Biochemical Tests

TEST FOR CARBOHYDRATES Molisch Test (-Naphthol Reaction)  general test for carbohydrates  reagent: 15% -naphthol in alcohol  positive result: violet ring at the junction of 2 liquids Seliwanoff’s Test (Resorcin-HCl Test)  specific test for fructose and ketohexoses  reagent: resorcinol in dilute HCl (Seliwanoff’s reagent)  rapid formation of hydroxymethylfurfural as compared to glucose and others that do so only when overheated  positive result: red color to red ppt Tollens Test (Phloroglucin-HCl Test)  test for aldehyde and ketone  reagent: 2% phloroglucin solution  positive result: silver mirror Bial’s Test (Orcin-HCl test)  test for pentoses  reagent: 50% saturated orcinol solution in alcohol  positive result: blue green color Tauber’s Benzidine Test  test for pentoses  reagent: benzidine solution  positive result: cherry red color Moore’s Test  action of strong alkalies  reagent: concentrated NaOH  positive result: caramel

Phenylhydrazine Reaction  test for sugars which produce specific osazone crystals (lactose and mannose)  glucose, fructose and mannose produce the same crystals  reagent: 1:1 phenylhydrazine and NaOAc  positive result: o glucosazone – needle shaped o lactosazone – fluffly ball shaped o maltosazone – sunflower shaped Fehling’s Test  test for reducing sugars  general test for monosaccharides  reagent: o Fehling’s A – copper sulfate solution o Fehling’s B – Rochelle salt in H2O (alkaline tartrate solution)  positive result: brick red ppt Benedict’s Test  test for reducing sugars  reagent: CuSO4 + Na2CO3 + Na3C6H5O7  positive result: brick red ppt Barfoed’s Test  test for distinguishing reducing monosaccharides from disaccharides  reagent: copper acetate solution, 50% HOAc  positive result: brick red ppt o monosaccharides – within 7 minutes of boiling o disaccharides – after 7 minutes of boiling Trommer’s Test  acid hydrolysis to generate monosaccharides from disaccharides  reagent: KOH added with CuSO4  positive result: bright red color

Nylander’s Test  test for glucose  reagent: bismuth subnitrate  positive result: black ppt Mucic Acid Test  test for lactose and galactose  reagent: concentrated HNO3 (oxidation)  positive result: mucic acid crystals Nitro-Chromic Acid Test  test for the presence of free CH-OH groups  reagent: concentrated HNO3 + 5% K2CrO4  positive result: blue color Iodine Test  test for starch  reagent: Lugol’s solution  positive result: deep blue color TEST FOR PROTEINS Biuret Test  test for Biuret (NH2-CO-NH-CO-NH2 group) from peptide linkages  reagent: NaOH + CuSO4 + potassium sodium tartrate  positive result: violet solution Pauly’s Diazo Test  test for histidine (H), tyrosine (Y)  reagent: sulfanilic acid in HCl  positive result: red color (azo dye)

Xanthoproteic Acid Test  test for phenyl group or benzene ring (phenylalanine, tyrosine, tryptophan)  reagent: HNO3  positive result: yellow to orange color Ninhydrin Test  test for -amino acids  reagent: phosphate buffer + ninhydrin solution  positive result: blue or purple product (Ruhemann’s purple) Millon’s Test  test for hydroxybenzene or phenol group  positive for tyrosine (Y)  reagent: mercuric sulfate in H2SO4  positive result: old rose ppt Glycoxylic Acid Test (Adamkiewicz Reaction)  test for indole ring  positive for tryptophan (W)  reagent: acetic acid + H2SO4  positive result: violet ring Lead Acetate Test (Reduced Sulfur Test)  test for sulfur in sulfhydryl group  positive for cysteine (C), methionine (M), cystine (2 cysteine molecules bonded with a disulfide link)  reagent: NaOH + 1% lead acetate solution  positive result: black ppt Sakaguchi Reaction  test for guanidine group (arginine)  reagent: NaOH and naphthol solution  positive result: red color

HISTOCHEMICAL TESTS

TEST FOR OILS

Reagent Dragendorff’s reagent

Component Tested alkaloids

Ferric chloride TS

tannins

Iodine TS Sudan III Tollens’ reagent

starch fats and oils lignin

Positive Result orange ppt green, blue, blue-green or black color dark blue color red orange globules bright pink color

Test Halpen or Bevan Test Perken Test Miloni Test Baudouin Test Serger Reaction

Fixed Oils Cottonseed oil Cottonseed oil Olive oil Sesame oil Vegetable oil

ALKALOIDAL REAGENTS GLYCOSIDE TESTS Test Liebermann-Burchard Test Kedde Test

Component Tested sterols

Keller-Kiliani Test

2-deoxysugars

Fehling’s Test

reducing sugars

unsaturated lactones

Positive Result green color with red and blue tones violet-blue color reddish-brown color that may turn blue or purple brick-red ppt

Reagent Tannic acid solution Mayer’s reagent Wagner’s reagent Valser’s reagent Dragendorff’s reagent Kraut’s reagent Hager’s reagent

TEST FOR TANNINS

Marme’s reagent Hydrolyzable Other Names

Pyrogallotannins

Composition

Gallic acid or related polyhydric compound esters

Hydrolysis

Readily hydrolyze into phenolic acids and sugar

Boiled with HCl + FeCl3 + heat + bromine H2O

Gallic or ellagic acids Blue or blue-green color Pyrogallol No ppt

Nonhydrolyzable Catechol tannins Pyrocatechol tannins Phlobatannins Only phenolic nuclei frequently linked to CHOs or CHONs Polymerize into insoluble, usually red-colored products known as phlobaphenes Red insoluble phlobaphenes Greenish black color Catechol Ppt

Sonnenschein’s reagent Scheibler’s reagent Rosenthaler’s reagent Shaer’s reagent Mecke’s reagent Marqui’s reagent Mandalin’s reagent Erdmann’s reagent

Component Tannic acid Hg in KI I in KI HgI2 K-Bi iodide Modified Dragendorff’s reagent Saturated picric acid solution Potassium cadmium iodide Phosphomolybdic acid Phosphotungstic acid Potassium arsenate, concentrated H2SO4 H2O2, concentrated H2SO4 Selenious acid, concentrated H2SO4 Formaldehyde, concentrated H2SO4 Sulfovanidic acid Concentrated HNO3, concentrated H2SO4

Positive Result Dark brown ppt Cream ppt Reddish-brown ppt Orange ppt Reddish brown ppt Brown ppt Yellow ppt

ADDITIONAL NOTES Antibiotic Structures

Penicillins Cephalosporins Aminoglycosides Tetracyclines Macrolides

-amino penicillanic acid (-lactam ring + thiazolidine ring) -lactam ring + dihydrothiazine ring Aminocyclitol ring Four fused cyclic ring Macrocyclic lactone ring

Blood Typing Blood Type A B AB (universal receiver) O (universal donor)

Antigen N-acetylgalactosamine D-galactose Both N-acetylgalactosamine and Dgalactosamine None