Biochemistry Basic Lab procedures
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BIOCHEMISTRY1st year laboratory test and their mechanismDIPESH YADAV
Molischs Test A)procedure:-1 ml of carbohydrate soln + 2-3 drops of -napthol + 2 ml conc. H2so4
B)Observation:-Violet ring at the junction of the two liquids C)Mechanism:- It is a general test for the detection of carbohydrates. The strong H2so4 Hydrolyses carbohydrate (poly- and disaccharides) to liberate monosac-Charides. The monosaccharide get dehydrated to form furfural (from pe-Toses) or hydroxy methylfurfural (from hexoses) which condense with -naphthol to form a violet coloured complex.CARBOHYDRATE
1)Procedure:- 2ml of carbohydrate soln + few drops of iodine soln Deep blue color:- with starch(non-reducing polysaccharides)
Purple color:-Dextrin soln
2)Mechanism:-Starch>soluble starch>Amylodextrin(purple)>Erythrodextrin(red)> Arcrodextrin(No colour)> maltose( end product of enzymatic hydrolysis of strach)Polysaccharides combine with iodine to form a coloured complex. -ve proceeds for benedicts test+ve proceeds for acid hydrolysis
>5 ml Bendicts reagent+ 1ml of carbohydrate soln>heat for 1-2 minutes>semiquantative quantative test because cuprous oxide gives different
colour with different concn of carbohydrate soln. 0.5-1%>>>>>>Green1-1.5%>>>>>>>>yellow1.5-2%>>>>>>>>>orange2-2.5%>>>>>>>>>>>Red composition of Benedicts reagent:- copper sulphate (BLUE SOLN), Sodium citrate(prevents cuprous ion) Sodium carbonate (mild alkali)Mechanism :- This is a test for the identification of reducing sugars, which form enediols (predominantly under alkaline conditions). The enediol forrms of sugars reduce cupric ions (cu++) of copper sulfate to coprous ions(cu+) which form a yellow precipiate of cuprous hydroxide or a brick red precipatate of cuprous oxide.
- Procedure:- 1ml of carbohydrate soln + 1ml of Barfoeds solnComposition of Barfoeds reagent
CLINICAL SIGNIFICANCE :- Thrombosis ,Degeneration, Necrosis Composition of Seliwanoffs reagent:- Resor cinol(ALDEHYDE COMPOUND) HCL(Dehydrates Ketohexose more rapidally to form furfural derivatives)Fructose-+VEGlucoseVEProcedure:-1ml of carbohydrate soln+ 3ml of Seliwanoff reagent heat for 30 sec.MECHANISM:- This is a specific test for ketohexoses. Concentrated HCL dehydrates Ketohexoses to form furfural derivatives which condense with resorcinol to give a cherry red complex.
Procedure:- 2ml of sugar soln+2ml of osazone reagent mix and heat over boiling water bath.
Osazone reagent :- 3 molecules of phenyl hyadrazine Osazone yellow crystals, crystalline derivatives of sugar and performed only with reducing sugarMechanism:-Phenylhydrazine in acetic acid, when boiled with reducing sugars forms osazones. The first two carbons(c1 and c2) are involved in this rxn. The sugars that differ in their configuration on these two carbons give the same type of osazones, since the difference is marked by binding with phenyl hydrazine. Thus, glucose, fructose, and mannose give the same type (needle shaped), maltose- sunflower shaped while lacose gives powder puff shaped
Procedure:-2ml of sucrose solution in test tube+ 5 drops of conc. Hcl boil for 1 min over small flame and cool the contents and add 40% NaOH soln.
Mechanism:- Sucrose is a non- reducing sugar. Hence, it does not give Benedicts and Barfoeds tests. Sucrose can be hydrolyzed by conc. HCL, to be con verted to converted to glucose and fructose which answer the reducing rxns. However, after sucrose hydrolysis, the medium has to be made alkaline (by adding 40% NaOH) for effective reduction process.Sucrose hydrolysis Test
BIOCHEMISTRY LAB TEST-Dipesh yadav (NEPALGUNG MEDICAL COLLEGE,MBBS-1)PROTEIN
Color rxn of protein
ReactionSpecific group or amino acidBiuret Two peptide linkagesNinhydrin rxnAlfa-amino acidsXanthoproteic rxnAromatic amino acid(phe, Tyr, Trp)Millions rxnPhenolic group(tyr)Hopkinss rxn(Aldehyde test)Indole ring (Trp)Sakaguchis test Guanidino group(Arg)Sulphur testSulfhydryl group(Cystine & Cystiene) Paulys testImidazole ring(His)Molischs test Carbohydrate moietyTest for organic phosphateCasein
ReactionMechanismBiuret In alkaline medium, peptide bond +cupric ion to form violet colored complexNinhydrin rxn1)AA + ninhydrinketo-acid+NH3+Co2+Hydrinadantin2) Hydrinadantin+NH3+NinhydrinRuhemanns purpleXanthoproteic rxn(Nitratation rxn) Phenyl gr. + HNO3nitrophenyl gr.(yellow)While adding alkali to the nitrophenyl soln gives orange colourMillions rxnPhenolic gr.+Hgso4mercurictyrosine complex which on nitration give red color with sod. nitrateHopkinss rxn(Aldehyde test)INDOLE gr.+Formaldehyde+(in presence of oxidizing agent H2SO4 with mercuric sulphate)violet colored complexSakaguchis test In alkaline medm(40% NaOH), -napathol+Guanidino Gr.complex which is oxidized by sod. Hypobromide to give red colour complex
Sulphur testWhen cysteine and cystine are boiled with NaOH, organic sulfur is converted to inorganic sodium sulfide. This reacts with lead aceatate to form black ppt. of lead sulfide. Methionine doesnot give this test, since sulfur of methionine isnot split by alkaliPaulys testDiazotised sulfanilic acid reacts with imidazole ring om alkaline medium to form a red colored complex.Molishs testThe proteins containing carbohydrate(Glycoprotein ) give +ve to this test.(ALBUMIN)Test for organic phosphate