Biochemistry Lab Midterm - Carbohydrates PDF

Biochemistry for Medical Laboratory Science MIDTERM LAB: CARBO PT. 1 — TESTS BASED ON FURFURAL & ITS DERIVATIVES CMLS A.Y. 24 – 25 1st Semester Ms. Danielle Marie Asprer Allarey-Susa PRE-LAB Examples:...

Biochemistry for Medical Laboratory Science MIDTERM LAB: CARBO PT. 1 — TESTS BASED ON FURFURAL & ITS DERIVATIVES CMLS A.Y. 24 – 25 1st Semester Ms. Danielle Marie Asprer Allarey-Susa PRE-LAB Examples: - Maltose (Glucose + Glucose) – CARBOHYDRATES abundant in germinating barley. - Sucrose (Fructose + Glucose) – also A polyhydroxy aldehyde, polyhydroxy known as table sugar / cane sugar / ketone, or a compound that yields beet sugar. polyhydroxy aldehydes and - Lactose or Milk Sugar (Galactose + polyhydroxy ketones upon hydrolysis. Glucose) – does not taste very sweet Commonly known as sugars because and is not fermented by yeast. most of them have a sweet taste. Serves as a major source of energy. 3. POLYSACCHARIDES Furnishes the carbon chains of compound synthesis by living cells. Found in nature and function either as: Bioorganic compounds: - Structural polysaccharide 1. Proteins ➔ Cellulose 2. Lipids - Storage polysaccharide 3. Nucleic Acids ➔ Starch Categorized as: ➔ Dextrin - Monosaccharide ➔ Glycogen - Disaccharide ➔ Insulin - Polysaccharide OBJECTIVES CLASSIFICATION OF CARBOHYDRATES To be able to identify the different types 1. MONOSACCHARIDES of carbohydrates using different specific chemical tests. Simple sugar. a. Test based on furfural and its - Highly soluble → in Water. derivatives - Less soluble → in Ethanol. b. Test based on the reducing properties - Insoluble → in Ether. of sugars They are either aldoses or ketoses. Free monosaccharides are all reducing Materials Used sugars. Equipment Hot Plate 2. DISACCHARIDES Materials Test Tube Rack Big & Small Beaker Test Tube Formed by two molecules of Test Tube Holder monosaccharides. Alcohol Lamp Wire Gauze Carlyle D. Substances 3% of: Xylose, Glucose, PROCEDURE Sucrose, Lactose, Fructose, Starch Distilled H2O 10% FeCl3 Molisch Reagent Bial’s Orcinol Reagent Seliwanoff’s Reagent Sulfuric Acid TEST BASED ON THE FORMATION OF Distilled H2O → 3% Glucose → 3% Lactose FURFURAL & ITS DERIVATIVES → 3% Starch → 3% Xylose Principle: Hot H2SO4 will dehydrate 1. Add 4 mL of solution each tube and 2 sugars into furfural and its derivatives. drops of Molisch reagent. 2. Incline the test tube and slowly add Furfural / Hydroxymethylfurfural + about 2 mL of concentrated H2SO4 Phenolic compounds (𝛂-naphthol, orcinol, - Sulfuric acid is denser than water resorcinol) → colored condensation and will form a lower layer. products [(+) for carbs] 1. MOLISCH TEST Results Solution Color Distilled H2O Presence of green ring 3% Glucose 3% Xylose Presence of purple ring 3% Lactose 3% Starch The general test for carbohydrates. 2. BIAL’S ORCINOL TEST Molisch reagent contains 𝛂-naphthol Determines the presence of pentoses, (+) = formation of purple ring. but not specific. The test tube is inclined, and Distinguishes pentose monosaccharide concentrated H2SO4 is added along the and hexose monosaccharide. side of the tube, causing the formation of The reaction is not specific for pentoses, a lower layer of acid. because other compounds like trioses, - The concentrated H2SO4 will uronic acids, and certain heptoses will dehydrate the sugar, allowing it to also give blue or green products. react with the alcohol forming furfural or hydroxymethyl-furfural. Carlyle D.  Pentoses + Orcinol → Blue Green Ketohexoses + (+) Cherry Red Furfural + Fe3 Ions Compound (HCl & Resorcinol) Condensation Product Hexoses + Orcinol → Yellow Brown Hydroxymethyl Furfural Pigment Aldohexoses + (—) Pale Pink + Fe3 Ions (HCl & Resorcinol) Condensation Product PROCEDURE PROCEDURE 3% Xylose → 3% Glucose → 3% Starch 3% Xylose → 3% Fructose → 3% Glucose → 3% Sucrose 1. Add 1 mL of solution each tube and 3 mL of Bial’s Orcinol reagent. 1. Add 1 mL of solution each tube and 4 mL 2. Carefully heat test tubes over a Bunsen of Seliwanoff’s reagent. Burner / alcohol lamp until the solution 2. Heat at boiling H2O for 5 minutes. begins to boil. 3. Record initial result. 3. Add 2 drops of FeCl3 solution. 4. Continue boiling for 10 minutes. Results Results Solution Color Type of Solution Initial Final Type of Mono Mono 3% Xylose Blue Green Pentose 3% No reaction None Xylose 3% Glucose Yellow Brown Hexose 3% Yellow Pale Aldohexose 3% Starch Glucose Pink All samples are (+) → w the 3% Pink / presence of sugar Fructose Red Cherry Red Ketohexose 3% Pale 3. SELIWANOFF’S TEST Sucrose Pink This test is used to differentiate ketohexoses from aldohexoses. Ketohexoses react faster with the solution of hydrochloric acid and resorcinol than aldohexoses. Carlyle D. Biochemistry for Medical Laboratory Science MIDTERM LAB: CARBO PT. 2 — TESTS BASED ON THE REDUCING PROPERTIES OF SUGARS CMLS A.Y. 24 – 25 1st Semester Ms. Danielle Marie Asprer Allarey-Susa OBJECTIVES Used for sugars with aldehydes. Benedict’s reagent: To be able to identify the different types - CuSO4 (copper sulfate) of carbohydrates using different specific - Na2CO3 (sodium carbonate) chemical tests. - Na3C6H5O7 sodium citrate a. Test based on furfural and its Positive compounds: derivatives - Brick Red Precipitate Cu2O (copper b. Test based on the reducing oxide) properties of sugars - Most aldehydes: ➔ Formic acid Materials Used ➔ Hydrazobenzene ➔ Phenols Equipment Hot Plate ➔ Phenylhydrazine Materials Test Tube Rack ➔ Pyrogallol Big & Small Beaker ➔ Uric acid Test Tube Test Tube Holder Alcohol Lamp PROCEDURE 2 Pasteur Pipette Wire Gauze Substances 3% of: Xylose, Glucose, Sucrose, Lactose, Fructose, Starch Distilled H2O Benedict’s Reagent Barfoed’s Reagent Tollen’s Reagent TESTS BASED ON THE REDUCING Urine → Juice PROPERTIES OF SUGARS 1. Add 2 mL of 3% glucose (mono; Reducing sugars must have a free aldohexose), 3% xylose (pentose), 3% aldehyde or ketone group. fructose (ketohexose), 3% lactose (di), All monosaccharides and disaccharide and 3% sucrose (di) samples in reduce, meaning it donates Hydrogen to separately labeled test tubes. Oxidizing reagents (cupric ions, 2. Add 2 mL of Benedict’s reagent, then mix dinitrosalicylic acid, and picric acid) → both. are all organic acids. 3. Place in boiling water for 2-3 minutes. Avoid prolonged heating. 1. BENEDICT’S TEST 4. Observe color of precipitate. Very sensitive test done under mildly alkaline conditions. Carlyle D.  Results PROCEDURE Solution Final Type of Time Sugar 3% Glucose 3:00 3% Brick Mono Xylose Red 2:40 3% Fructose 1:30 3% Glucose → 3% Fructose → 3% Lactose → 3% Starch → 3% Xylose 3% Brownish Lactose Red Di 3:00 1. Add 1 mL of sugar per tube and 3 mL of 3% Blue Barfoed’s reagent, then mix both. Sucrose 2. Place in boiling water for 10 minutes. 3. Observe colored precipitate. All test analytes except sucrose have a red powdery precipitate formed at the bottom of the test tube. Results - Sucrose did not react since it is not a Solution Final Type of Time reducing sugar. Sugar Monosaccharides oxidize faster than disaccharides. Control Blue None None H2O 2. BARFOED’S TEST 3% Glucose Used to distinguish monosaccharide, disaccharides, and oligosaccharides. 3% Brick Mono 2-3 mins Xylose Red Very sensitive test under acidic conditions. 3% Barfoed’s reagent: Fructose - Cupric acetate in dilute acetic acid 3% Di Barfoed’s reagent oxidizes: Lactose - Monosaccharides (hydrolyzed) Blue None - Disaccharides 3% Poly - Oligosaccharides (less oxidized) Starch ➔ Oxidized if prolonged heating Carlyle D.  3. TOLLEN’S TEST Guide Questions Monosaccharide (+) – Gray-Black Will disaccharides & polysaccharides Precipitate 1. give positive results for the Molisch Disaccharide (+) – Gray-Black Test? Elaborate answer. Precipitate with silver mirror - Deposition of AG (silver) Tollen’s reagent: Which of the following carbohydrate - Ammoniacal solution of silver 2. tests would give a positive result for Aldehyde will give a positive result. maltose? Describe the color change. PROCEDURE Can Seliwanoff’s Test be used to 3. distinguish sucrose from fructose? Explain the answer. Insulin is a polysaccharide composed of fructose units. Which test should 4. be used to best identify the 3% Sucrose → 3% Glucose → 3% Lactose presence of fructose? 1. Add 5 drops of sugar per tube and 2 mL of Tollen’s reagent. 2. Boil for 5 minutes. 3. Observe the color of precipitate. Results Solution 3% Sucrose 3% Glucose 3% Lactose Carlyle D. Biochemistry for Medical Laboratory Science MIDTERM LAB: NUCLEIC ACIDS CMLS A.Y. 24 – 25 1st Semester Ms. Danielle Marie Asprer Allarey-Susa PRE-LAB Isolation of RNA from Yeast NUCLEIC ACIDS 1. Heating with Alkali is essential. Extracts the 1 nucleic acid and 2 water Found in all living cells. soluble proteins. - No living cells do not contain nucleic Inactivates the nucleases. acid. 2. Acid Extraction Plays a vital role in cellular organization and function. Nucleic acid is separated from 1 Essential in: associated protein and 2 other interfering substances by acid extraction at pH 4-5. - Cell division - Reproduction 3. Treatment with Alcohol & Concentrated HCl - Transmission of hereditary functions. Main information carrying molecules of To precipitate the RNA. the cell. 4. Repeated washing with Alcohol and They determine the inherited other organic solvents characteristics of living cells. To remove substances that may interfere Involved in protein synthesis of different with the chemical tests tissues. Components of molecules that functions Hydrolysis of Nucleotides yields to a as: mixture of: - Co-factors. - Sugar - Coenzymes ➔ Ribose Solubility: - Nitrogenous Bases - Mildly soluble → in Cold Water. ➔ Purine - Insoluble → in Alcohol. ➔ Pyrimidine - Instantly mixes → in Weak Alkali - Phosphate (basic) + Acid = Alkali Metal Salts (as the precipitate) OBJECTIVES Precipitated from alkaline solution by the addition of acid. To isolate RNA from yeast. To test properties of the isolated RNA. Carlyle D.  Materials Used 3. Pour the mixture in a beaker and dilute with 0.2% NaOH solution to make 50 mL. Equipment Hot Plate Water Bath 4. Cover the beaker with a watch glass to avoid evaporation. Materials Test Tube Rack 5. Heat the beaker in a water bath with a Big & Small Beaker Test Tubes constant temperature of 90°C for 30 Test Tube Holder minutes. Alcohol Lamp Cheesecloth 6. Filter the solution thrice using Filter Paper cheesecloth and once through a filter Funnel Yeast paper. White Sand 7. Allow the filtrate to cool → RNA EXTRACT 2 Pasteur Pipette (RE). Mortar & Pestle 8. Perform the following tests on the filtrate Substances 0.2% NaOH (sodium (RE). hydroxide) 10% NaOH 1% CuSO4 (copper 2. QUALITATIVE TESTS: TEST FOR sulfate) 20% & 10% H2SO4 NUCLEOPROTEINS (BIURET’S TEST) (sulfuric acid) Acidic RNA Extract Nucleoproteins are any proteins that are NH4OH (ammonia water) Ammonium Molybdate structurally associated with nucleic Bial’s Orcinol Reagent acids either DNA or RNA. 0.1% of: Xylose & Glucose 5% AgNo3 (silver nitrate) PROCEDURE 1. ISOLATION OF RNA FROM YEAST 1. Add 1 mL of the filtrate (RE) in a test tube. A complicated method, needing to break 2. Then add 1 mL of 10% NaOH solution. the cell wall. 3. Add 10 drops of 1% CuSO4 solutions. Method: Enzyme Treatment or Glass 4. Note the color produced. Bead Disruption Heating with an Alkali will produce: Purple indicates there are - Nucleic acids nucleoproteins in a test tube. - Water soluble proteins Blue indicates there are no - Inactivated nuclease nucleoproteins in the test tube (original color of copper sulfate). PROCEDURE 2.1 QUALITATIVE TESTS: MILD ACID 1. Mix and grind 2g of yeast with 2g of HYDROLYSIS white sand together in a mortar and pestle. PROCEDURE 2. Then add 15 mL of freshly prepared 0.2% NaOH to make a smooth creamy 1. Add 20 mL of 10% H2SO4 to the remaining paste. filtrate in a beaker. Carlyle D. 2. Boil the solution gently on a hot plate for 2.1.3 TEST FOR PURINES 5 minutes. 3. You will then have your Acidic RNA PROCEDURE Extract (RE). 1. Add 3 mL of 10% NH4OH (ammonia 2.1.1 TEST FOR INORGANIC PHOSPHATES water) to 2 mL of the filtrate (Acidic RE) in a test tube. PROCEDURE 2. Add 2-3 drops of 5% AgNo3 solution to it. 3. Note the color of the precipitate. 1. Add 1 mL ammonia to 1 mL of the filtrate (Acidic RE). Tabulated Results 2. Acidify it using 1-2 drops of 10% H2SO4 Tests Observation Conclusion then add 2 mL of ammonium molybdate. 3. Note the color of the precipitate. Biuret Test Purple Presence of for Nucleo- solution & Nucleo- proteins precipitate proteins 2.1.2 TEST FOR PENTOSE (RIBOSE) Phosphates Cloudy White (Acid solution & Presence of PROCEDURE Hydro- white Phosphates lyzate) precipitate → Acidic RE 0.1% xylose – Test for blue green Sugar 0.1% glucose – Presence of yellow brown Pentoses Acidic RE – blue green 1. Label 3 test tubes and place the following solutions: Test for Cloudy White Purines solution & Presence of - T.T #1 → 1 mL of 0.1% xylose white Purines - T.T #2 → 1 mL of 0.1% glucose precipitate - T.T #3 → 1 mL of Acidic RNA Extract Conclusion: All tests produced (+) 2. Add 3 mL of Bial’s Orcinol reagent to reaction each test tube. 3. Put test tubes in boiling water for 5 Guide Questions minutes until the color changes. 4. Compare color reactions. 1. What is the purpose of the sand in the experiment? The sand’s purpose in the experiment is to help break down the yeast cells and release its cell contents that are rich in nucleic acid alongside NaOH that increases the permeability of the yeast itself. Carlyle D. 2. Why is it necessary to isolate the nucleic acid from the yeast? The reason as to why it is necessary to isolate nucleic acid from the yeast is so that it can be rid of impurities and have a clear analysis for DNA and RNA samples. Additionally, nucleic acid that has been isolated from the yeast can be used for various laboratory techniques, such as PCR, southern blotting, or even restriction enzyme digestion. Carlyle D.