EXPERIMENT 3 & 4 - REAGENTS

Biuret

MAIN REAGENT:

• Copper (II) sulfate (CuSO₄)

  • Source of (Cu²⁺) that reacts with the nitrogen atom in the peptide bond, producing the purple/violet complex.

• Sodium hydroxide (NaOH)

  • Provides an alkali medium

Sakaguchi

MAIN REAGENT:

• a-naphthol

  • reacts with guanidino group

• Sodium hydroxide (NaOH)

  • provides the alkaline medium.

• Sodium hypochlorite (NaOCl)

  • modifies a-naphthol into a more reactive intermediate. 

    • Promotes the condensation reaction, producing a stable red-colored chromogen.

Ninhydrin

MAIN REAGENT:

• Ninhydrin (2,2-dihydroxyindane-1,3-dione)

  • produces the purple/blue color in the reaction when heated.

• Triketohydrindene

  • oxidizes a-amino acid.

Xanthoproteic

MAIN REAGENT:

• Conc. Nitric Acid (HNO3)

  • strong acid

  • reacts with the phenyl rings present in tyrosine and tryptophan,

  • which causes nitration to NO2 

• Sodium Hydroxide (NaOH)

  • base that ionizes the solution,

  • turning the solution orange 

Hopkin’s Cole

MAIN REAGENT

• Sulfuric Acid (H2SO4) is a dehydrating agent that allows glyoxylic acid to react easily with the indole group present in the solution. 

• Hopkins-Cole Reagent; 

  • Glyoxylic acid (from acetic acid) reacts with the indole group of the amino acid such as tryptophan. In the presence of H₂SO₄, the reaction produces a violet/purple ring

Molisch

MAIN REAGENT: α-naphthol dissolved in ethanol

• Ethanol

  • acts as a bridge solvent

  • helps ⍺-naphthol spread evenly, because it is miscible with water and ⍺-naphthol.

WITH: Conc. sulfuric acid (H₂SO₄)

Anthrone

MAIN REAGENT: Anthrone (C₁₄H₁₀O)

WITH: Conc. sulfuric acid (H₂SO₄)

Iodine

MAIN REAGENT:

Lugol’s Iodine Solutio: Iodine (I₂) dissolved with Potassium Iodide (KI) in water

Mucic Acid

MAIN REAGENT: Conc. nitric acid (HNO₃)

• strong oxidizing agent

Benedict’s

COMPOUNDS IN REAGENT:

• Copper(II) sulfate (CuSO₄)

  • source of copper ions, blue color

    
    

• Sodium carbonate (Na₂CO₃)

  • provides alkaline environment

    
    

• Sodium citrate (Na₃C₆H₅O₇) or (C₆H₅Na₃O₇)

  • keeps copper in solution

Barfoed’s

MAIN REAGENT:

• weak acidic solution:

  • Copper (II) acetate + 1% acetic acid (diluted, ethanoic)

Bial’s Orcinol

MAIN REAGENT: Orcinol + HCl + FeCl₃

WITH: Conc. sulfuric acid (H₂SO₄)

Seliwanoff

MAIN REAGENT: Resorcinol

WITH: Hydrochloric acid (HCl)

Fehling

MAIN REAGENTS:

• Fehling’s A: Copper(II) sulfate solution

  • clear, blue solution that contains CuSO₄ dissolved in water.

  • This copper compound provides Cu²⁺ ions that are reduced if a reducing substance is present.

• Fehling’s B: Alkaline solution of sodium potassium tartrate (Rochelle salt) and sodium hydroxide

  • a colorless solution made from potassium sodium tartrate (Rochelle salt) and sodium hydroxide (NaOH).

  • Tartrate keeps the copper ions in solution without forming a precipitate.

  • NaOH provides the alkaline medium needed for the reaction to happen.

• Keeping them separate and only mixing when ready creates an active reagent that reacts with reducing sugars.

• Without the tartrate in Fehling’s B, the test wouldn’t work because Cu²⁺ would be unavailable for reduction.