3 hydroxyl groups) forms ester linkages with fatty acids.Function: storage (especially in animals)

PhospholipidsType of lipid that contains a phosphorus atom attached to the glycerol backboneThey are known to be the main lipid component of the cell membrane2 types of phospholipids:Phosphoacylglycerols- glycerol is the backboneSphingomyelins- sphingosine instead of glycerolGlycolipidsA lipid to which a sugar moiety is bondedA carbohydrate is bound to an alcohol group of a lipid by a glycosidic linkageCeramides are the parent compounds for glycolipids

SphingolipidsDo not contain glycerol but instead they contain sphingosine. Found both in plants and animals & they are abundant in the nervous system

OilsCoconut oil= composed mainly of lauric acid & is slow to oxidize and is resistant to rancidificationCanola oil= low in saturated fatOlive oil= composed mainly of oleic acid and palmitic acid and other fatty acidsPalm oil= has 30 phenolic compounds & has high saturated vegetable fatsSesame oil= high proportion of polyunsaturated linoleic fatty acidCorn oil= high smoke poinyFour tests for lipidsGrease-spot testSaponification testAcrolein testUnsaturation test

Grease-spot testGeneral test for the presence of lipids

Positive: oil & lecithinNegative: water & dichloromethane

Saponification testGeneral test for ester bonds

Positive: oil & melted fatNegative: water

SaponificationHydrolysis reactionHydrolysis using a base such as sodium hydroxide or potassium hydroxideProducts are:GlycerolSodium/Potassium salts- salts are soaps

Saponifiable lipidsContains an ester functional groupCan behydrolyzedunder basic conditions Includes triacyglycerols, phospholipids, glycolipids, sphingolipids and the waxes

Acrolein testGeneral test for fats or glycerin

Positive result= strong odorUnsaturation testGeneral test for the presence of double bonds and their degree of saturation

Objectivesto characterize lipids using the grease-spot test, saponification test, acrolein test and unsaturation test.MethodologyTee, Margarita R.3PSY5

Grease Spot Test Tee, Margarita R.3PSY5 apply a drop of the appropriate substance to the labeled areas

Warm the filter paperTee, Margarita R.3PSY5With the use of pencil, four areas of a filter paper was labeled vegetable oil, lecithin, H2O and dichloromethane

Observations after dropping and after heating

Saponification Test Tee, Margarita R.3PSY5 8 drops of appropriate substance placed on their corresponding test tube

Add 10 drops of 3M NaOH on each test tubes

Place in boiling water bath for 15-20 mins

Place in test tube rack and allow to cool to room temperatureTee, Margarita R.3PSY5

3 large-sized test tubes labeled oil, fat and H2O

add 5ml of distilled water to each test tube

Stopper the test tubes and mix vigorouslyTee, Margarita R.3PSY5

Record observations after shaking whether an emulsion, micelle or clear solutionAcidify with few drops of H2SO4

Tee, Margarita R.3PSY5

record the pH of the material, either acidic or basic, using blue and red litmus paper

Mix with stirring rod

Note the color of the material on top

Acrolein Test Tee, Margarita R.3PSY5 Add 5 drops of an oil sample or a small piece of solid lipid

Heat over a Bunsen burner for a few minutes

Allow to coolTee, Margarita R.3PSY5

1 Kg of KHSO4 in a test tubeStrong odor

Unsaturation Test Tee, Margarita R.3PSY5 Place 3ml of DCM on each test tube

Add 10 drops of appropriate substance on their corresponding test tube

Mix thoroughly

Under the fume hood, add dropwise of 5% bromine-dichloromethane to each test tube while stirring until reddish brown color appears

3 large-sized test tubes labeled oil, fat and glycerolTee, Margarita R.3PSY5

Tee, Margarita R.3PSY5

Number of drops of bromine-dichloromethane and the net number of dropsRESULTSTADY, Kristine Claire D.GREASE- SPOT TEST: After droppingGRPOILLECITHINDCMHO1TranslucentOpaqueOpaque*Translucent2TranslucentOpaqueOpaque*Translucent3TranslucentOpaqueOpaque*Translucent4TranslucentOpaqueOpaqueTranslucent5TranslucentTranslucentDisappearedTranslucent6TranslucentOpaqueOpaqueTranslucent7TranslucentOpaqueOpaqueTranslucent8TranslucentOpaqueDisappearedTranslucent9TranslucentOpaqueOpaqueTranslucent10TranslucentOpaqueOpaqueTranslucent11TranslucentOpaqueOpaqueTranslucent12TranslucentOpaqueOpaque*Translucent35GREASE- SPOT TEST: After heatingGRPOILLECITHINDCMHO1TranslucentOpaqueOpaqueOpaque (dried)2TranslucentOpaqueOpaqueOpaque (dried)3TranslucentLight stainDisappearedWrinkles 4TranslucentSlight yellow stainOpaqueOpaque (dried)5TranslucentTranslucentNo stainDisappeared6TranslucentOpaqueOpaqueWrinkles7TranslucentOpaqueDisappearedDried8TranslucentOpaqueNo stainDisappeared*9TranslucentOpaqueOpaqueDisappeared*10TranslucentOpaqueOpaqueOpaque (dried)*11TranslucentOpaqueOpaqueOpaque (dried)12TranslucentOpaqueOpaque (dried)OpaqueSAPONIFICATION TEST: After shakingGRPOILMELTED FATHO1Bubbles with white interphase; cloudy solutionBubbles with yellow interphase; cloudy solutionLight yellow soln2EmulsionMicelleLight yellow liquid3Murky yellow emulsionCloudy yellow micelleLight brown soln4Yellow cloudy liquidCloudy white liquidColorless Solution5EmulsionMicelleColorless Solution6Bubbles formedNo bubbles formedClear Solution7EmulsionMicelleClear Solution8White foam on topWhite and yellow foam on topFaint brown soln9Bubbles formedBubbles formedPale yellow soln10Bubbles formedBubbles formedLight yellow soln11EmulsionMicelleOrangish Solution12Cloudy SolutionCloudy solution with yellow precipitateClear SolutionSAPONIFICATION TEST: Material on topGRPOILMELTED FATHO

1Dirty white liquidYellow solidsNone2White pptYellow pptNone3Bubbles Yellow spots-4BubblesBubbles yellow layer-5Bubbles, white filmBubbles, yellow powderNone6Light yellowYellow None7Cloudy whiteYellowColorless8White substanceYellow formationColorless9White pptVivid yellow pptNone10Dirty white liquidYellow solidsNone11White pptYellow pptNone12BubblesBubblesNoneSAPONIFICATION TEST: Acid or BaseGRPOILMELTED FATHO

1AcidAcidAcid2AcidAcidAcid3AcidAcidAcid4AcidAcidAcid5AcidAcidAcid6AcidAcidAcid7AcidAcidAcid8AcidAcidAcid9AcidAcidAcid10AcidAcidAcid11AcidAcid-12AcidAcidAcidUNSATURATION TEST: Br in DCMGRPGLYCEROLOILMELTED FAT

1---221155-325100414---511 6626 620135607229527816206092520181030130291185850 122528922UNSATURATION TEST: Net # of dropsGRPGLYCEROLOILMELTED FAT

142802520134-307516420130175858186011040722735804449057100100-11105042-1204-3REPORTER: Terrence Julian B. PestaoDiscussionPESTAO, #29Grease Spot TestA simple test in determining the presence of lipidsReagents: DCM, H2O, Lecithin, Oil sample

Principle: Vaporization of volatile substances

Visible Result: Translucent Stains

Positive results in: Oil Sample and Lecithin

PESTAO, #29

Grease Spot TestVolatilityUnsaturationNumber of CarbonsPESTAO, #29

Triglyceride

PESTAO, #29LECITHIN

PESTAO, #29Saponification TestInvolves saponification hydrolysis using a strong base Lipids (fat, oil)

Glycerol and Soap

PESTAO, #29Saponification Test

PESTAO, #29

PESTAO, #29Saponification TestReagents: NaOH (strong base), H2SO4

Principle: base hydrolysis

Visible Result: Formation of bubbles / precipitate

Positive results in: Melted Fat and Oil

PESTAO, #29Saponification Number/ Value (a.k.a. Koettstorfers Number) is the needed mg of NaOH (or KOH) to neutralize the fatty acids from the hydrolyzed 1g of the lipid

Can give notions regarding the structure of fatty acids

Molecular weightChain Length

The higher the saponification number (), the lower the molecular weight () (more fatty acids are freed per gram of fat after hydrolysis)PESTAO, #29

PESTAO, #29ReferencesRetrived:http://www.florinag.ch/index.php?sid=OV15cjStGNEbKLsL3baR56UxgZ1755qvyPOlQReb&c5p=495&c5l=en. (November 26, 2015).

Campbell, M.K. & Farrell, S.O. (2012/. Biochemistry. (7th ed.). California, USA: Brooks/Cole, Cengage Learning.

ConclusionVillanueva, Kervin B.ObjectivesTo characterize a fat or an oil sample using the grease-spot test, saponification test, acrolein test, and unsaturation test.The groups were able to characterize a fat or an oil sample using the grease-spot test, saponification test, acrolein test, and unsaturation test.The grease-spot test as a general test for the presence of lipids, would give a positive result in both the oil sample and lecithin but a negative result in water and DCM(Dichloromethane).The saponification test as a test for the presence of ester bonds, would give a positive result in both the oil sample and melted fat but a negative result in water.

The unsaturation test as a test for the presence of double bonds and the degree of saturation, would have different number of drops of bromine-dicholoromethane in unsaturating the glycerol, oil and melted fat until they colorize to a red-brown solution.

The acrolein test as a test for the presence of glycerol, would give a positive result in lecithin.

Sources of Errorinaccurate measurement of the reagents usedobservational error

ReferencesBook ReferencesBoyer, R. (2006). Concepts in Biochemistry,3rdedition. John Wiley and Sons(Asia) Pte.LtdCampbell, M. (2015). Biochemistry. Cengange Publishing (Singapore).Legaspi, G. (2009). Essentials of Biochemistry Laboratory. UST Publishing (Philippines).Menguito, C., et al. (2010). Basic Laboratory Experiments in Biochemistry. College of Science, University of Santo Tomas.Vance, D. (2008). Biochemistry of lipids, lipoproteins and membranes. Elsevier B.V.Online ReferencesCaballero, B. MD, PhD (2006) Lipids. Retrieved on February 20, 2011 from http://ocw.jhsph.edu/courses/humannutrition/PDFs/Lecture4