Postlab.pdf

Qualitative Tests for Proteins Biuret Test Biuret reagent (blue) – copper (II) sulfate, sodium hydroxide, potassium sodium tartrate (stabilizer) Detects peptide with at least two peptide bonds Positive: violet-colored complex; more peptide bonds, greater color intensity Nin...

Qualitative Tests for Proteins Biuret Test Biuret reagent (blue) – copper (II) sulfate, sodium hydroxide, potassium sodium tartrate (stabilizer) Detects peptide with at least two peptide bonds Positive: violet-colored complex; more peptide bonds, greater color intensity Ninhydrin Test Ninhydrin solution (colorless) Detects amines, α-amino acids, proteins with free amino groups (e.g., lysine) Positive: purple solution Hydroxyproline and proline: yellow solution Millon’s Test Millon’s reagent (colorless): mercuric nitrate and mercurous nitrate dissolved in nitric acid and distilled water Detects tyrosine and phenolic functional groups Positive: red or pink-colored precipitate Xanthoproteic Test Reagent: concentrated nitric acid and sodium hydroxide Detects aromatic functional groups (tyrosine and tryptophan) Positive: yellow or orange solution Phenylalanine – negative; too stable (positive after extended heating) Hopkins-Cole Test Reagent: glyoxylic acid and sulfuric acid Detects indole (tryptophan) Positive: purple ring Fohl’s Test Lead acetate or lead sulfide test Reagent: sodium hydroxide and lead acetate Detects cysteine and cystine Positive: black precipitate Methionine - negative Pauly’s Test Reagent: sulfanilic acid, HCl, sodium nitrite, sodium carbonate Detects tyrosine and histidine, amines, phenols, imidazole Positive: red-colored complex Sakaguchi Test reagent: 1-naphthol, sodium hypobromite Detects guanidino group, arginine Positive: red-colored complex  HIMALA MIRACLE Pauly’s test Positive Negative Fohl’s test Negative Positive Sakaguchi test Negative Positive  Biuret – negative Xanthoproteic test – positive Either dipeptides or free amino Aromatic amino acids present acids (tyrosine, tryptophan, phenylalanine may be present) Ninhydrin – positive Hopkins-Cole test – positive Amino acids are present Tryptophan is present Sakaguchi – positive Pauly’s test – positive Arginine is present Tyrosine or histidine possible present Fohl’s – negative No cysteine Millon’s test – negative Tyrosine is confirmed absent  Confirmed present Arginine Tryptophan Histidine Enzyme Activity of Invertase  Invertase Sucrose to glucose and fructose Monitored using Benedict’s test – detects fructose and glucose but not sucrose Sucrose is non-reducing; fructose and glucose are reducing Reducing sugars – brick-red precipitate in Benedict’s test  Effect of Concentration 10% sucrose optimum concentration More than 10% - no observable change Effect of Temperature Optimum temp – room temp Effect of pH Optimum pH – pH 5 Greater than pH 5 – decreased activity  optimum activity at pH 4.5 and 55°C (Neumann and Oliver Lampen, 1967) Qualitative Tests for Carbohydrates Molisch Test Molisch reagent (α-naphthol) and sulfuric acid Detects all carbohydrates except tetroses and trioses Positive: purple ring Sulfuric acid forms furfural from pentoses and hydroxymethylfurfural from hexoses Iodine Test Detects starch (amylose) Positive: blue-black solution Benedict’s Test Benedict’s reagent (blue) – copper (II) sulfate, sodium carbonate, sodium citrate Alkaline or basic condition Detects reducing sugars (hemiacetal functional group) Barfoed’s Test Barfoed’s reagent (blue) – copper (II) acetate, acetic acid Acidic condition Detects monosaccharides; all monosaccharides are reducing Reducing disaccharides – slower Positive: brick-red precipitate Fehling’s Test Fehling’s reagent A: copper (II) sulfate B: potassium sodium tatrate and sodium hydroxide Detects aldehydes, aldoses, and alpha-hydroxy-ketones (e.g., fructose) Positive: brick-red precipitate Seliwanoff’s Test Reagent: resorcinol in concentrated HCl Detects ketoses Positive: cherry red color HCl causes formation of furfural derivatives Bial’s Test Reagent: orcinol, concentrated HCl, FeCl3 Detects pentoses Positive: blue or green color HCl causes formation of furfural from pentoses  Cassava starch Fructose Iodine test Positive Negative Benedict’s Negative Positive Barfoed’s Negative Positive Fehling’s Negative Positive Seliwanoff’s Negative Positive  Glucose Sucrose Benedict’s Positive Positive (slow) Barfoed’s Positive Positive (slow) Fehling’s Positive Positive (slow) Seliwanoff’s Negative Positive  Galactose Lactose Benedict’s Positive (fast) Positive (slow) Barfoed’s Positive Negative  Reducing sugars have hemiacetal functional group Sucrose – nonreducing Glucose – reducing Lactose – reducing  Molisch – sample is a Seliwanoff’s – sample is not a carbohydrate ketose Iodine – sample is not starch Fehling’s – reducing sugar; Barfoed’s – sample is a aldose monosaccharide Bial’s – not a pentose Benedict’s – sample is a reducing Sample is most likely an sugar aldohexose