CHEM LAB M6

• Amino acids


  • Building blocks of proteins
  • Contains both an amino (-NH2) group and a carboxyl (-COOH) group
  • There are 20 naturally occurring amino acids
  • At their isoelectric point (pI) or neutral pH state, they exist as zwitterions
  • Except for glycine, all are optically active due to the presence of an asymmetric alpha Carbon atom
  • All naturally occurring proteins are in L-amino acid configurations while D-amino acids are usually present in bacterial cell wall and antibiotics
• Amino acid structure


  • Alpha carbon - central carbon
  • R group - side chain
  • NH2 - amino group
  • COOH - carboxyl group
• Amino acid solubility


  • Generally soluble in water but insoluble in non-polar organic solvents due to their property as zwitterions
  • Their solubility depends on the nature of the R group
  • Lack of solubility in non-polar organic solvents is due to lack of strong strong attraction between the solvent and amino acid
• Ninhydrin test


  • Blue color = alpha amino acid
  • Yellow color = imino acid = Proline
  • Amino acid undergoes oxidative deamination with the ninhydrin reagent, forming the liberation of CO2, NH3, an aldehyde, and hydrindantin
  • Almost all amino acids contain a free amino group which reacts with the ninhydrin reagent to form a blue-to-purple colored solution
  • Only imino acid (proline) has no free amino group to react with the reagent
• Xanthoproteic acid test


  • Orange color = aromatic amino acid = tryptophan or tyrosine
  • Aromatic rings become nitrated (nitric acid), creating a yellow solution and a strong base (NaOH) turns the solution orange
• Histidine test


  • Blue/violet = histidine
  • Bromination of histidine in acid solution (acetic acid) results in yellow solution then the ammonium carbonate neutralizes it resulting to blue-violet
• Sakaguchi test


  • Red = monosubstituted guanidine = arginine
  • Guanidine reacts with alpha naphthol and sodium hypochlorite (oxidizing agent) forming a red colored complex
  • Oxidation using NaOH and alpha naphthol reagent
  • Under alkaline conditions, the oxidizing agents react with arginine and treatment with hypochlorite resulting to red
• Lead sulfide test


  • Black Precipitate = Sulfur = Cystine
  • Amino acids release sulfur in the presence of NaOH at high temperatures which then reacts with the lead acetate to form a black precipitate
  • The NaOH and cysteine yields sodium sulfide, with heat, due to the partial conversion of organic sulfur to inorganic sulfide
  • The lead acetate solution precipitates it to form lead sulfide
• Adamkiewicz-hopkins or Acree-Roseanheimt test

  • Purple ring = tryptophan
  • The indole ring of tryptophan reacts with formalin and concentrated sulfuric acid to form an intermediate compound that then undergoes further oxidation to form a purple-colored chromophore
  • The formalin results in a condensation reaction with 2 tryptophan molecules
  • Sulfuric acid causes the 2 distinct layers and the violet ring indicates the presence of tryptophan which are the condensation products of the reaction
• Proteins


  • Polymers of amino acids held together by peptide bond, formed by amide linkage
  • Exists in different levels of structure, from the simplest covalently linked amino acids in primary structure, to more than one protein chain tied together in quaternary structure
  • Disruption of protein structure can cause temporary or permanent inactivation of protein
  • This disruption may be caused by changes in pH, addition of metals that interact with disulfide bridges, addition of organic poisons, or extreme temperatures
• Casein


  • Gives milk its white color
  • 80% = known amino acids
  • 20% = glutamic acid
  • One of the important dietary nutrients found in milk
  • Primary milk protein suspended in colloidal form
  • It will be precipitated and isolated using acetic acid which will then be subjected to qualitative tests
• Reagents


  • 10% acetic acid - used to precipitate milk
  • 50% ethyl alcohol-50% ethyl acetate - for dissolving fats and lipids
  • 0.6 M NaOH - balancing the solution to be more alkaline, making casein easier to dissolve
• Isolation of casein


  1. Mix liquid skim milk with acetic acid until complete precipitation

  2. Warm the mixture to 50-60 degrees C

  3. Allow the solid to settle and decant completely to collect casein

  4. Then add ethyl alcohol-ethyl acetate mixture to dissolve fats

  5. Decant the mixture

  6. Add distilled water and NaOH and stir to dissolve completely

  7. Use the liquid portion for the tests
• Biuret test


  • Proteins, in alkaline medium, reacts with dilute copper sulfate that form purple colored compound/complex
  • Copper (II) ions forms violate chelate complex = presence of proteins
  • More complex peptide bonds = increasing intensity in color
• Ninhydrin test


  • Reacts with free amino acids in a solution
  • Detects ammonia groups or primary and secondary amines
  • Blue color is caused by Ruhemanns complex (diketohydrin), detecting alpha amino acid
  • Yellow color indicates the presence of imino acid = Proline
• Isoelectric point (pI)


  • The pH at which majority of molecules have neutral or 0 net charge
  • pI < pH = like charged molecules emit a repulsive force, meaning the solution dissolves with minimal precipitation
  • pI > pH = solution molecules emit a greater attractive force, meaning the solution coagulates along with fats, rather than precipitation and isolation of the protein
  • Casein pI has a pH value of 4.6 (3.8 - 5.4)
  • At pH 4.6, the solution reaches saturation of precipitates or maximum precipitation