Biochemistry lab manual

1. Prepared by: Abere Habtamu November, 2017 Debre Markos University Natural and computational science college Department of chemistry Biochemistry laboratory manual Prepare By: Abere Habtamu Chemistry Department, E-mail: abere.habtamu2010@gmail.com 1

2. Prepared by: Abere Habtamu November, 2017 Experiment 1 Definition of a Laboratory A laboratory is a place, building or part of a building used for scientific and related work that may be hazardous. The work conducted in a laboratory may include teaching or learning, research, clinical or diagnostic testing and analysis. A laboratory may have associated areas including preparation, instrumentation, decontamination, wash-up and storage rooms, or a workshop in an engineering area (eg mechanical, electrical, aeronautical and civil engineering). Laboratories are commonly used for scientific disciplines ranging from biology, chemistry, physics, botany and zoology to medicine, psychology, dentistry, engineering, agriculture and veterinary science. Access to Laboratories Laboratories are considered to be high risk environments when compared to other areas in the University (e.g. offices, tutorial rooms, lecture theatres etc). As a result, entry to any laboratory is to be restricted to individuals who are authorised by the laboratory supervisor or laboratory manager, to enter. The Supervisor shall ensure that any person given authority to enter receives appropriate: (a) information regarding hazards and related risks that are present; (b) safety measures to be adopted (eg local rules, SWPs, suitable protective clothing and equipment..Etc),.and (c) supervision. Laboratory Hazards Defined simply, a hazard is anything that may cause injury, harm or damage. The hazards encountered in a laboratory are many and varied and may result in short term or long term health effects if individuals are exposed to these hazards. When planning any work in a laboratory the risk of exposure to laboratory hazards is an important consideration during the risk assessment 2

3. Prepared by: Abere Habtamu November, 2017 and risk management process. Laboratory hazards fall generally into one of five categories: ?Biological - eg pathogenic microorganisms, animals, biological tissues, blood and other body fluids (human and animal) ?Chemical - eg corrosive, flammable, toxic ?Physical - eg noise, radiation, manual handling ?Electrical/Mechanical - eg high voltage apparatus, machinery with moving parts ?Psychological - eg emotional stress, workplace bullying Laboratory Safety Laboratory safety rules and safe work practices or procedures (SWPs) should be established by Faculties, Schools and Disciplines to cover specific operational needs and to reduce the risks associated with laboratory hazards. As a condition of entry to a laboratory, all individuals must complete a laboratory safety induction and receive specific training in local safety rules and laboratory procedures relating to their work (including relevant SWPs). Individuals are required to comply with laboratory safety rules and procedures at all times whilst in the laboratory. Individuals who act contrary to the rules and procedures should be excluded from the laboratory. So, the following safety rules must be followed without exceptions: ?No authorized experiments are to be performed ?Do not touch any equipment, chemicals and/or other materials ?Do not drink or eat food and gum in lab ?Always work in a well ventilated area ?Read carefully instructions of equipments before use them ?Keep hands from eye, face, mouth and body while using chemicals and lab equipments ?Wash your hands with soap and water after performing all experiments 3

4. Prepared by: Abere Habtamu November, 2017 ?Long hair must be tied back and clothing must be secured and shoes must completely cover the foot. ?Contact lenses are not allowed ?Lab coat, gloves and safety goggles should be worn during lab experiments ?If chemicals splash in your eyes or on your skin by accident, wash with water immediately ?All chemicals in the lab are dangerous hence, don’ t taste or smell ?Always pour acid to water ?Do not use highly flammable reagents near open flame ?Never remove chemicals or lab equipments from lab area ?Examine glassware before each use and never use cracked and dirty glassware ?Don’ t enter hot glassware in cold water ?Notify the instructor immediately in case of accident! How to write laboratory reports Overview This document describes a general format for lab reports that you can adapt as needed. Lab reports are the most frequent kind of document written in engineering and can count for as much as 25% of a course yet little time or attention is devoted to how to write them well. Worse yet, each professor want something a little different. Regardless of variations, however, the goal of lab reports remains the same: document your findings and communicate their significance. With that in mind, we can describe the report's format and basic components. Knowing the pieces and purpose, you can adapt to the particular needs of a course or professor. A good lab report does more than present data; it demonstrates the writer's comprehension of the concepts behind the data. Merely recording the expected and observed results is not sufficient; you should also identify how and why differences occurred, explain how they affected your experiment, and shows your understanding of the principles the experiment was designed to 4

5. Prepared by: Abere Habtamu November, 2017 examine. Bear in mind that a format, however helpful, cannot replace clear thinking and organized writing. You still need to organize your ideas carefully and express them coherently. Typical Components •Title Page •Conclusion •Abstract •References •Introduction •Appendices •Methods and Materials (or Equipment) •Experimental Procedure •Results •Discussion 1. The Title Page Needs to contain the name of the experiment, the names of lab partners, and the date that you did the laboratory as well as the number of experiment. Titles should be straightforward, informative, and less than ten words. 2. The Abstract Summarizes four essential aspects of the report: the purpose of the experiment (sometimes expressed as the purpose of the report), key findings, significance and major conclusions. The abstract often also includes a brief reference to theory or methodology. The information should clearly enable readers to decide whether they need to read your whole report. The abstract should be one paragraph of 100-200 words. Quick Abstract Reference Must have: 1.Purpose 2.Key result(s) 5

6. Prepared by: Abere Habtamu November, 2017 3.Most significant point of discussion 4.Major conclusion May Include: 1.Brief method 2.Brief theory Restrictions: ONE page 200 words MAX. 3. The introduction is more narrowly focused than the abstract. It states the objective of the experiment and provides the reader with background to the experiment. State the topic of your report clearly and concisely, in one or two sentences: Quick Introduction Reference Must Have: 1.Purpose of the experiment 2.Important background and/or theory May include: 1.Description of specialized equipment 2.Justification of experiment's importance Example: The purpose of this experiment was to identify the specific element in a metal powder sample by determining its crystal structure and atomic radius. These were determined using the Debye-Sherrer (powder camera) method of X-ray diffraction. 6

7. Prepared by: Abere Habtamu November, 2017 A good introduction also provides whatever background theory, previous research, or formulas the reader needs to know. Usually, an instructor does not want you to repeat the lab manual, but to show your own comprehension of the problem. For example, the introduction that followed the example above might describe the Debye-Sherrer method, and explain that from the diffraction angles the crystal structure can be found by applying Bragg's law. If the amount of introductory material seems to be a lot, consider adding subheadings such as: Theoretical Principles or Background. Note on Verb Tense Introductions often create difficulties for students who struggle with keeping verb tenses straight. These two points should help you navigate the introduction: •The experiment is already finished. Use the past tense when talking about the experiment. "The objective of the experiment was..." •The report, the theory and permanent equipment still exist; therefore, these get the present tense: "The purpose of this report is..." "Bragg's Law for diffraction is ..." "The scanning electron microscope produces micrographs ..." 4. Methods and Materials (or Equipment) Can usually be a simple list, but make sure it is accurate and complete. In some cases, you can simply direct the reader to a lab manual or standard procedure. 7

8. Prepared by: Abere Habtamu November, 2017 5. Experimental Procedure Describes the process in chronological order. Using clear paragraph structure, explain all steps in the order they actually happened, not as they were supposed to happen. If your professor says you can simply state that you followed the procedure in the manual, be sure you still document occasions when you did not follow that exactly (e.g. "At step 4 we performed four repetitions instead of three, and ignored the data from the second repetition"). If you've done it right, another researcher should be able to duplicate your experiment. 6. Results Are usually dominated by calculations, tables and figures; however, you still need to state all significant results explicitly in verbal form, for example: Quick Results Reference 1.Number and Title tables and graphs 2.Use a sentence or two to draw attention to key points in tables or graphs 3.Provide sample calculation only 4.State key result in sentence form Using the calculated lattice parameter gives, then, R = 0.1244nm. Graphics need to be clear, easily read, and well labeled (e.g. Figure 1: Input Frequency and Capacitor Value). An important strategy for making your results effective is to draw the reader's attention to them with a sentence or two, so the reader has a focus when reading the graph. In most cases, providing a sample calculation is sufficient in the report. Leave the remainder in an appendix. Likewise, your raw data can be placed in an appendix. Refer to appendices as necessary, pointing out trends and identifying special features. 8

9. Prepared by: Abere Habtamu November, 2017 7. Discussion Is the most important part of your report, because here, you show that you understand the experiment beyond the simple level of completing it. Explain. Analyse. Interpret. Some people like to think of this as the "subjective" part of the report. By that, they mean this is what is not readily observable. This part of the lab focuses on a question of understanding "What is the significance or meaning of the results?" To answer this question, use both aspects of discussion: Analysis Interpretation What do the results indicate clearly? What is the significance of the results? What What have you found? ambiguities exist? What questions might we Explain what you know with certainty based raise? Find logical explanations for problems in on your results and draw conclusions: the data: Since none of the samples reacted to the Although the water samples were received on 14 Silver foil test, therefore sulfide, if present at August 2000, testing could not be started until 10 all, does not exceed a concentration of September 2000. It is normally desirably to test as approximately 0.025 g/l. It is therefore quickly as possible after sampling in order to unlikely that the water main pipe break was avoid potential sample contamination. The effect the result of sulfide-induced corrosion. of the delay is unknown. More particularly, focus your discussion with strategies like these: Compare expected results with those obtained. If there were differences, how can you account for them? Saying "human error" implies you're incompetent. Be specific; for example, the instruments could not measure precisely, the sample was not pure or was contaminated, or calculated values did not take account of friction. Analyze experimental error. Was it avoidable? Was it a result of equipment? If an experiment was within the tolerances, you can still account for the difference from the ideal. If the flaws result from the experimental design explain how the design might be improved. 9

10. Prepared by: Abere Habtamu November, 2017 Explain your results in terms of theoretical issues. Often undergraduate labs are intended to illustrate important physical laws, such as Kirchhoff's voltage law, or the Müller-Lyer illusion. Usually you will have discussed these in the introduction. In this section move from the results to the theory. How well has the theory been illustrated? Relate results to your experimental objective(s). If you set out to identify an unknown metal by finding its lattice parameter and its atomic structure, you'd better know the metal and its attributes. Compare your results to similar investigations. In some cases, it is legitimate to compare outcomes with classmates, not to change your answer, but to look for any anomalies between the groups and discuss those. Analyze the strengths and limitations of your experimental design. This is particularly useful if you designed the thing you're testing (e.g. a circuit). 8. Conclusion Can be very short in most undergraduate laboratories. Simply state what you know now for sure, as a result of the lab: Quick Conclusion Reference Must do: 1.State what's known Justify statement Might do: 10

11. Prepared by: Abere Habtamu November, 2017 1.State significance 2.Suggest further research Example: The Debye-Sherrer method identified the sample material as nickel due to the measured crystal structure and atomic radius (approximately 0.124nm). Notice that, after the material is identified in the example above, the writer provides a justification. We know it is nickel because of its structure and size. This makes a sound and sufficient conclusion. Generally, this is enough; however, the conclusion might also be a place to discuss weaknesses of experimental design, what future work needs to be done to extend your conclusions or what the implications of your conclusion are. 9. References Include your lab manual and any outside reading you have done. Check the site's documentation page to help you organize references in a way appropriate to your field. 10. Appendices Typically include such elements as raw data, calculations, graphs pictures or tables that have not been included in the report itself. Each kind of item should be contained in a separate appendix. Make sure you refer to each appendix at least once in your report. For example, the results section might begin by noting: "Micrographs printed from the Scanning Electron Microscope are contained in Appendix A." 11

12. Prepared by: Abere Habtamu November, 2017 Experiment 2. Preparation of Solutions from solid NaCl Purpose: This experiment provides practical experience in preparing solutions using the concentration units of molarity and molality. Introduction A solution is a homogeneous mixture of two or more chemical substances. If we have a solution made from a solid and a liquid, we say that the solid is dissolved in the liquid and we call the solid the solute and the liquid the solvent. Initially, we will consider only solutions of a solid in water. We normally think of a solute as a solid that is added to a solvent (e.g., adding table salt to water), but the solute could just as easily exist in another phase. For example, if we add a small amount of ethanol to water, then the ethanol is the solute and the water is the solvent. If we add a smaller amount of water to a larger amount of ethanol, then the water could be the solute. If a solution has a small amount of solute in a large amount of solvent, we say that the solution is dilute (or that we have a dilute solution). If a solution has a large amount of solute for a certain amount of solvent, we say that the solution is concentrated (or that we have a concentrated solution). We see that the terms dilute and concentrated are not precise and are merely used to give a rough indication of the amount of solute for a given amount of solvent. The amount of solute in a given amount of solvent (or solution) is called the concentration of the solution. The concentration of a chemical solution refers to the amount of solute that is dissolved in a solvent. The concentration of a solution can be expressed in many ways such as molarity, molality, percentage composition, normality, mole fraction etc. Percent Composition by Mass (%) This is the mass of the solute divided by the mass of the solution (mass of solute plus mass of solvent), multiplied by 100. Example: Determine the percent composition by mass of a 100 g salt solution which contains 20 g salt. Solution: 20 g NaCl / 100 g solution x 100 = 20% NaCl solution 12

13. Prepared by: Abere Habtamu November, 2017 Molarity (M) Molarity is probably the most commonly used unit of concentration. It is the number of moles of solute per liter of solution (not necessarily the same as the volume of solvent!). Example: What is the molarity of a solution made when water is added to 11 g CaCl2 to make 100 mL of solution? Solution: 11 g CaCl2 / (110 g CaCl2 / mol CaCl2) = 0.10 mol CaCl2 100 mL x 1 L / 1000 mL = 0.10 L molarity = 0.10 mol / 0.10 L molarity = 1.0 M Normality (N) Normality is equal to the gram equivalent weight of a solute per liter of solution. A gram equivalent weight or equivalent is a measure of the reactive capacity of a given molecule. Normality is the only concentration unit that is reaction dependent. Example: 1 M sulfuric acid (H2SO4) is 2 N for acid-base reactions because each mole of sulfuric acid provides 2 moles of H+ ions. On the other hand, 1 M sulfuric acid is 1 N for sulfate precipitation, since 1 mole of sulfuric acid provides 1 mole of sulfate ions. Part A: Preparation/Testing of 0.1M NaCl Apparatus Electronic balances, volumetric flasks, graduated cylinder and Erlenmeyer flasks will be used for preparing the solutions. Chemicals: NaCl and water 13

14. Prepared by: Abere Habtamu November, 2017 Procedure Calculate the amount of solid solute needed for a solution of given volume and concentration. Prepare the solution and test to see if your concentration is correct. For help with the calculations, refer to the Data and Results sheet. 1. Find the number of moles needed to make 50 mL (0.050 L) NaCl of 0.10 M solution using a 50-mL volumetric flask. Record, 2. From the molar mass of sodium chloride, NaCl and the number of moles from Step 1, find the mass in grams of solute needed. Record, 3. Weigh the mass of solute calculated from Step 2 and carefully transfers solid NaCl to a clean 50-mL volumetric flask. 4. Add about half the volume of distilled water needed and swirl the flask. When most of the solid has dissolved add the rest of the water stopping below the mark on the flask. To add the remaining water use the water wash bottle. Insert the stopper and invert the flask a few times for uniform mixing. Exercise. Outline the preparation of a. 0.2 M of 125ml of MgSO4.9H2O solution b. 250 ml of 0.45 M HCl solution 14

15. Prepared by: Abere Habtamu November, 2017 Experiment 3 Preparation of solution from liquid sample Purpose To become familiar with the techniques of preparing a solution. To gain an understanding of the concepts of molarity, solution, solute and solvent. Equipments Volumetric flask-250 ml and 100ml, HCl solution 37 % by volume, wash bottle, pipette, burette (50 ml), burette clamp, and ring stand, Erlenmeyer (250 ml), wash bottle. Chemical: HCl, water Theory There are two components of a solution. These are Solute & Solvent. Solute is the matter that dissolved. Solvent is the matter that dissolves the solute. Molarity is the number of moles of solute that dissolved in 1 liter solution. The molar concentration of a solution can be found in different ways. Dilution is the process of adding solvent to a solution. Since this makes the volume of the solution larger but the number of moles of solute remains the same, the concentration of the solution decreases and the solution is said to have been diluted. Consider a solution of molarity MO (molarity of the original solution) and volume VO litres. The number of moles of solute in this solution is given by nO = MOVO. Suppose more solvent is added to the solution and the new volume is VD (volume of the diluted solution). The molarity of the solution will decrease to a value MD. Since no solute has been added, the number of moles of solute remains nO. Therefore, for the diluted solution nO = MDVD = MOVO. In the equation n = MV, V must be in litres. However, in MDVD = MOVO, VO and VD can be in any units provided they are in the same units. Example: How many millilieters of 5.5 M NaOH are needed to prepare 300 mL of 1.2 M NaOH? 15

16. Prepared by: Abere Habtamu November, 2017 Solution: 5.5 M x Vo = 1.2 M x 0.3 L V0 = 1.2 M x 0.3 L / 5.5 M V0 = 0.065 L V0 = 65 mL So, to prepare the 1.2 M NaOH solution, you pour 65 mL of 5.5 M NaOH into your container and add water to get 300 mL final volume. For dilute solutions, it can be assumed that the volume of a diluted solution is equal to the sum of the volume of the original solution and the volume of solvent added (VD = VO + VS). Procedure Part A: Preparing 100ml of 0.1M HCl solution from 37 % stock HCl solution. Calculate the amount of HCl mass needed to prepare 100ml 0.1M. (Molar mass of HCl is 36.5 g/mol) Calculate the volume needed of HCl from the 37 % HCl solution by volume and using pipette pour the calculated volume to 100 ml volumetric flask. (Density of HCl is 1.19 g/ml). Pour distilled water into the flask until the level is exactly 100ml Table1: Preparing 100ml of 0.1M HCl solution. Results MHCl [mol/l]_____________ VHCl [ml] _______________ nHCl [mol] _____________ MA,HCl [g/mol] _____________ mHCl [g] _______________ dHCl [g/ml] _______________ VHCl,pure [ml] _______________ 16

17. Prepared by: Abere Habtamu November, 2017 Experimental 4 Standardization of HCl solution with Primary standard Substance (Borax) and Determination of normality of sodium hydroxide solution by a standard solution of hydrochloric acid. Purpose: To determine the concentration of HCl using a primary standard, borax. To determine the amount of NaOH using a secondary standard solution, HCl. Introduction: Titration Titration is a standard laboratory method of quantitative/chemical analysis which can be used to determine the concentration of a known reactant. Because volume measurements play a key role in titration, it is also known as volumetric analysis. A reagent, called the titrant, of known concentration (a standard solution) and volume is used to react with a measured quantity of reactant (analyte). Using a calibrated burette to add the titrant, it is possible to determine the exact amount that has been consumed when the endpoint is reached. The endpoint is the point at which the titration is stopped. This is classically a point at which the number of moles of titrant is equal to the number of moles of analyte, or some multiple thereof (as in di- or tri- protic acids). In the classic strong acid-strong base titration the endpoint of a titration is when the pH of the reactant is just about equal to 7, and often when the solution permanently changes color due to an indicator. There are however many different types of titrations. Many methods can be used to indicate the endpoint of a reaction; titrations often use visual indicators (the reactant mixture changes colour). In simple acid-base titrations a pH indicator may be used, such as phenolphthalein, which turns (and stays) pink when a certain pH is reached or exceeded. Methyl orange can also be used, which is red in acids and yellow in alkalis. In titrimetric method reagents can be classified as primary and secondary substances. Primary Standard A primary standard is a substance which satisfies the following requirements: 1. It must be easy to obtain, to purify, to dry (preferably at 110-120oC) and to preserve in pure state. 17

18. Prepared by: Abere Habtamu November, 2017 2. The substance should remain unaltered during weighing i.e., it should not be hygroscopic, or oxidized by the air, or affected by carbon dioxide. 3. The substance should be capable of being tested for impurities by qualitative and other tests of known sensitivity. 4. It should have a high equivalent so that the weighing errors may be negligible. 5. The substance should be readily soluble under the experimental conditions. 6. The reaction with the standard solution should be stoichiometric and practically instantaneous. In practice, it is difficult to obtain a primary standard, and a compromise between the above ideal requirements is necessary. The commonly employed primary standards include sodium carbonate, sodium tetraborate, potassium hydrogen phthalate, constant-boiling-point hydrochloric acid, potassium hydrogen iodate, Borax, and benzoic acid. Secondary Standard A substance, which fulfills the requirement that it can be weighed accurately to provide a known amount of reactant but which is not a pure substance, is called secondary standard. It may be used for standardizations, and whose content of the active substance has been found by the comparison against a primary standard. Hydrochloric acid reacts with sodium hydroxide according to the equation: NaOH + HCl ͢͢ NaCl + H2O The equivalent weight of both HCl and NaOH is equal to their molecular weights and as both the acid and alkali are strong. Any indicator may be used. Chemicals required: Hydrochloric acid solution, Sodium hydroxide solution of unknown normality, borax and phenolphathalien. Apparatus Electronics balance, Small beaker or conical flask, burette and pipette, stand and burette clamp Part A: Standardization of Hydrochloric Acid by Disodium Tetraborate (Borax) Procedures Weigh about 4.8 g of pure borax (disodium tetraborate) accurately. Dissolve it in distilled water and make up to 250 ml in a volumetric flask. Pipette 25 ml of this solution into a conical flask, 18

19. Prepared by: Abere Habtamu November, 2017 add a few drops of methyl orange solution and titrate against the hydrochloric acid provided. Take unknown solution of HCl to the burette and titrate against borax solution. Determine the molarity of hydrochloric acid solution. (Molecular mass of borax is 381 g molL-1) Colour change: yellow to reddish orange Na2B4O7⋅ 10 H2O (aq) + 2 HCl(aq) → 2 NaCl(aq) + 4 H3BO3(aq) + 5H2O(l) Part B Titration of NaOH with HCl Procedure: 1. Transfer known volume of the sodium hydroxide solution, with a pipette, to a conical flask then add one or two drops of phenolphthaline. The solution has the pink color. 2. Add the acid from the burette gradually with continuous swirling of the solution in the conical flask, and near the end point, the acid is added drop by drop. Continue the addition of the acid until the color of the solution discharged. 3. Repeat the experiment three times 19

20. Prepared by: Abere Habtamu November, 2017 Experiment 5 Protein Objectives: To examine the chemical properties of proteins Theory Proteins are the most abundant organic molecules of living cells and serve as structural units of muscle, skin, hair and other tissues. Proteins are polymers with molecular weights ranging from 5000 to 1 million or more. On acid hydrolysis proteins yield a series of organic compounds of low molecular weights known as amino acids. Some 20 amino acids are usually found as the building blocks of proteins. In proteins molecules, the amino acid units are covalently bonded by amide linkages known as peptide bonds (NHCO-) to form long chains or polypeptides. Proteins are important nutrients which are obtained from several types of food. The digestion of proteins in stomach gives rise to amino acids and these in turn provide the building blocks for the synthesis of proteins that we need to build and maintain our body. Man is not capable of synthesizing certain amino acids and must therefore obtain these, so called essential amino acids, in one form or another in the diet. The presence of the amino and carboxyl functional groups in proteins makes these substances amphoteric (i. e act as acids or base). Proteins generally coagulate and harden when heated or treated with acids. The protein is said to be denatured and the hardening is due to changes in the stereochemistry of the constituent protein molecules. Treatment of protein with conc. Nitric acid results in the development of yellow colour. This test which is known as the xanthoprotic test is based on the nitration of benzene ring of certain amino acids such as tyrosine, phenylalanine, and tryptophan. Proteins containing free amino groups react with nitrous acid and liberate nitrogen gas. Casein: The principal protein in milk is casein and it is the chief ingredient of cheese. Cow’ s milk contains about 3% casein. Casein has an approximate molecular weight of 375,000 and is made up of at least 16 amino acids. 20

21. Prepared by: Abere Habtamu November, 2017 Procedure A.Isolation and reaction of casein Place 50 ml of non fat milk in a 125 ml Erlenmeyer flask, place the flask in water bath and warm it to 40-45oC. Add 25 drops of glacial acetic acid to the warm milk. Filter the casein precipitate by suction. Wash the casein first with 5 ml water and then with 5 ml alcohol. Transfer the product on to a piece of paper and press it with another piece and spread it out. Transfer the casein to another dry piece of paper and let it stand for at least half hour and then record the weight of the dried casein. Test on casein a.Solubility Test: - pour 5 ml of concentrated hydrochloric acid in a test tube and add to it 0.1 g of casein, shake and warm. Describe the test and significance. Repeat the solubility test using 1 ml of 2% NaOH b.Xanthoprotic Test: Add 10 drops of nitric acid and 0.1 g of casein into a small test tube. Observe and note the changes that take place. Explain the significance of this test with respect to amino acid composition of casein. c.Nitrous Acid-Casein Reaction: suspend 0.1 g of casein in 2 ml of 10 % HCl solution. Cool the mixture by placing the test tube in an ice bath and then add 1 ml of 5% sodium nitrite solution. Keep the test tube in the ice bath and note if gas bubbles appear. What are these bubbles? Explain by means of a chemical equation how these bubbles arise? B.Properties of Egg Albumin Obtain 10 ml of white of an egg and divide it equally into four portions and conduct the following test:- a.Xanthoprotic Test: Pour the first portions of the albumin into a test tube containing 5 ml of water. Shake well and add 10-15 drops of concentrated nitric acid. Warm gently and note the colour change. Cool the solution and neutralize it with 5 ml of 10 % sodium hydroxide. Record your observations in your note book. b.Test for sulfur Put 1 ml of albumin in a test and add to it 7 ml of 10 % sodium hydroxide solution. Place the test tube for 15 minutes in a water bath heated to boiling. Cool the solution 21

22. Prepared by: Abere Habtamu November, 2017 and acidify it by adding the provided hydrochloric acid solution drop wise till it turns blue litmus paper to red. Place the test tube with the acidified solution in the boiling water bath, but this time place a piece of moist lead acetate paper over the mouth of the test tube. Observe the change in the colour of the lead acetate paper. Note and explain your observations. c.The Biuret Test: To a 2 ml portion of the albumin add 4-5 ml of 10 5 sodium Hydroxide solution followed by 4-5 drops of 2 % aqueous copper sulphate solution. Shake the mixture well and observe the colour. d.Precipitation with salts of Heavy Metals: prepare a dilute solution of the albumin by shaking 2 ml of the albumin with 15 ml of water. Divide this into three portions. Add slowly 2 ml of 10 % solution of copper sulfate, ferric chloride and lead acetate solutions to the three test tubes respectively. Record your results. Exercise 1.Show by chemical equations formation of a dipeptide from glycine gly-gly and the tripeptide cys-ala-gly? Get the structure of the amino acids from your textbook. 2.Why does the casein precipitate out after addition of glacial acetic acid? 3.What is the basis for the Xanthoprotic and the Biuret tests? 22

23. Prepared by: Abere Habtamu November, 2017 Experiment 6 Carbohydrates Objectives: To examine the chemical properties of carbohydrates Theory It is interesting to note that there is probably more carbohydrate in the biosphere than all other organic matter combined. This is mainly because of the extraordinary abundance of two polymers of D-glucose namely cellulose and starch. Monosaccharides, or simple sugars, are made up of a single polyhydroxy aldehyde or ketone unit. The most abundant monosaccharide is the simple six carbon sugar D- glucose. Oligosaccharides contain from two to ten monosaccharide units joined in glycoside linkage. Oligosaccharides as their name indicates are long chains of monosaccharides. Examples of monosaccharide’ s include the simplest member glyceraldehydes, followed by erythro ribose, glucose, mannose, fructose etc. the sugars that have three carbon in the chain are called triose and as the carbon chains are extended by the addition of carbon atoms, we have successively tetrose, pentoses, hexose, heptoses and octose. Furthermore, if the carbonyl group is at the end of thecahin the monosaccarides is called an aldose, where as if it is at any other positions the sugar is a ketose. Furthermore pentose and hexoses manily exist in cyclic forms. Oligosaccharides may be illustrated by common disaccharides like lactose (found in milk), sucrose, (found in cane sugar), cellobiose (in cellulose) and maltose(product of enzyme amylase on starch) Test for carbohydrates 1.Solubility Tests: By placing 0.1 g each of the following carbohydrates in test tubes, determine their solubility in water and ethanol: Glucose, Sucrose and starch. Tabulate your results. Prepare 25 ml each of 5% aqueous solution of glucose and sucrose and conduct the following tests:- 2.The Molisch Test: Keep 2 ml of conc. Sulfuric acid in a test tube. In another test tube. Measure 2 ml of the above glucose test solution and add to it 2 drops of the Molisch reagent (15% solution of α -naphthol in ethanol). Pour the sulfuric acid down the inside of glucose solution by holding the test tube holding the latter at an angle of about 30 while 23

24. Prepared by: Abere Habtamu November, 2017 pouring so that two separate layer are formed. Do not shake the test tube. The purple colored ring formed at the junction of the two liquids is typical of carbohydrates. 3.Fehling’s Test for reducing Sugars: place 4 ml of Fehling’ s solution in a test tube and heat it to gentle boiling. Add the glucose solution, two to three drops at a time followed by one minute of heating. Observe any colour change and continue adding the solution till the blue color disappears. The total volume of glucose solution added should not exceed 5 ml. Repeat the test with sucrose solution and record your results. Explain your results in the discussion section of your report. 4.Osazone Formation: Monosaccharide’ s and reducing disaccharides react with phenylhydrazine to yield osazones. (Write the equation). Osazones are crystalline derivatives of sugars which are extremely useful in the identification and characterization of sugars. The following simple procedure helps on prepare osazone derivatives of simple sugars. Bring a water bath to boiling. To each of two test tubes add 2 ml of phenyl hydrazine reagent and 5 ml of the 5% glucose solution to test tube 1 and 5 % sucrose solution to test tube 2. Mix the solutions well by shaking. Place the test tubes in water bath and note how long it took for each sugar to form a precipitate. Note the colour and crystal shape of the respective osazones. Exercise 1.The properties of glucose are better accounted for the cyclic hemiacetal and it has been shown that 90 % of glucose exists in this form. Draw the hemiacetal cyclic structure formed between the aldehyde group at carbon 1 and the hydroxyl group at carbon 5? 2.Write the structures of glucose and lactose, and show how one might distinguish between the two in the laboratory? 3.What is a Fehling’ s solution? Why is the Fehling’ s test very useful in clinical chemistry? 4.Write the equation for the reaction between phenylhydrazine and glucose? What is mechanism of the reaction? 24

25. Prepared by: Abere Habtamu November, 2017 Experiment 7 Preparation of Soap Objective: to prepare ordinary soap and to examine its properties Theory The art of making soap has known since ancient times and is still used today industrially. Fats are esters of fatty acids and glycerol. That is why fats are sometimes referred to as glycosides. When these esters are hydrolyzed by use of base such as sodium hydroxide they give rise to sodium salt of long chain fatty acids and glycerol. The process of converting fats to salt of fatty acids and glycerol is known as saponification. Thus the hydrolysis of fat with alkali yields glycerol and three molecules of the salt of the fatty acid (soap). The most widely occurring Easters in nature are fats from animal and vegetable origin. Animal Tallow, lard, palm oil, coconut oil, olive oil, peanut oil, cotton seed oil, corn oil etc. are but a few of the naturally occurring glycoside. Natural fats and oils are mixtures of glycosides. For instance, tallow containg myristic, almitic, stearic, oleic and linoleic esters of glycerol. The difference between fats and oils is caused by the fact that fats are composed of glycerides of saturated acids, while oils are made up of unsaturated fatty acids (caused by the presence of double bond). The extent of unsaturation in fat or oil is expressed in terms of its iodine value, which is defined as the number of grams of iodine which will add to 100 g of fat or oil. Thus a high iodine value as in vegetable oil indicates a high degree of instauration. The soap molecules possess detergent or cleansing properties because of their ability to form aggregates with fat soluble materials in which the long, fatty acid chains surround the “ dirt ” in such a way as to enclose it with in a cluster of soap anions leaving the hydrophilic carboxyl groups as a peripheral water solubilizing envelope. The resulting “ Dirt + soap ” complex can then be carried away by the water. Procedure Note how a reflux set up is arranged by observing the set up prepared by your instructor and arrange your own reflux set up. In a round bottom flask place 10 g of beef tallow, 20 ml of 20% sodium hydroxide solution, 15 ml 95% ethanol and 2 boiling chips. Ethanol helps the fat to 25

26. Prepared by: Abere Habtamu November, 2017 dissolve easily. Light the Bunsen burner using gentle flame and reflux for about 1 h. A homogenous soap solution now forms. Turn the heat off, wait for a few minutes to let the flask to cool a bit, and then pour the warm solution in to a beaker containing 100 ml of brine solution (i.e. saturated salt solution). Cool the beaker in an ice bath. Separate the solid soap from the mixture by decanting the liquid or filtering using suction filtration. Transfer the solid soap to a small beaker and place the baker over a wie gauze and heat it till the soap becomes soft. Turn the heat off, wait for a few minutes so that the beaker cools down to room temperature and then put the beaker in an ice bath so that the soap becomes harder. Remove the soap from the beaker using spatula and conduct the following tests: Test Solution 1: Dissolve 1 g of the soap you prepared in 50 ml of water The following solutions are available in the laboratory Test solution 2: is 2% solution of commercially available ordinary soap. Test solution 3: is 2 % detergent solution (such as Omo or Roll etc). Perform the following experiments on the above three test solutions and note the results in your report book. 1.Test the alkalinity of each of the above solutions by means of litmus paper. 2.Place 2 ml of test solution 1, into 4 test tubes. Add 3-5 drops of calcium chloride solution to the first test tube, 5% magnesium chloride to the second, 5% ferric chloride to the third and 6 m hydrochloric acid to the fourth. Shake each test tube well and note your observations and present the result in a table. Repeat this experiment with test solutions 2 and 3. Explain the different behaviors of the test solutions in the discussion section of your report. Quiz Questions 1.Why is glycerol very soluble in water? 2.What is the mechanism by which soap removes dirt? 3.Which part of soap molecule associates with “ dirt” and which part with water? 4.Why is better to use a reflux set up for a reaction that needs the solution to be boiled for a long period? 26

27. Prepared by: Abere Habtamu November, 2017 Capsicum annum or berbere Columbus, on his discovery of the new world found capsicum (chillies), which is America’ s most important contribution to the spices. Capsicums soon spread throughout the old world. That is how we got the berbere that we very much like to spice our food with. If the powdered chilled is extracted with hexane the colorful components known as capsanthins are extracted. The structure of the major pigment of capsanthin is shown bleow. The pungent or hot components known as capsaicins are extracted with the more polar solvent alcohol. These are vanillyl amides of differing side chain: capsaicin (1), dihydrocapsaicin (2), nordihydrocapsaicin (3), homodihydrocapsaicin (4), homocapsaicin (5) the major one being capsaicin. 27