biochem

• Experiment 1 | Qualitative Tests for Identification of Functional Groups in Organic Compounds
• Unsaturated


  Contains double or triple bonds (alkenes, alkynes)
• Saturated


  Contains only single bonds (alkanes)
• Benzene = UNSATURATED
• von Baeyer's test


  1. Reagent used = KMnO4 Potassium permanganate (violet color)

  2. Positive Result = brown precipitate

  3. Reducing Product = MnO2 manganese dioxide
• Bromine Test


  1. Reagent used = Bromine solution (dark red-orange)

  2. Positive Result = instant disappearance of bromine color
• Alcohols


  Organic compounds containing the hydroxyl (-OH) functional group attached to a saturated carbon atom

  
• Phenols


  Type of alcohol where -OH group is directly attached to a benzene ring

  
• Bordwell-Wellman Test


  1. Reagent Used = K2Cr2O7 Potassium dichromate dissolved in conc. sulfuric acid (H2SO4) (orange yellow)

  2. Positive Result = greenish color

  3. For Phenols, positive result = brown color
• Lucas Test


  1. Reagent Used= Lucas reagent (mixture of conc. Hydrochloric acid and zinc chloride) (HCl+ZnCl2)

  2. Positive Result = Emulsion/Formation of 2nd layer

  3. Primary – no reaction

  4. Secondary – slow reaction

  5. Tertiary – fast/Immediate reaction
• Ferric Chloride Test


  1. Reagent Used = 2% ferric chloride solution (orange)

  2. Positive Result = violet
• EXPERIMENT 1 OUTLINE
• Tests in Experiment 1

  • Test for Unsaturation (Alkenes and Alkynes)
  • Test for Alcohols and Phenols
  • Test for Aldehydes and Ketones
  • Test for Carboxylic Acid
• Aldehydes & Ketones


  Share carbonyl functional group (C=O)

  
• Aldehydes


  At least one Hydrogen Bonded to carbonyl carbon; the other attachment may be a carbon or a hydrogen

  
• Ketones


  Two carbon atoms attached to carbonyl group and no hydrogen

  
• 2,4-DNP Test


  1. Reagent used = 2,4 Dinitrophenylhydrazine

  2. Positive Result = yellow-orange precipitate

  3. Product = 2,4-dinitrophenylhydrazone
• Tollen's Test


  1. Reagent used = Tollen's reagent

  2. Positive Result = silver mirror (on the wall of clean test tube)
• Carboxylic acids


  Contains a carbonyl group but with one hydroxyl (-OH) group attached to carbonyl carbon

  
• Sodium Bicarbonate Test


  1. Reagent used = saturated sodium bicarbonate solution

  2. Positive result = brisk effervescence
• Experiment 2 | Properties of Proteins
• Outline of Experiment 2

  • Protein: Definition & Composition
  • Denaturation of Proteins
  • Color Reaction
• Proteins are responsible for a lot of physiological functions, making it the most abundant macromolecules of the body. It accounts for about 15% of total cell's mass.
• Protein


  A protein when it has at least 50 amino acid residues, if less than it is called a peptide
• Alpha-amino acid


  General structure is composed of a carboxyl group, amino group, and a side chain. The side chain (R-group) gives the amino acid's identity.
• Classification of amino acids according to the polarity of their side chains

  • Non-polar amino acids
  • Polar neutral amino acids
  • Polar acidic amino acids
  • Polar basic amino acids
• Peptide bonds


  Connects peptides and proteins in amino acids. Formed when carboxyl group of one amino acid reacts with the amino group of another amino acid.
• Native conformation


  Specific three-dimensional structure of protein. It can be globular (spherical), fibrous (elongated).
• Denaturation


  The unfolding and changing of structure in protein that alters its biological function. Denaturation can be reversible or irreversible.
• Coagulation


  The clumping of molecules. This clumping is called the coagulum.
• Coagulation


  1. Alcohol | Reagent used = Absolute ethanol

  2. Heat | Treatment =Heating

  3. Both results yield COAGULUM
• Precipitation of proteins


  1. Heavy Metal Salts

  2. Strong Mineral Acids

  3. Alkaloidal Reagents
• Egg Albumin used in all tests
• Color Reactions


  Used to transform colorless chemical compounds into colored derivatives which can be detected visually to determine the functional groups in the amino acids present.
• Biuret Test for Peptide Bonds (Piotrowski's Test)

  1. Checks for proteins

  2. Reaction: Cupric ions (Cu2+) in alkaline medium form a violet complex with peptide bonds

  3. Color Change: Original blue reagent turns violet

  4. Results: Violet, pink, or pale violet indicates a positive test

  5. Intensity: stronger violet means more peptide bonds

  6. Other compounds: also works for peptides, proteoses, and peptones

  7. Requirement: At least 2 bonds needed

  8. NOTE: Individual amino acids won't react.
• Ninhydrin Test for Free Alpha-Amino Group

  1. Checks for proteins w/ free alpha-amino groups

  2. Reaction: Free alpha-amino groups react with ninhydrin

  3. Products: Produces CO2, NH3, aldehyde, and hydrinantin

  4. Color Change: NH3 reacts with ninhydrin to form Ruhemann's pruple

  5. Positive Result= deep blue/violet color

  6. Proline= yellow color

  7. Asparagine and Glutamine = brown color
• Xanthoproteic Test (Phenyl-containing Amino Acids)


  1. Checks for amino acids with phenyl rings (phenylalanine, tyrosine, tryptophan)

  2. Reaction: Phenyl rings undergo nitration w/ conc. Nitric acid (HNO3) when heated

  3. Color change: produces a yellow color

  4. Alkaline Medium: Yellow intensifies

  5. Positive Result: Yellow to orange color
• Adamkiewicz Reaction (Tryptophan)


  1. Checks for tryptophan

  2. Reaction: Tryptophan's indole group reacts with glyoxiclic acid and conc. Sulfuric acid (H2SO4)

  3. Color change: forms a purple complex

  4. Positive Result: Violet ring indicates tryptophan is present
• Reduced Sulfur Test (Cysteine)


  1. For cysteine

  2. Reaction: Boil cysteine with strong alkali

  3. Product: Forms Na2S (sodium sulfide)

  4. Indicator: add lead acetate to get lead sulfide, a black precipitate

  5. Positive Result: Black precipitate (cysteine; present)

  6. NOTE: Methionine doesn't react because its sulfur is harder to break.
• Carbohydrate Moiety


  Amino acids & non-amino acid groups called prosthetic groups, like carbohydrates and phosphate