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Biochem Lab Finals Reviewer (expt5-7)


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Biochem Lab Finals Reviewer (expt5-7)

  1. 1. BIOCHEM LAB FINALSActivity 5A “Characterization of Lipids”A.SOLUBILITY-Lipids are immiscible in water-Immiscible in ordinary solvents-Miscible in Chloroform (CHCl3), Ether(C2H5O C2H5), Boiling alcohol (C2H5OH) andother ORGANIC SOLVENTS.B.TRANSLUCENT SPOT FORMATION-Lipids don’t absorb heat to evaporate whenthey in contact with paper or other surfaces.Factors affecting formation of translucentspot: Volatility and Number of Carbon atomsOrder of Formation of TS1.Linseed2.Coconut3.Peanut4.Corn5.Olive6.Cod LiverC.REACTION OF FATS-Fatty acids are carboxylic acids (organicacid)/weak acids-Neutral but when exposed to air becomesacidic due to hydrolysis result is formation ofvolatile fatty acids.D.ACROLEIN FORMATION-qualitative test for the presence ofGLYCEROL, free or esterified, based uponits oxidative dehydration to ACROLEINwhen heated with Potassium Bisulfate(KHSO4).Chemical Reaction:Activity 5B “Characterization of Lipids”EMUSIFICATION OF FATS-1 drop Coconut Oil + 1mL water-1 drop Coconut Oil + 1mL water + 0.5%Sodium Carbonate (Na2CO3) =emulsification taken place-1 drop Coconut Oil + 1mL albuminActivity 5C “Qualitative Test for Cholesterol”Preparation: Homogenize 20g Brain/Livertissue with 30mL acetone (C3H6O). Filterhomogenate on funnel. Suspend residue in30mL acetone and filter (Repeat twice).Combined filter contains neutral fats andcholesterol. Evaporate 25mL acetone filteron steam bath, dissolve in 5mL warm ethylalcohol (C2H5OH). Place in centrifuge tubeand add 1mL of 1% digitonin (C56H92O29) in90% alcohol. Put stopper set aside for 3hrs.white ppt. of cholesterol digitonin will betested by SALKOWSKI and LEIBERMANN-BURCHARD. Neutral fats are onsupernatant, after digitonin precipitation.Test for unsaturation.SALKOWSKI TEST-double bond in cholesterol rings + conc.Inorganic acid = color product-Conc. H2SO4 dehydrating agent-BLUISH color bet. CHCl3 and H2SO4LIEBERMANN-BURCHARD TEST
  2. 2. (Acetic Anhydride(CH3CO)2O or Ac2O)-H2SO4)-due to HYDROXYL GROUP (-OH) ofcholesterol + acetic anhydride + Conc.H2SO4 = DEEP GREEN COLORActivity 6A “Qualitative Test for Diff. Typesof Carbohydrates”MOLISCH TEST-sugar = (+)Reagent: a-naphthol and conc. H2SO4Reaction: PURPLE RING bet. H2SO4 andsugarPositive: Glucose, Galactose, Fructose,Maltose, Lactose, Sucrose, Glycogen,StarchANTHRONE TEST-reducing and non-reducing sugars = (+)Reagent: Anthrone if sol’n is milky addglacial CH3COOH or 50% H2SO4Reaction: BLUE-GREEN COLORPositive: Glucose, Galactose, Fructose,Maltose, Lactose, Sucrose, Glycogen,StarchPHENYLHYDRAZINE TEST (OSAZONEFORMATION)-presence of aldehyde or ketone group inreducing sugars and (-OH group)Reagent: PhenylhydrazineReaction: YELLOW-PALE ORANGECRYSTALSPositive: Glucose, Galactose, Lactose,MaltoseActivity 6B “Qualitative Test forCarbohydrates”MOORE’S TEST (Influence of ConcentratedAlkali)-based of liberation of aldehydes whichsubsequently polymerizes to form resinoussubs. “CARAMEL”-test for reducing sugars except SucroseReagent: Conc. NaOHReaction: YELLOW-ORANGE-FINALLY TODARK BROWN liberating a caramel odor,more marked upon acidificationPositive: Glucose, Galactose, Maltose,Fructose, LactoseNegative: Sucrose, Glycogen, StarchFEHLING’S TEST-aldoses are easily oxidized to yieldcarboxylic acids-Cupric ion complexed with tartrate ion isreduced to Cuprous oxide (brick red ppt.)-Tartrate ion complexing agent keepscopper ion solution. If absent cuprichydroxide would precipitate from basicsolution-Reduction of Cu2+> Cu+by reducing sugarReagent: Fehlings I and Fehlings II = deepblue solutionReaction: ORANGE-RED PPT. of Cu2OPositive: Glucose, Galactose, Maltose,Fructose, LactoseNegative: Sucrose, Glycogen, Starch
  3. 3. BENEDICT’S TEST-used to detect presence of allmonosaccharides and generally all reducingsugarsReagent: Sodium Citrate (Na3C6H5O7),Sodium Carbonate, Sodium hydroxide andCopper sulfateReaction: GREEN, YELLOW, ORANGE,RED-BRICK RED PPT.Positive: Glucose, Galactose, Maltose,Fructose, LactoseNegative: Sucrose, Glycogen, StarchNYLANDER’S TEST-used to detect presence of reducing sugarsexcept sucroseReagent: Sol’n of Rochelle Salt (potassiumsodium tartrate), KOH or NaOH andBismuth Subnitrate in waterReaction: BLACK PPT.Positive: Glucose, Galactose, Maltose,Fructose, LactoseNegative: Sucrose, Glycogen, StarchPICRIC ACID TEST-detects presence of reducing sugarsReagent: Saturated Picric acid solution(C6H3N3O7) and 5 drops 10% Na2CO3Reaction: MAHOGANY RED SOL’NPositive: Glucose, Galactose, Maltose,Fructose, LactoseNegative: Sucrose, Glycogen, StarchBARFOED’S TEST-reducing monosaccharidesReagent: Cupric Acetate (Cu(OAc)2) andAcetic acid (CH3COOH), HClReaction: REDDISH PPT. of copper (I)oxidePositive: Glucose (Dextrose), Galactose,Fructose (Levulose)Negative: Maltose, Lactose, Surcose,Glycogen, StarchSELIWANOFF’S TEST-dehydrated ketoses react with resorcinol toproduce deep cherry red color-aldoses react to produce faint pinkReagent: Resorcinol and Conc. H2SO4Reaction: DEEP CHERRY RED=Fructoseand SucroseFAINT PINK=Glucose, Galactose, Maltose,LactoseIODINE TEST-reacts with starch to form deep purple color-heating and addition of sodium thiosulfatemakes sol’n lighter in colorReagent: Iodine in Potassium Iodide (I2KI)and Sodium Thiosulfate (Na2S2O3)Reaction: DEEP PURPLE COLORMUCIC ACID TEST-oxidation of galactose and hydrolysis oflactose will yield insoluble mucic acid
  4. 4. -mucic acid crystal or galactaric acid melts@ 213 degree Celsius insoluble in alcoholand nearly in cold waterReagent: Mucic acid (C6H10O8) and HNO3Reaction: CRYSTAL FORMATION inGalactose and LactoseActivity 7 “Salivary Digestion”REACTION OF SALIVASaliva at rest = pH 6-7Stimulated = pH 8 up not exceed 12-PhenolphthaleinColorless-acidPurple-basic pH 8-9-Congo RedBlue violet- pH 3Red- pH 5-Litmus paperBlue-Red = AcidRed-Blue = BasicINORGANIC MATTER-Chlorides- Silver Nitrate (AgNO3)-Phosphates-Ammonium Molybdate((NH4)2MoO4)-Sulfates-Barium Chloride (BaCl3)-Calcium-Ammonium oxalate ((NH4)2C2O4)or Potassium oxalate (K2C2O4)Other inorganic matters: Sodium,magnesium, iodine, bicarbonate, potassiumORGANIC MATTERMucus, Amylase, Lipase, Ptyalin, Proteins,Antimicrobial enzymes