Qualitative Tests for Proteins

Study Notes

Qualitative Tests for Proteins

Biuret Test: Detects the presence of peptide bonds in proteins.
- Reagent: Biuret reagent (blue) containing copper (II) sulfate, sodium hydroxide, and potassium sodium tartrate.
- Positive Result: Violet-colored complex, with the intensity of the color increasing with the number of peptide bonds.
Ninhydrin Test: Detects the presence of free amino groups in proteins.
- Reagent: Ninhydrin solution (colorless).
- Positive Result: Purple solution.
- Exception: Hydroxyproline and proline produce a yellow solution.
Millon’s Test: Detects the presence of tyrosine and phenolic functional groups.
- Reagent: Millon’s reagent (colorless) containing mercuric nitrate and mercurous nitrate dissolved in nitric acid and distilled water.
- Positive Result: Red or pink-colored precipitate.
Xanthoproteic Test: Detects the presence of aromatic functional groups, such as those found in tyrosine and tryptophan.
- Reagent: Concentrated nitric acid and sodium hydroxide.
- Positive Result: Yellow or orange solution.
- Exception: Phenylalanine is negative, but it can become positive with extended heating.
Hopkins-Cole Test: Detects the presence of tryptophan, which contains an indole group.
- Reagent: Glyoxylic acid and sulfuric acid.
- Positive Result: Purple ring.
Fohl’s Test: Detects the presence of cysteine and cystine.
- Reagent: Sodium hydroxide and lead acetate.
- Positive Result: Black precipitate.
- Exception: Methionine is negative.
Pauly’s Test: Detects the presence of tyrosine, histidine, amines, phenols, and imidazole groups.
- Reagent: Sulfanilic acid, HCl, sodium nitrite, and sodium carbonate.
- Positive Result: Red-colored complex.
Sakaguchi Test: Detects the presence of arginine, which contains a guanidino group.
- Reagent: 1-naphthol and sodium hypobromite.
- Positive Result: Red-colored complex.

Qualitative Tests for Carbohydrates

Molisch Test: Detects the presence of carbohydrates except tetroses and trioses.
- Reagent: Molisch reagent (α-naphthol) and sulfuric acid.
- Positive Result: Purple ring.
- Mechanism: Sulfuric acid forms furfural from pentoses and hydroxymethylfurfural from hexoses.
Iodine Test: Detects the presence of starch (amylose).
- Positive Result: Blue-black solution.
Benedict’s Test: Detects the presence of reducing sugars, which contain hemiacetal functional groups.
- Reagent: Benedict’s reagent (blue) containing copper (II) sulfate, sodium carbonate, and sodium citrate.
- Positive Result: Brick-red precipitate.
Barfoed’s Test: Detects the presence of monosaccharides.
- Reagent: Barfoed’s reagent (blue) containing copper (II) acetate and acetic acid.
- Positive Result: Brick-red precipitate.
- Note: Disaccharides are also reducing sugars, but their reaction is slower.
Fehling’s Test: Detects the presence of aldehydes, aldoses, and alpha-hydroxy-ketones.
- Reagent: Fehling’s reagent (A: copper (II) sulfate, B: potassium sodium tartrate and sodium hydroxide).
- Positive Result: Brick-red precipitate.
Seliwanoff’s Test: Detects the presence of ketoses (ketones) - specifically fructose.
- Reagent: Resorcinol in concentrated HCl.
- Positive Result: Cherry red color.
- Mechanism: HCl causes the formation of furfural derivatives.
Bial’s Test: Detects the presence of pentoses.
- Reagent: Orcinol, concentrated HCl, and FeCl3.
- Positive Result: Blue or green color.
- Mechanism: HCl causes the formation of furfural from pentoses.

Enzyme Activity of Invertase

Invertase: Enzyme that breaks down sucrose into glucose and fructose.
- Monitoring: Benedict’s test can be used to monitor the reaction, as it detects fructose and glucose but not sucrose.
- Reducing Sugars: Fructose and glucose are reducing sugars, producing a brick-red precipitate in Benedict’s test.
Effect of Concentration:
- Optimum Concentration: 10% sucrose.
- Above Optimum: No observable change in activity.
Effect of Temperature:
- Optimum Temperature: Room temperature.
Effect of pH:
- Optimum pH: pH 5.
- Above Optimum: Decreased activity.

HIMALA and MIRACLE

HIMALA: A hypothetical substance that is positive for Sakaguchi, Fohl’s, and Pauly’s tests.
MIRACLE: A hypothetical substance that is negative for Sakaguchi, Fohl’s, and Pauly’s tests.