Vitamins & Nucleic Acids & Urinalysis PDF

VITAMINS - Vitamin C is a reducing agent, therefore when the iodine solution reacts with the starch to produce a blue-black color, the vitamin C reduces the complex formed between the iodine and thus the blue-black color vanishes. - The amount of iodine added is direc...

VITAMINS - Vitamin C is a reducing agent, therefore when the iodine solution reacts with the starch to produce a blue-black color, the vitamin C reduces the complex formed between the iodine and thus the blue-black color vanishes. - The amount of iodine added is directly proportional to the amount of vitamin C. Meaning, that as the iodine solution added increases the amount of vitamin C also increases - C6H8O6 + I2 —--> 2I- + C6H6O6 + 2H+ Chemicals used Chemical Purpose Vitamin C (Ascorbic Acid) The target analyte in the experiment. It is a reducing agent that reacts with iodine. Iodine Used as a titrant to determine the concentration of vitamin C. It reacts with vitamin C to form iodide ions. Starch Acts as an indicator in the titration. It forms a blue-black complex with iodine. Hydrochloric Acid (HCl) Used to acidify the solution, creating a suitable environment for the reaction between vitamin C and iodine. Lemon/Lime Juice Contains vitamin C, which can be used for the "magic writing" experiment. NUCLEIC ACIDS Chemical used Chemicals Purpose NaOH Lyses the yeast cells to release the RNA. Glacial Acetic Acid Neutralizes the solution after lysis to prevent RNA degradation. Ethanol Precipitates the RNA from solution. HCl Used to acidify the ethanol solution to  enhance RNA precipitation. Ether Removes any remaining contaminants from the precipitated RNA. Cold water, hot water, Ethanol, 6M HCl, 6M Used to test the solubility of RNA in various NaOH solvents. CuSO4 Used in the biuret test to detect the presence of peptide bonds. Methyl Red, Congo Red, Alizarin Acid-base indicators used to determine the pH of the RNA solution. H2SO4 Used to hydrolyze the RNA into its constituent nucleotides. Molisch Reagent Used to detect the presence of carbohydrates, including pentoses. Bial’s Reagent Used to specifically detect the presence of ribose (a pentose sugar found in RNA). Ammonium Molybdate ((NH₄)₂MoO₄) Used to detect the presence of phosphate groups in the hydrolyzed RNA. AgNO3 Used in the murexide test to detect the presence of purines. Bromine Water Used in the Wheeler-Johnson test to detect the presence of pyrimidines. Ba(OH)2 Used to neutralize the solution after the bromine water treatment in the Wheeler-Johnson test. Isolation of Yeast RNA - The goal of this step is to isolate and precipitate pure RNA - It involves the lysis of yeast, precipitation of unwanted proteins, filtration, precipitation of RNA and washing. Solubility Solvent Solubility Cold water Low  Hot water High Ethanol Low HCL Low NaOH High Solubility of most solid or liquid solutes increases with increasing temperature Hydrolysis of Yeast RNA Hydrolysis is a chemical reaction that breaks down a compound into its constituent parts by the addition of water. In the case of yeast RNA, this process involves the cleavage of the phosphodiester bonds that link the nucleotides together. RNA is hydrolyzed or “split” in order to assess its composition RNA + H₂O → Nucleotides pH Methyl red Red (6.2) Congo red Blue (5.0) Alizarin Yellow (6.8) Tests Principle 1. Biuret Test - The cupric (Cu2+) ions in the biuret reagent bind to the nitrogen atoms in the peptide bonds of proteins forming a violet-colored copper complex. 2. Molisch Test - A purple ring forms at the interface when molisch’s reagent and sulfuric acid are added to a carbohydrate solution 3. Bial’s Orcinol Test - When orcinol reacts with pentoses in the presence of hydrochloric acid, it produces a blue-green color 4. Ammonium Molybdate Test - Formation of yellow PPT via the stated reaction. When a mixture containing phosphate is heated with concentrated HNO3 & Ammonium Molybdate solution, a canary yellow precipitate of Ammonium Phosphomolybdate is formed.  5. Test for Purines (Murexide Test) - Formation of gelatinous PPT 6. Test for Pyrimidine (Wheeler-Johnson Test) - This test is based on the reaction of pyrimidines with specific reagent that leads to a purple color. Tests Summary Tests Test for presence Color produced Biuret Test Peptide bonds (proteins) Violet Molisch test Carbohydrates Purple ring Bial’s Orcinol Test Pentoses / Ribose Blue-green Ammonium Molybdate Test Inorganic phosphate Yellow PPT / Canary yellow Test for Purines Purines Gelatinous PPT Test for Pyrimidine Pyrimidines (Uracil & Purple Cytosine) URINALYSIS Chemical used Reagent Purpose Concentrated NaOH Used to create an alkaline environment for reactions, such as in the urea test. Bromine Water Reacts with urea to produce nitrogen gas, indicating its presence. Phosphotungstic Acid Reduces in the presence of uric acid, producing a blue color. Obermayer Reagent (concentrated HCl + Oxidizes indican to indigo blue, indicating its 10% FeCl3) presence.  Saturated Picric Acid Reacts with creatinine to form an orange-colored compound. 10% NaOH Used to create an alkaline environment for reactions, such as in the creatinine test. 20% Na2CO3 Used to create an alkaline environment for reactions, such as in the uric acid test. Exton’s Reagent Precipitates proteins, indicating their presence in the urine. Lugol’s Solution Reacts with acetone to form iodoform crystals, indicating the presence of ketone bodies. Benedict’s Reagent Reduces in the presence of glucose, producing a color change. Tincture of Alcoholic Iodine Mixture Oxidizes bile pigments, producing colored derivatives. Guaiac Powder Oxidizes in the presence of blood, producing a blue color. Hydrogen Peroxide Used in the occult blood test to oxidize guaiac powder. Acetic Acid Used to create an acidic environment for the occult blood test. Qualitative test for normal constituents Tests Principle 1. Test for Urea – Urea is the main end product of protein metabolism in man and other animals. The ammonia released from the deamination of amino acids are never removed through the urea cycle which occurs in the liver – The urea formed is then carried by the blood to the kidneys where it is excreted – The test for urea involves the production of N2 gas when urea reacts with NaOBr 2. Test for Uric Acid – Uric acid is the final product of purine oxidation in the body. It is derived from the degradation of nucleoproteins in food and of the body cells – In this test, the uric acid reduces phosphotungstic acid to give blue colored compounds – Uric acid is a reducing agent in alkaline medium. It reduced phosphotungstic acid to tungsten blue.  3. Indican Test – Indican (Potassium indoxyl sulfate) results from the decomposition of the amino acid tryptophan. – Urine indican when hydrolyzed yields indoxyl which is oxidized by ferric ions to indigo blue in the chloroform layer. – Indican converted to indoxyl with HCl and the created indoxyl converted to indigo blue colored compound with FeCl3 4. Test for Creatine – Urine creatinine comes from creatinine of the muscles, a waste product formed in the normal breakdown of muscle tissue – Creatinine reacts with picric acid in the alkaline medium to form a reddish-orange colored complex of creatinine picrate. Tests Summary Positive Negative Test for Urea Brisk effervescence of N2 Absence of effervescence gas Test for Uric Acid Formation of blue color Absence of blue color Indican Test Indigo in the chloroform layer Absence of color change in chloroform layer Test for Crea Presence of reddish-orange Absence of color change color Qualitative Test for Pathological Constituents Tests Principle 1. Test for Ketone Bodies (Gunning’s Test) a. The ketone bodies are formed in the liver and transported to peripheral tissues. Certain abnormal conditions cause an increase in blood ketone bodies above the normal levels and consequently increased in urine. b. The Gunning’s test depends on the reaction of acetone and alcoholic iodine resulting in the formation of iodoform crystals. 2. Test for glucose (Benedict's Test) a. Diabetes mellitus is a condition whereby the body cannot normally utilize sugar and consequently a large amount is excreted in the urine. b. Benedict's test is based on the fact that in a strong alkaline solution and in the presence of heat, reducing sugars will reduce cupric ions to cuprous oxide. 3. Test for Albumin (Exton’s Test) a. The kidneys, in the filtering process retain large protein molecules in the blood, presence of protein in the urine may indicate a disorder in the filtering mechanism. b. In this test the sulfosalicylic acid precipitates the proteins irreversibly c. Negatively charged sulfasalicylic acid neutralizes the positive charge on proteins causing denaturation, and hence precipitation of proteins. 4. Test for Bile Pigments (Smith’s Test) a. Bile is synthesized in the liver, stored in the gallbladder and is released into the intestinal tract during digestion. However in certain pathologic conditions the bile is not excreted in the intestine but accumulates in the blood and excreted in the urine. b. Tests for bile pigments (bilirubin, bilicyanin, biliverdin) depend upon oxidation of these bile pigments resulting in colored derivatives. 5. Test for Occult Blood a. The presence of red blood cells in the urine may indicate hemorrhage in the kidney or urinary tract. b. The test is based on the peroxidase and the liberated oxygen oxidizes organic substances such as benzidine and guaiac powder forming a green or blue color. Tests Summary Positive Negative Test for Ketone Yellowish six-pointed star or None Bodies six-sided plate (Gunning’s Test) Test for Glucose Green / yellow Traces of Blue solution None (Benedict’s Test) PPT reducing sugar Orange red PPT Moderate Brick red PPT Large amount of reducing sugar Test for Albumin Presence of precipitate Absence of precipitate (Exton’s Test) Test for Bile Presence of emerald/green layer Absence of green layer Pigments (Smith’s Test) Test for Occult Presence of blue ring / layer Absence of blue layer Blood