biochem

• Carbohydrates are the most abundant biomolecule in nature
• In plants, carbohydrates provide structural integrity in stems and roots in the form of cellulose
• In plants, carbohydrates act as a storage of glucose in the form of starch for the production of biochemical energy
• In humans, carbohydrates provide energy for the cell through the oxidation of monosaccharides
• In humans, carbohydrates are part of the structural framework of nucleotides
• In humans, carbohydrates act as messengers in cell communication and cell recognition
• Carbohydrates are polyhydroxylated aldehydes or ketones or molecules that yield polyhydroxylated aldehydes or ketones upon hydrolysis
• Monosaccharides are the simplest type of carbohydrates and can be classified based on the type of carbonyl group present or based on the number of carbons present in its open chain structure
• Monosaccharides can form glycosidic linkages with one another to form more structurally and biochemically complex carbohydrates such as oligosaccharides and polysaccharides
• Functional groups found in carbohydrates are the carbonyl group (as aldehyde or ketone) and the hydroxyl group
• Carbohydrates have high boiling and melting points and are generally soluble in polar solvents such as water due to intermolecular hydrogen bonding
• Carbohydrates undergo different chemical reactions due to the presence of different and multiple functional groups
• At the end of the activity, students must be able to classify carbohydrates based on the carbonyl functionality and based on the number of carbons, using qualitative chemical tests
• Materials needed for the experiment include reagents like glucose, amylose, maltose, xylose, fructose, galactose, lactose, sucrose, Molisch reagent, conc. sulfuric acid, iodine solution, Benedict’s reagent, Barfoed’s reagent, Seliwanoff’s reagent, Bial’s Orcinol reagent, and instruments like a water bath
• Safety precautions include wearing PPE and lab goggles at all times, washing hands thoroughly with soap and water before leaving the laboratory, and disposing gloves properly after performing the experiment
• Procedure involves preliminary preparations like labeling test tubes and placing sugar samples, conducting tests like Molisch Test, Iodine Test, Benedict’s Test, Barfoed’s Test, Seliwanoff’s Test, and Bial’s Test, and waste disposal guidelines for different types of waste generated
• Lipids are macromolecules that are insoluble in water and are not polymeric in nature
• Most components of biological membranes are lipids
• Lipids may function as hormones, antioxidants, pigments, growth factors, and vitamins
• Egg is a good source of nutrition for humans and provides a complete diet for a developing embryo
• An average egg contains approximately 74% water, 13% protein, 11% fat, and 1% ash
• Egg white does not contain fats, while egg yolk contains fats and is composed of approximately 33% of the egg
• Procedure for isolating and analyzing lipids from egg involves cracking open eggs, separating egg yolk, adding chloroform-methanol solution, filtering the mixture, extracting lipids using NaCl solution, evaporating and collecting lipids, and conducting tests like Libermann-Burchard Test
• Libermann-Burchard Test:

  • Place 20 drops of each lipid sample into two separate test tubes
  • Place 20 drops of distilled water into another test tube (blank)
  • Add 10 drops of acetic anhydride into each test tube then swirl gently
  • Allow 10 drops of conc. H2SO4 to slide down the walls of each test tube until it reaches the samples one at a time
  • Check for the formation of a blue-green-colored product to confirm the presence of cholesterol
• Test for Phosphate:

  • Mix 20 drops of each lipid sample with 5 mL of KNO3:Na2CO3 (3:1) solution into an evaporating dish
  • Heat the mixture until it turns to a grayish or colorless liquid or when a white or gray ash is obtained
  • Add 6 mL of warm water after cooling, then add 3 M HNO3
  • Heat the solution in a water bath at 65°C
  • Add 6 mL of 2.5% ammonium molybdate and heat in a boiling water bath
  • Check for the formation of a yellow precipitate to confirm the presence of a phosphate group
• Ninhydrin Test:

  • Place 15 drops of each lipid sample into two separate test tubes and prepare a blank in a separate test tube
  • Add 16 drops of ninhydrin in ethanol solution into each test tube
  • Heat the test tubes in a boiling water bath
  • Check for the formation of a blue or violet-colored product to confirm the presence of amino groups
• Molisch Test:

  • Place 15 drops of each lipid sample into two separate test tubes and prepare a blank in a separate test tube
  • Heat the samples until the liquid has evaporated
  • Add 15 drops of distilled water into the test tubes with dried samples
  • Add 5 drops of Molisch reagent into each test tube
  • Tilt the test tubes and allow 15 drops of conc. H2SO4 to flow down
  • Check for the presence of a purple ring at the interface to confirm the presence of carbohydrates
• Kraut’s Test:

  • Place 15 drops of each lipid sample into two separate test tubes and prepare a blank in a separate test tube
  • Heat the samples until the liquid has evaporated
  • Add 15 drops of distilled water into the test tubes with dried samples
  • Add 20 drops of Kraut’s reagent and swirl gently
  • Heat the test tube and check for the formation of a dark orange to red precipitate to confirm the presence of choline
• Acrolein Test:

  • Place 5 drops of each lipid sample into two separate test tubes and prepare a blank in a separate test tube
  • Add a pinch of potassium hydrogen sulfate (KHSO4) into each test tube
  • Heat the test tubes in an open flame or in a boiling water bath
  • Check for the formation of a burnt fat odor to confirm the presence of glycerol
• Qualitative Analysis of Amino Acids and Proteins:

  • Amino and carboxylic acid groups are functional groups in amino acids, building blocks of proteins
  • Proteins are polymers composed of amino acids linked by peptide bonds
  • Proteins have primary, secondary, tertiary, and quaternary structures
  • Denaturation alters protein structures and can be caused by heat, light, or chemical reagents
  • Objectives include determining protein presence, identifying amino acids, differentiating coagulable proteins, and understanding chemical tests
• Ninhydrin Test:

  • Perform on glycine, arginine, tryptophan, NAC, albumin, and gelatin
  • Heat the test tubes and check for a blue or violet-colored product to confirm amino group presence
• Biuret Test:

  • Perform on glycine, arginine, tryptophan, NAC, albumin, and gelatin
  • Check for the formation of a blue-violet solution to confirm protein presence
• Sakaguchi Test:

  • Perform on glycine, arginine, tryptophan, NAC, albumin, and gelatin
  • Check for the formation of a red-colored solution
• Hopkins-Cole Test:

  • Perform on glycine, arginine, tryptophan, NAC, albumin, and gelatin
  • Check for the formation of a deep violet or purple ring at the junction of two layers
• Lead Sulfide Test:

  • Perform on glycine, arginine, tryptophan, NAC, albumin, gelatin, and a strand of hair
  • Check for the formation of a black precipitate
• Heat Coagulation Test:

  • Perform on albumin and gelatin
  • Check for the appearance of dense coagulum after heating
• Simple carbohydrates have an empirical formula of CnH2nOn or Cn(H2O)n
• Carbohydrates can be classified based on the carbonyl functionality and the number of carbons
• Carbohydrates can be classified into monosaccharides, disaccharides, and polysaccharides
• Molisch Test:

  • Positive test for all carbohydrates
  • Positive result is a purple ring at the interphase of the two layers
  • Reagent used is α-naphthol with sulfuric acid