Introduction of Biochemistry Lab PDF

University of Science ‫جامعة العلوم والتكنولوجيا‬ and Technology ‫كلية الطب والعلوم الصحية‬ Faculty of Medicine and ‫وحدة الكيمياء الحيوية‬ Health Sciences Dr/ Sond...

University of Science ‫جامعة العلوم والتكنولوجيا‬ and Technology ‫كلية الطب والعلوم الصحية‬ Faculty of Medicine and ‫وحدة الكيمياء الحيوية‬ Health Sciences Dr/ Sondos Alsoufi Index No. of weeks Lab safety 1 Reactions of Carbohydrates: 1 a. Reactions of monosaccharides. 1 b. Reactions of disaccharides. 1 c. Reactions of polysaccharides. 1 - Scheme for identification of carbohydrates. 1 Med term exam. 1 Reactions of proteins: 1 Reactions of lipids: 1 Enzymes kinetic. 1 Final practical exam. 1 Total number of weeks. 11 pg. 1  Introduction of biochemistry lab ------------------------------------------------------------------------------------------------- Reactions of Carbohydrates Introduction: Definition: Carbohydrates are aldehyde or ketone derivatives of polyhydric alcohols or any substance derived from them  Classification of Carbohydrates: A. Monosaccharides: Contain one sugar unit. e.g (glucose-fructose) B. Disaccharides: Contain two sugar units.e.g (sucrose-maltose-lactose) C. Oligosaccharides: Contain more than 2 – 10 sugar units. e.g (maltotriose-raffinose) D. Polysaccharides: Contain more than 10 sugar units. e.g (starch- glycogen-cellulose…) 1. Molisch`s test: Used as a general test to detect carbohydrates (Special test of Carbohydrates ). Reactions: 1. C5H10O5 (pentose) + (conc.) H2SO4 → C5H4O2 + 3 H2O 2. C5H4O2 (furfural) + 2 C10H8-OH (α-naphthol) → colored product Concentrated acid dehydrates the sugar to form furfural (in the case of pentoses) or furfural derivatives (hexoses and heptoses which then condense with α-naphthol to give a reddish violet colored complex. pg. 2  Introduction of biochemistry lab ------------------------------------------------------------------------------------------------- Test Observation Result 1. Molisch`s test:  1 ml of solution(any sugar)  2 or 3 drops of alpha A reddish violet ring at naphthol, the junction of two This solution is  mix. layers carbohydrates  2 ml concentrated H2SO4 along the side of the test tube carefully. Not carbohydrates Notes: carbohydrates  Instead of a violet ring in the Molisch test, appearance of dark brown color indicates charring of sugar due to the heat generated during the addition of acid (acid water interaction generates heat)  Appearance of a green color suggest excess use of Molisch reagent than required or due to the presence impurities in the reagent. pg. 3  Introduction of biochemistry lab ------------------------------------------------------------------------------------------------- A/ Reactions of Monosaccharides: Cannot be hydrolyzed into simpler carbohydrates They are divided into aldoses and ketoses based on the functional group present in them. Example of monosaccharides: Glucose (Aldose) Fructose (Ketose) pg. 4  Introduction of biochemistry lab ------------------------------------------------------------------------------------------------- 2.Barfoed's test : Barfoed's Test is a chemical test used for detecting the presence of reducing sugars (monosaccharides). Reducing sugars: Sugars containing free aldehyde or ketone groups can reduce other reagents (e.g. they can reduce copper (II) acetate to copper(I)oxide (Cu2O), which forms a reddish brown precipitate.) (Disaccharides may also react, but the reaction is much slower). Reactions: Reducing sugars (monosaccharides) + 2 Cu+2 + 2H2O→ Oxidized Sugar + Cu2O (red ppt)+ 4H+ Test Observation Result 2. Barfoed's test;  2 ml glucose  2 ml Barfoed's reagent Red precipitate This test is specific for  Mix monosaccharides.  keep in boiling water bath for 4 minutes.  Cool under tap water. (Repeat Test with fructose) Not monosaccharides monosaccharides pg. 5  Introduction of biochemistry lab ------------------------------------------------------------------------------------------------- 3. Seliwanoff's test: Seliwanoff’s Test is utilized for the differentiation of aldoses (glucose) and ketoses (fructose). A ketose is said to be present if the mixture turns red. Ketoses are more rapidly dehydrated than aldoses when heated. The dehydrated ketose then reacts with the resorcinol (Present in test reagent) to produce a deep cherry red color. Aldoses may react slightly to produce a faint pink color Reactions: H2SO4 Ketose sugar (Frucose) Cherry red color product. -3H2O Test Observation Result 3. Seliwanoff's test:  3 ml Seliwanoff's Cherry red This test is specific for fructose (the reagent color ketose sugars).  1 ml fructose.  Boil for 4 minutes in water bath Note: If boiled for longer duration, even glucose gives light red color. Repeat with glucose No red color glucose Glucose Fructose pg. 6  Introduction of biochemistry lab ------------------------------------------------------------------------------------------------- B/ Reactions of Disaccharides. Disaccharides are the carbohydrates which on hydrolysis form two monosaccharide units. Maltose, lactose and sucrose are the common disaccharides. The former two are reducing disaccharides, sucrose is non-reducing.  Example of disaccharides :  Maltose, Lactose (reducing sugars ).  Sucrose (Non-reducing sugars). 1. Benedict's test: Benedict's reagent contains blue copper (II) sulphate (CuSO4) 5H2O which is reduced to red copper(I) oxide by aldehydes, thus oxidizing the aldehydes to carboxylic acids (Oxidized sugar).The copper oxide is insoluble in water and so precipitates. The color of the final solution ranges from green to brick red depending on the concentration of sugar.. Reactions: Cupric (Blue) + Sugar → Cuprous (red) + Oxidized sugar. Test Observation Result 1. Benedict's test: Perform with maltose & Red color Maltose and lactose are lactose. reducing sugars.  1 ml sugar solution + 3 ml Benedict's reagent.  Mix.  Boil for 4 min.  Sucrose is a non- (Repeat with sucrose). No red color reducing sugar. pg. 7 Introduction of biochemistry lab -------------------------------------------------------------------------------------------------  The color of the precipitate give an idea about the concentration of the sugar solution as shown below:  Blue – absence of reducing sugar  Green – up to 0.5 gm%  Yellow – > 0.5 to 1.0 gm%  Orange – > 1.0 to 2.0 gm%  Brick red – ≥ 2 gm% Thus, Benedict’s test is described as a semi-quantitative test. sucrose Maltose or lactose pg. 8  Introduction of biochemistry lab ------------------------------------------------------------------------------------------------- C/ Reactions of Polysaccharides: The most commonly available polysaccharide is Starch, Starch is formed of hundreds of glucose chains. 1. Iodine Test: Test Observation Result 1.Iodine Test  add 1 ml of starch solution Deep blue color Starch gives a blue color with  2-3 drops of diluted Iodine iodine solution solution  mix  Deep blue color appears which then disappears on heating and then reappears on cooling.  Starch forms a adsorption complex with iodine to give a blue color. The blue color disappears on heating due to the breaking of the Iodine starch adsorption complex and appears on cooling due to reformation of the adsorption complex pg. 9 Introduction of biochemistry lab ------------------------------------------------------------------------------------------------- Scheme for Identification of Carbohydrate Molisch's Test No Violet Ring Violet Ring.......................................... Iodine test Blue Color No blue color Starch Barfoed' Test No Red ppt Red ppt Benedict's test Seliwanoff's test Cherry red color No Reddish or orange color Fructose Sucrose No change Glucose Reddish or orange color Lactose or Maltose pg. 10 Introduction of biochemistry lab ------------------------------------------------------------------------------------------------- Reactions of Proteins Introduction: Proteins are polymers of amino Acids. Amino acids are organic compounds, which contain both an amino group (NH2) and a carboxyl group (COOH). The term protein used to describe molecules, which contain 50 amino acids or more united by peptide bonds. 1. Biuret test: Principle: When substances contain two or more peptide bonds (e.g. Proteins), they react with the biuret reagent (copper sulfate in alkaline condition), a violet color complex is formed. The colored product is the result of coordination of Cu2+ with nitrogen atoms peptide (Cu2+ reacts with the peptide bond). pg. 11  Introduction of biochemistry lab ------------------------------------------------------------------------------------------------- Test Observation Result 1.Biuret test:  This test detects the presence of peptide  1 ml protein Violet color bonds. Thus, this test is specific for solution proteins. The minimum requirement for a positive  1ml of Biuret biuret test is the presence of at least 2 solution. peptide bonds.  This test detects the presence of free amino  Mix group in the protein. pg. 12  Introduction of biochemistry lab ------------------------------------------------------------------------------------------------- 2. Coagulation test:- Test Observation Result 2.Coagulation test:-  1ml protein Coagulation Albumin  heat by water flame. 2. Silver nitrate test:- Test Observation Result 3.Silver nitrate test:- White ppt Albumin  1 ml protein  5 drops of Silver nitrate Brown ppt Casein (AgNO3). Peptone No change pg. 13  Introduction of biochemistry lab ------------------------------------------------------------------------------------------------- General scheme for identification of carbohydrates and proteins Molisch's Test No Violet Violet Ring Ring Iodine test Biuret Test Blue Color No blue color Violet Color Starch Barfoed's Test Silver Nitrate Test No Red ppt Red ppt Brown ppt. No ppt. Benedict's Seliwanoff's Casien Peptone test test No Reddish Cherry red White ppt. color color Albumin Sucrose Fructose Reddish No change color Glucose Lactose or Maltose pg. 14 Introduction of biochemistry lab ------------------------------------------------------------------------------------------------- Reactions of Lipids Introduction: Lipids: are group of compound related to fatty acids, they are insoluble in water but soluble in Organic Solvents (such as ether, chloroform…). Fatty Acids: are mono carboxylic organic acids. CH3-CH2-CH2-CH2-CH2-CH2-CH2=CH2-CH2-CH2-CH2-CH2-CH2-CH2- CH2-COOH Palmitoliec acid 1- Solubility test Principle: Lipids are water insoluble and soluble in organic solvents. We perform this test with water, by heat, with Chloroform, with Ether, and with Alcohol. pg. 15  Introduction of biochemistry lab ------------------------------------------------------------------------------------------------- Test Observation Result Solubility test Fat is insoluble in 1-With water water and is lighter Fat-and-water-remains  Take a small amount of fat (e.g. separate. in weight. butter) in a test tube  enough distilled water to cover the same. 2- Heat Fats melts, but Forms the distinct upper layer while water-forms-the lower portion. 3- With Chloroform Fat is soluble in  Take a small amount of fat in a Fat-completely chloroform (organic test tube dissolves-in chloroform. solvent).  Add some chloroform until cover the fat completely.  Shake. 4- With Ether  Take a small amount of fat in a Fat-completely Fat is in ether test tube dissolves in ether. (organic solvent  Add some ether until cover the soluble). fat completely.  Shake. 5-With Alcohol  Take a small amount of fat in a Fat-dissolves-in Fat is soluble in test tube Alcohol. alcohol (organic  Add some alcohol (absolute) solvent). until cover the fat completely.  Shake. pg. 16  Introduction of biochemistry lab ------------------------------------------------------------------------------------------------- 2- Glycerol test: Test Observation Result Glycerol test  2 drops oil  1ml sulphuric acid conc. Black color Glycerol is present.  Then heat by water bath for 3min. 3- Copper acetate test: Test Observation Result Copper acetate test  2 drops oil Blue color in Saturated fatty acid  2ml ether then lower layer shake.  Add 2ml copper Green color in Unsaturated fatty acid acetate solution. upper layer GOOD LUCK pg. 17