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 ANALYSIS OF REAL SAMPLE (Chem3118) LAB MANUAL Abdu Hussen Ali Email: abdelmelik9@gmail.com JULY 2021 TULUAWLIYA, ETHIOPIA ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Preface and Acknowledgments Analysis of Real Sample is dealing with sampling, preservation and preparation of samples for the determination of the major, trace elements, inorganic compounds (speciation) and organic compounds; biological samples; food and beverages; water and waste water samples; soils and related samples. It’s also concerned with developing the tools used to examine these properties. Thus, it is important that students of chemistry do experiments in the Lab to more fully understand the theories they study. The manual helps students understand the timing and situations for the various techniques. Each experiment is presented with concise objectives, a comprehensive list of techniques, and detailed lab introduction, step-by-step procedures and discussion questions at the end of each lab. It is also important that you carefully prepared for each experiment by reading the related text material before coming to the lab. This way you can maximize the laboratory experience. I encourage you to discuss ideas for improvements or suggestions for new experiments with me. This manual was completed with support of chemistry staff members of Mekdela Amba University. Therefore, I am thankful for their efforts. 2|Page By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 TABLE OF CONTENT Content Page preface and Acknowledgments 1 Table of Content 3 Course Policies and Information 4 Learning Outcomes 4 Laboratory Precautions 5 Laboratory Maintenance 6 Some Laboratory Equipments 8 Experiment-1:Spectrophotometric determination of iron in meat through derivation with ferrozine 12 Experiment-2: Determination of vitamin c content in fruit juice 16 Experiment-3: Spectroscopic analysis of caffeine and benzoic acid in soft drink 22 Experiment-4: Determination of acid content of vinegar 28 Experiment-5: Determination of fluoride ion using an ion selective electrode 34 Experiment-6:Analysis of turbidity, colour, ph, and alkalinity of water Experiment-7:Determination of permanent hardness due to Ca 2+ and Mg 40 2+ in tap water by EDTA method 47 Experiment-8:Determination of chemical oxygen demand (COD) of wastewater using open reflux method 52 Experiment-9: Soil sample collection and preparation for heavy metal analysis 59 Experiment-10: Determination of manganese in soil sample using FAAS 66 Experiment-11: Determination of soil organic matter by walkley and black method 70 Experiment-12:Determination of extraction efficiency of some organic solvents using soxhlet extractor 75 Experiment-13:Determination of the lipid content of snack food using soxhlet extraction method REFERENCES 80 85 3|Page By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Course Policies and Information In this laboratory, you will be working as a team with two or three persons in each group. During the first lab period your instructor will assign you to a group. You will then introduce yourself to group members and get to know other members of your group. Then your instructor will read you important safety rules. In this meeting of the laboratory, you will also be given your first lab experiment and the rest of this lab period you will work on a plan of action for the first experiment. Each student in the group must have a lab Notebook and bring it to the lab every week. You should keep a good notebook with all the calculations and the results because your instructor will grade your Lab notebooks at the end of each experiment. Finally, each member of your group has to write a 5 or 6-page lab report after completing the experiment. This is going to be a individual report therefore, even if your results are the same. The reports you write must be your own work. If your instructor finds out that your report is exactly the same with another member of your group, you will not receive any credit for that report and he/she may consider it as cheating. Learning Outcomes At the end of this course students should be able to: x Select appropriate sampling and preservation of a particular real sample x Identify preparation methods for analysis of metals by different methods x Perform experiments on water, soil and air x Familiarize the students with the techniques of sampling, storage, and analysis of real samples. Format of the Lab Report You should prepare your lab reports by handwriting. They should include tables and illustrations where necessary. Typically, a lab report should contain the following sections: title page, introduction, experimental section, results and discussion, Conclusion and references. Your title page should be a separate page including the title of the project which might simply be the name of the experiment, your name, name of the course and the date the report is due. 4|Page By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 LABORATORY PRECAUTIONS A. Inside the Laboratory 1. Do not eat, drink beverages or chew gum in the laboratory. Do not use laboratory glassware as containers for food or beverages 2. Wear safety goggles and aprons 3. Always keep the working area clean and orderly 4. Know the locations and operating procedures of all safety equipment. 5. Notify the instructor immediately of any unsafe condition you observe B. Handling Chemicals 1. All chemicals in the laboratory are to be considered dangerous. Do not touch, taste or smell any chemical unless specifically instructed to do so 2. Check the label on chemical bottles twice before removing any of the contents. 3. Never return unused chemicals to their original containers. 4. Acid must be handled with extreme care. ALWAYS ADD ACID SLOWLY TO WATER. 5. Handle flammable hazardous liquids over a pan to contain spills. Never dispense flammable liquids anywhere near an open flame or source of heat. C. Handling glassware and Equipment 1. Always lubricate glassware (tubing, thistle tubes, thermometers, etc.) before attempting to insert it in a stopper. 2. When removing an electrical plug from its socket, grasp the plug, not the electrical cord. Keep your hands dry when working with electricity. 3. Do not immerse hot glassware in cold water, it may shatter. 4. Report damage electrical equipment immediately. D. Heating Substances 1. TURN OFF THE GAS AT GAS OUTLET VALVE after using. 2. Never leave a lit burner unattended. Never leave anything that is being heated or is visibly reacting unattended. 3. Use tongs or heat-protective gloves when holding or touching heated apparatus. 5|Page By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 LABORATORY MAINTENANCE 1. Make sure your Laboratory space(s) is cleaned. Also clean all the equipment’s and returned them to their assigned positions. Failure to do so will result to a zero grade for the experiment. NO exceptions please. 2. All glassware must be cleaned before it is put away. 3. Use sponges to clean bench tops and wiping of non-hazardous materials. 4. Laboratory instructors are the ONLY one allowed to clean up corrosive or toxic materials 5. Sweep up broken glassware with a broom and collect with the dust pan and then place in the special container provided for glasses. 6. No debris of any type should be left in the sink. Put all debris in allocated containers 7. Make sure all drawers are properly closed and locked when necessary. GENERAL INFORMATION 1. Dispense organic solvents, strong acids and bases and other volatile solvents in the fume hoods. 2. No fee will be collected for broken equipment or glassware. Each broken glassware and equipment’s will be replaced with two of similar type by the culprit. LABORATORY TECHNIQUES 1. Use proper utensils such as crucible tongs to hold or move hot items. 2. Make sure there are no flammable materials near you when lightning a burner 3. Add boiling chips to liquids before heating them up. This will help to prevent bumping or boil over. 4. Place test tubes in a slanting position away from yourself and others when heating liquids. Heat liquids at the surface of the liquid. 5. Do not heat up a closed system 6. Heat all substances that emit noxious fumes under the hood 7. Use funnel to transfer liquids into a narrow neck container 8. Use a bulb or pump to pipette a liquid. Never use your mouth 9. Avoid smelling anything unless instructed to do so. While sniffing, gently waffle the material towards your nose when allowed to do so 6|Page By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 10. Do not return excess reagent to its original container 11. Do not experiment with the chemicals in the laboratory except those that your are scheduled to do. 12. Do not use your pipette or spatula to remove samples from the stock container. Use the one provided by the laboratory technologist 13. Correctly label test tubes or other containers indicating their contents 14. Strong acids and bases should be added to water and not vice versa. EMERGENCIES AND FIRES 1. Laboratory instructors are in-charge of all emergencies. Follow instructions as directed 2. All laboratory users should learn how to locate the following materials: safety shower, eyewash, blankets, fire extinguishers, first aid kit, fire alarm 3. Laboratory users should notify the laboratory instructors of any fire. 4. Turn off all gas jets if it is the source of the fire 5. All laboratory users should learn how to use the fire extinguisher ACCIDENTS AND INJURIES 1. The chemistry department does not treat injuries or illness. Any injury or illness will be referred to the University of Mekdela Amba. 2. It is the responsibility of the laboratory instructor(s) on duty to prevent further injury by taking the appropriate action after the incident. Arrangement should be made to immediately transport the victim to the Medical Center. If the injury is minor and the student can walk to the Medical Center, such student should be accompanied by another person to the Medical Center. 3. An accident report form must be filled at all times even when the victim declines Medical treatment. 7|Page By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 SPECIAL WASTE 1. The laboratory will provide label containers for hazardous waste. Read the label very well and dispose the waste appropriately. 2. At no time should organic or toxic wastes such as mercury, lead, chromium be dumped down the drain. 3. Ask when in doubt about proper disposal of waste. SOME LABORATORY EQUIPMENTS 8|Page By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 9|Page By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 10 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 11 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 EXPERIMENT-1 Spectrophotometric Determination of Iron in Meat through Derivation with Ferrozine Objectives: To determine the iron content of food samples using derived UV-Vis Spectrophotometry Introduction Determination of the concentration distribution of soluble reactive species is key to understanding biogeo-chemical processes in natural settings. Iron is one of the most reactive elements in aquatic environments, and its cycling is coupled to that of the major biogeo-chemical elements (C, O, S and P) and trace elements such as heavy metals. It is present in the hydrosphere under two oxidation states, II and III, which are thermodynamically stable under anoxic and oxic conditions, respectively. Chromogens are chemicals that react with compounds of interest and form colored products that can be quantified using spectroscopy. Several chromogens that selectively react with minerals are available. In this case, ferrozine is used to measure ferrous iron in an ashed food sample. The meat samples are first ashed to dissociate the iron bound to proteins, and thus ash residue is solubilized in dilute HCl. The acid is necessary to keep the mineral in solution. Ferrozine complexes with ferrous iron but not with ferric iron. Prior to the reaction with ferrozine, the solubilized ash is first treated with ascorbic acid to reduce all forms of ferric iron to the ferrous form. This step is necessary with ashed samples as this procedure would be expected to reduce all the iron present in the meat. Spectroscopic analysis is based on the change in the intensity of the colour of a solution with variations in concentration. These methods represent the simplest form of absorption analysis. The human eye is also used to compare the colour of the sample solution with a set of standards until a match is found. An increase in sensitivity and accuracy results when a spectrophotometer is used to measure the absorbace. Basically, it measures the fraction of an incident beam of light which is transmitted by a sample at a particular wavelength. Iron oxides are dissolved in hot, diluted 12 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 ascorbic acid. Ascorbic acid rapidly reduces Fe(III) to Fe(II) in acidic solution. Ferrozine forms a stable and colored complex with Fe(II) in the pH range 4-10, and this makes a sensitive determination of iron possible by means of spectrophotometry. Principle of Method Ferrous iron in extracts or ashed samples reacts with ferrozine reagent to form a stable colored product which is measured spectrophotometrically at 562 nm. Iron is quantified by converting absorbance to concentration using a standard curve. Materials and Instruments Meat sample, 16 Test tubes (18 × 150 mm) porcelain crucible, volumetric flask (250 mL), pipettes (10 mL,25 mL), muffle furnace, hot plate, spectrophotometer, analytical balance Chemicals and reagents ¾ Ferrozine, ascorbic acid, ammonium acetate and ferric stock solution ¾ Ferrozine reagent (0.493 g of ferrozine in water and dilute to liter in a volumetric flask ¾ Ascorbic acid (0.02% in 0.2 N HCl, made fresh daily) ¾ Ammonium acetate (30% w/v) ¾ Iron stock solution (10 μg iron/mL) ¾ Solutions of 0.1 N and 0.2 N HCl PROCEDURE Ashing 1. Place 5 g sample into the crucible and weigh accurately. Make a triplicate of measurement 2. Heat on the hot plate until the sample is well charred and has stopped smoking. 3. Ash in muffle furnace at 550 Ԩ until the ash is white. 13 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Iron Measurement 1. Prepare working standards of 10, 8, 6, 4 and 2μg iron/mL from a stock solution of 1 μg iron/mL using serial dilution. In addition, prepare a blank solution. 2. Dissolve ash in small amount of 1 N HCl, and dilute to 50 mL in volumetric flask with 0.1 N HCl. 3. Put a triplicate of 0.50 mL of appropriately diluted samples and standards into 10 mL test tubes. 4. Add 1.25 mL ascorbic acid (0.02 % in 0.2 N HCl, made fresh daily). Mix the solution thoroughly and let it set for 10 minutes. 5. Add 2.0 mL 30 % ammonium acetate and mix the solution well (pH needs to be >3 for color development) 6. Add 1.250 mL ferrozine (1mM in water). Mix the solution and let set in dark for 15 minutes 7. Group the contents of the two standard water blanks and use this to zero the spectrophotometer at 562 nm (single beam instrument) or place in the reference position (dual beam instrument). 8. Take your readings three times Data and Calculations I. Calculation of percentage of ash Calculating the percentage of ash using % Ash = ࢃ૛ିࢃ૚ ࢃ࢙ ‫ כ‬૚૙૙ Where, W1: weight of crucibles W2: weight of crucibles with ash sample Ws: weight of sample 14 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 II. Absorbances of Standards and sample (μg iron/mL) Trial- 1 Trial-2 Average Unknown 2 4 6 8 10 Absorbances of replicate (Rep) Samples Rep 1 = ___________________ Rep 2 = ___________________ Rep 3 = ___________________ Calculation of total iron in sample: 1. Plot the standard curve and determine the content of iron (μg iron/mL) in the dissolved ash solution. 2. Calculate the iron (μg iron/g) in the sample using ࣆࢍ࢏࢘࢕࢔ ൈ૞૙࢓ࡸࢇ࢙ࢎ࢙࢕࢒࢛࢚࢏࢕࢔ ࢓ࡸ࢕ࢌ࢙࢕࢒࢛࢚࢏࢕࢔ ࢓ࢇ࢙࢙࢕ࢌ࢓ࢋࢇ࢚࢚ࢇ࢑ࢋ࢔ =ൈ ࣆࢍ࢏࢘࢕࢔ ࢍ࢓ࢋࢇ࢚ Where x stands for mass of iron per dry mass in the sample Questions 1. What is the purpose on adding ascorbic acid in the solution? 2. Why the sample are ashed before analysis? 3. Which chemical are used to form stable colour products to be spectoro-photometrically determined? 15 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 EXPERIMENT-2 Determination of Vitamin C content in Fruit Juice Objective: To determine the content of vitamin c in fruit juice Introduction Vitamin C, known chemically as ascorbic acid, is an important component of a healthy diet. The history of Vitamin C revolves around the history of the human disease scurvy, probably the first human illness to be recognized as a deficiency disease. Its symptoms include exhaustion, massive hemorrhaging of flesh and gums, general weakness and diarrhea. Resultant death was common. Scurvy is a disease unique to guinea pigs, various primates, and humans. All other animal species have an enzyme which catalyzes the oxidation of L- gluconactone to L-ascorbic acid, allowing them to synthesize Vitamin C in amounts adequate for metabolic needs. L-Ascorbic Acid -- Vitamin C The RDA (Recommended Daily Allowance) for Vitamin C put forward by the Food and Nutrition Board of the National Research Council is 60 mg/day for adults. It is recommended that pregnant women consume an additional 20 mg/day. Lactating women are encouraged to take an additional 40 mg/day in order to assure an adequate supply of Vitamin C in breast milk. Medical research shows that 10 mg/day of Vitamin C will prevent scurvy in adults. There has been much controversy over speculation that Vitamin C intake should be much higher than the RDA for the prevention of colds and flu. Vitamin C and the Common Cold, that humans should be consuming around 500 mg of Vitamin C a day (considered by many doctors to be an excessive amount) to help ward off the common cold and prevent cancer. Vitamin C is a six carbon chain, closely related chemically to glucose. It is a simple, inexpensive, four-step process for synthesizing ascorbic acid from glucose. This method has been used for 16 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 commercial synthesis of Vitamin C. Vitamin C occurs naturally primarily in fresh fruits and vegetables. Vitamin C content of some foodstuffs are described below: Vitamin-C (mg/100g) Foods 100 – 350 Chili peppers, sweet peppers, parsley, and turnip greens 25 – 100 Citrus juices (oranges, lemons, etc.), tomato juice, mustard greens, spinach, brussels sprouts 10 – 25 Green beans and peas, sweet corn, asparagus, pineapple, < 10 Eggs, milk, carrots, beets, cooked meat cranberries, cucumbers, lettuce Vitamin C is a water-soluble, antioxidant vitamin. It is important in forming collagen, a protein that gives structure to bones, cartilages, muscles, and blood vessels. Vitamin C also aids in the absorption of iron, and helps maintain capillaries, bones, and teeth. It is the most common electroactive biological compound and one of the most ubiquitous vitamins ever discovered. Rich sources include blackcurrant, citrus fruit, leafy vegetables, tomatoes, green and red peppers. Ascorbic acid is known for its reductive properties. Hence, it is used on a large scale as antioxidant in food and drinks. Due to its content variation caused by the thermal lability, vitamin C represents an important quality indicator that contributes to the antioxidant properties of food. Traditional methods for ascorbic acid assessment involve titration with an oxidant solution: dichlorophenol indophenol (DCPIP), potassium iodate or bromate. Chromatographic methods, particularly HPLC with electrochemical detection, has turned out to be a selective and sensitive method for ascorbic acid assessment in foodstuffs and biological fluids. Fluorimetric methods and UV-VIS absorbance-based determinations were also used for ascorbic acid estimation. However, the determination of vitamin C concentration in a solution by a redox titration using iodine is simple and best method rather than the above method. Vitamin C, more properly called ascorbic acid, is an essential antioxidant needed by the human body. As the iodine is added during the titration, the ascorbic acid is oxidised to dehydroascorbic acid, while the iodine is reduced to iodide ions. ascorbic acid + I2 → 2 I− + dehydroascorbic acid 17 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Due to this reaction, the iodine formed is immediately reduced to iodide as long as there is any ascorbic acid present. Once all the ascorbic acid has been oxidised, the excess iodine is free to react with the starch indicator, forming the blue-black starch-iodine complex. This is the endpoint of the titration. The method is suitable for use with vitamin C tablets, fresh or packaged fruit juices and solid fruits and vegetables. The amount of ascorbic acid in a fruit juice sample will be determined by titrating a weighed amount of the sample with iodine. The iodine will immediately react with the ascorbic acid until all of the ascorbic acid has been exhausted. The next drop of iodine cannot be reduced to iodide (I-) and, thus, reacts with the starch causing the solution to turn blue-black. Thus, the amount of iodine necessary to bring about the color change is an indicator of the amount of ascorbic acid present in the sample. In a titration procedure a solution of unknown analyte concentration is mixed with a solution with a known concentration of a compound that reacts with the analyte. (The analyte is the compound being analyzed; in this experiment it is ascorbic acid.) Measuring the amount of known solution required to just completely use up the analyte allows the calculation of the concentration of analyte in the unknown solution. (The known solution is called the "titrant". In this experiment, the titrant is the iodine solution.) Usually a burette is used to measure the amount of the known solution required. Material and Instrument ¾ burette and stand ¾ volumetric flask ¾ pipette ¾ measuring cylinders ¾ conical flasks Chemical and Reagent ¾ 2% Lugol’s iodine solution, acetic acid ¾ commercial fruit juice ¾ Starch solution ¾ Sodium thiosulfate 18 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Standardization of Iodine A solution of iodine can be standardised by titration against a known concentration of sodium thiosulfate according to the following equation. I2(aq) + 2S2O32–(aq) → 2I–(aq) + S4O62–(aq) A solution of sodium thiosulfate has already been prepared for you. The exact concentration is written on the label. You are going to use this solution to determine the exact concentration of the I2 solution used for determining the concentration of vitamin C in your fruit. 1. Collect about 200 mL of a solution of I2 in a clean dry stoppered 250 mL conical flask. Prepare the burette for titration and as shown by your demonstrator, by washing with water and then three times with a small amount of the I2 solution. Remember to restopper the iodine solution in your 250 mL flask. 2. In a clean and dry 250 mL conical flask collect 120 mL of the sodium thiosulfate solution. 3. Pipette a 25.00 mL aliquot of the sodium thiosulfate solution into a clean 250 mL conical flask. Add a half a Ni spoonful of Vitex reagent and 10 drops of 0.2 M acetic acid. Titrate with the I2 solution until you get a permanent colour change for at least 30 seconds. The endpoint is a light blue colour. Record your initial and final volumes of the titration. 4. Repeat at least twice. Vitamin C Determination Your demonstrator will allocate you a fresh juice OR a preserved juice. Fresh fruit juice preparation Begin with step (6) if you have been allocated a preserved juice. 1. Weigh 2 clean and dry Petri dishes on the top loading balance. Record their masses. 2. In duplicate, accurately weigh about 5 g of a fruit using a top loading balance. Record the exact mass. 3. For each sample, cut the fruit into small pieces, place it in the microcloth and squeeze the fruit juice into a clean 250 mL conical flask via a funnel. 4. Rinse the cloth twice with an additional 5 - 10 mL of deionized water. Squeeze the water through the cloth and allow the filtrate to mix with that from step (3). 19 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 5. Use a spatula to scrape the remaining pulp onto the pre-weighed Petri dish and reweigh. Record the mass. Add half a Ni spoonful of Vitex and 10 drops of 0.2 M acetic acid. Continue with step (10). Preserved fruit juice 6. Weigh a 10 mL measuring cylinder on the top loading balance and record its mass. 7. Measure 5 mL of fruit juice in the pre-weighed 10 mL measuring cylinder and reweigh it. 8. Place the fruit juice into a clean 250 mL conical flask and dilute with 20 mL of deionised water. If necessary, filter off any pulp using the microcloth and rinse with deionised water into a clean 250 mL conical flask. Add a spatulaful of Vitex and 10 drops of 0.2 M acetic acid. 9. Repeat steps (6) - (7) to obtain a duplicate sample. 10. Fill the burette with the standardised I2 solution and titrate your fruit juice until a permanent pale blue colour persists for at least 30 seconds. Record your initial and final volumes of I2. Data and Calculation Part 1: Standardization of I2 Titration 1 Titration 2 Initial volume (mL) Final volume (mL) Titre (mL) Average= Concentration of thiosulfate ion = 2.040 × 10–3 M (from bottle) ¾ Calculate the concentration of your I2 solution? 20 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Part 2: Vitamin C Determination Sample 1 Smple 2 Mass of juice Initial volume (mL) Final volume (mL) Titre (mL) [I2] = ------------ (from Part 1) Reaction: I2(aq) + ascorbic acid → 2I–(aq) + dehydroascorbic acid Therefore 1 mol of I2 reacts with 1 mol of ascorbic acid Molar mass of ascorbic acid (C6H8O6) = 176.12 g mol–1 ¾ Calculate the mass of vitamin C in mg per g of fruit or mg per mL of fruit juice for your assigned sample? Content of Vitamin C in fruit juice = ‫ܖܗܑܜ܉ܚܜܑܜܕܗܚ܎܌܍ܖܑܕܚ܍ܜ܍܌܋ܖܑܕ܉ܜܑܞ܎ܗܛܛ܉ܕ‬ ‫ܖ܍ܓ܉ܜ܍ܔܘܕ܉ܛ܍܋ܑܝܒ܎ܗܛܛ܉ܕ‬ Questions 1. Why are I2 is added to each of our flasks during titrating in this experiment? What is the function? 2. Why were the fruit juices centrifuged and filtered? 3. Using your average milligrams of Vitamin C per gram or milliliter of product as the "correct" value, determine the percent error in the manufacturer or text’s claim (show calculations)? 4. What can you conclude about the labeling of this preserved juice or reference value? How do your account for any discrepancies? Does the manufacturer or reference overstate or understate the amount of Vitamin C in the product? If so, why might they do this? Explain below. 21 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 EXPERIMENT-3 Spectroscopic analysis of caffeine and benzoic acid in soft drink Objective: To analysis caffeine and benzoic acid in soft drink Introduction Soft drinks come under the category of junk food products. This is because of their nutritional value is less and fat, sugar, salt and calories contents are high. Soft drinks are manufactured from carbonated water, sugar cane syrup, caffeine and extract of kola nut and coca leaves. A soft drink is a beverage that contains carbonate water, sweetener, Flavoring agents, Preservatives like salts of benzoic acid, Caffeine, Coloring agents etc. Caffeine, is an alkaloid (C8H10O2N4.H2O) found in coffee, tea, cacao, and some other plants. Caffeine is added to soft drinks as a flavoring agent, and from dietary sources is the most frequently and widely consumed central-nervous-system stimulant today. Caffeine is a non-polar organic compound, which goes by many names including caffeine, guaranine, mateina etc. The molecular weight of caffeine is 196.19 g/mol, with a melting point of 238 Ԩ and a sublimation point at 178 Ԩ. The chemical name for caffeine is 1, 3, 7trimethylxanthine or 1, 3, 7-trimethyl-2, 6-dioxopurine. The caffeine drug increases the blood pressure, stimulates the central nervous system, promotes urine formation and stimulated the action of the heart and lungs. Caffeine is used in treating migraine because it constricts the dilated blood vessels and thereby reduces the pain. It also increases the potency of analgesics such as aspirin, and it can somewhat relieve asthma attacks by widening the bronchial airways. Caffeine produces increased mental alertness and reduces fatigue and increases the heart rate slightly. It is relatively nontoxic, but clearly has addictive potentials. Other withdrawal symptoms in heavy users include fatigue and difficulty in concentration. Over use can lead to insomnia, gastrointestinal disturbances and hypertension. Caffeine is also useful in pain medication. Butalbital, sedative barbiturate drug, is typically combined with other ingredients such as aspirin, acetaminophen, caffeine or codeine, to create a medication used to relieve headache or muscle pain in the neck and shoulders. It works by decreasing the activity of the central nervous system. Propoxyphene is also a drug containing caffeine and other ingredients such as aspirin and acetaminophen. This drug is used to treat mild to moderate pain. It is a narcotic 22 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 that works by depressing the activity of the central nervous system to create an anesthetic or pain killing effect. Benzoic acid (C6H5COOH) is the simplest of carboxylic acids of the aromatic series. It is used as a food preservative. Food preservation is the prevention of chemicals decomposition and the development of harmful bacterial in foods. Generally affected by the sterilization of the food (that is by the destruction of bacterial in it) is by heating in sealed vessels or making the conditions unfavorable for the development of bacterial. Yeast, a unicellular micro-organism producing zymase, converts sugars (hexose) into alcohol and carbon dioxide. This is because benzoic acid inhibits the growth of yeast and moulds. Benzoic acid is also a white crystal sparingly soluble in cold water, moderately soluble in hot water, having melting point of 122.4 Ԩ and sublimes if rapidly heated. High amount of benzoic acid (added as preservative) is harmful for liver and it disturbs carbohydrate metabolism which may lead to accumulation of fat causing obesity and impairment of liver also affects removal of toxic waste materials from body which leads to several metabolic disorders. Thus consumption of soft drinks having large quantity of benzoic acid causes severe health hazards. Both caffeine and benzoic acid are aromatic in nature and absorb ultraviolet radiation. So for the estimation of Caffeine and benzoic acid spectrophotometric method has been used. 23 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Material and instrument ¾ UV/Visible spectrophotometer ¾ Chemical balance ¾ Measuring cylinder ¾ Magnetic stirrer ¾ Filter ¾ Beakers ¾ Thermometer ¾ Separatory funnel ¾ Funnel ¾ Erlenmeyer flask Chemical and Reagents ¾ Benzoic acid standard ¾ Caffeine standard ¾ Sodium carbonate ¾ Methylene chloride ¾ Magnesium sulphate ¾ Potassium chlorate ¾ Hydrochloric acid ¾ Ammonia solution Sampling ¾ The following soft drinks are taken from a supermarket in: Fanta, Pepsi, Miranda and coca cola. Preparation of a stock solution 1. A 0.01 g of benzoic acid standard and caffeine standard were weighed separately and dissolved into two different 100 mL volumetric flasks and topped with distilled water to the 100 mL mark. 2. Prepare 5 ppm, 10 ppm, 15 ppm, 20 ppm, and 25 ppm from the stock solution. 3. Pipette 0.5 mL of stock solution into 50 mL volumetric flask and diluted with distilled water to the 50 mL mark. 24 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 4. Pipette 0.75 mL of stock solution into 50 mL volumetric flask and diluted with water to the 50 mL mark. 5. Pipette 1mL stock solution into 50 mL volumetric flask and diluted with distilled water to the 50 mL mark. 6. Pipette 1.25 mL of stock solution into 50 mL volumetric flask and was topped with distilled water to the 50 mL mark. Extraction of caffeine 1. A brand of soft drink was recorded and 150 mL was carefully measured into a conical flask. 2. To this, 2.0 g of sodium carbonate was added to it. The mixture was then tested with a red litmus paper and the litmus registered a blue color. 3. A 50 mL of methylene chloride was added to the mixture in the conical flask and the flask swirled gently for at least 5 minutes and poured into a separating funnel and allowed to settle for about 5 -10 minutes. 4. The organic layer was drained into a clean 250 mL conical flask. 5. A fresh 50 mL sample of methylene chloride was added to the mixture in the separating funnel and the flask stoppered. 6. The funnel was gently inverted a few times to allow the remaining caffeine to be extracted into the methylene chloride layer. 7. The lower layer was then separated and combined with the first extract. 8. The total was treated with 5g anhydrous magnesium sulphate to remove water. 9. The methylene chloride was filtered through a cotton pad into a 250 mL Erlenmeyer flask. 10. The extract in the Erlenmeyer flask was placed on a water bath to evaporate methylene chloride. A small amount of the precipitate was placed on a watch glass and mixed with 2 -3 drops of concentrated hydrochloric acid. 11. Few crystals of potassium chlorate were mixed well with a glass rod and mixture evaporated to dryness on a water bath. 12. The watch glass was cooled and moistens with a drop of 2M ammonia solution. 13. Residue turned purple which was an indication of the presence of caffeine. 14. The rest of the precipitate was diluted with methylene chloride and taken to the UV for the absorbance to be taken. 25 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Isolation of Benzoic Acid 1. 150 mL of soft drink was poured into a conical flask and acidified with 2 drops of dilute hydrochloric acid. 2. 50 mL of methylene chloride was added and the flask swirled gently for at least 5 minutes. 3. The mixture was transferred into 250 mL separating funnel and allowed to settle for about 5 10 minutes. 4. The organic layer was drained into a beaker and allowed to evaporate on a water bath, leaving a residue of benzoic acid. 5. The residue was diluted with methylene chloride and sent to the UV for the absorbance to be taken. Data and Calculation Part 1: Standard caffeine and benzoic acid solution Standard concetration UV absorbace Standard concetration UV absorbace of caffeine (ppm) of benzoic acid (ppm) 5 5 10 10 15 15 20 20 25 25 26 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Part 2: Caffeine and benzoic acid in soft drink Name of soft drink Average absorbace (benzoic Average absorbace acid) (Caffeine) Mirinda Fanta Pepsi Coca cola ¾ Calculate the concentration of benzoic acid and caffeine in each soft drink? Questions 1. What is the difference between caffeine and benzoic acid? 2. What is the purpose of caffeine and benzoic acid found in soft drink? 3. Calculate and compare the amount of benzoic acid and caffeine in each soft drink? 27 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 EXPERIMENT-4 Determination of acid content of vinegar Objective: To determine the acetic acid content of vinegar Introduction Vinegar is an acidic liquid, which is made both naturally and synthetically, from the oxidation of ethanol, CH3CH2OH, in an alcohol-containing liquid such as wine, fermented fruit juice (e.g. cider) or beer. It has been used since ancient times as an important cooking ingredient, e.g. in salad dressings and on fish and chips. The key chemical component of vinegar is acetic acid, CH3COOH (systematic name: ethanoic acid). The trivial name, acetic acid, is derived directly from the word for vinegar, which, for example, in Italian is called aceto. The word vinegar itself derives originally from the Latin vinum aegrum, meaning “feeble wine”. In fact, when a wine has “gone off” and has acquired a sour taste, this is due to the oxidation of the ethanol in the wine to acetic acid. (The “corking” of wine, i.e. tainting of the wine by compounds transferred from or through the cork, is due to a totally different chemical process.) The acetic acid content of vinegar can vary widely, but for table vinegar it typically ranges from 4 to 8 % v/v. To determine the amount of acetic acid in vinegar (typically 4-8% by mass) we will use titration. Titration is a common analytical method used to measure the amounts of compounds in solution. The glassware you will be using is called a buret. The buret holds one of the reactants, called the titrant, and conveniently adds it into a reaction vessel which contains the second reactant. The titrant in this experiment will be a sodium hydroxide (NaOH) solution. In this experiment we titrate acetic acid with sodium hydroxide (a strong base). The reaction of acetic acid with sodium hydroxide is shown below: HC2H3O2 (aq) + NaOH (aq) → NaC2H3O2(aq) + H2O (l) acetic acid sodium acetate This equation is an acid-base reaction; also know as a neutralization reaction. The acetic acid (HC2H3O2) found in the vinegar will react with the NaOH until all of the acetic acid is neutralized. When an acid, such as acetic acid reacts with a base like NaOH, the products are a salt (NaC2H3O2, 28 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 sodium acetate) and water (H2O). If you know the concentration of the sodium hydroxide solution and the volume that you need to add to the acid, then you can figure out how much acetic acid is in the vinegar. In an acid-base titration, the point at which both acid and base have been completely consumed can be detected and is known as the “equivalence point”. The amount of one reactant (the analyte) can be calculated from the known concentration and the volume of reactant in a standard solution (the titrant) using the balanced chemical equation. The end point in this experiment will be detected with an acid/base indicator. An acid/base indictor is a coloured substance with two or more different colors depending on the value of the pH of the solution. The standard solution may be prepared in two ways – the direct or indirect method. In the direct method, a precisely weighed quantity of the pure solute (primary standard) is dissolved and diluted to a known volume in a volumetric flask. The concentration of the standard solution is then calculated from the known mass of the solute and the known volume of the solution. If the solute used to prepare the standard solution is pure and the solution is stable (does not decompose), then the compound is referred to as a primary standard. However, often it is not possible to obtain the solute in sufficiently pure form to be suitable as a primary standard. For example, NaOH(s) reacts with gases (H2O and CO2) in the air which means that NaOH (s) is not pure enough to be used as a primary standard. In this case the standard solution is prepared by an indirect method. A solution is prepared at approximately the desired concentration and it is then standardized against another primary standard to determine its exact concentration. Material and Instrument ¾ 50 mL beaker ¾ 100 mL volumetric flask (with cap) ¾ 50 mL burette ¾ 250 mL Erlenmeyer flask ¾ 25 mL pipette ¾ Titration apparatus 29 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Chemical and Reagent ¾ KHC8H4O4 ¾ phenolphthalein indicator solution ¾ Distilled water ¾ Sample of commercial grade vinegar (may be obtained from home) Preparation of the Potassium Hydrogen Phthalate Standard For the titration of the vinegar in this experiment the following specific reaction will be used to calculate the acetic acid content of the vinegar sample: HC2H3O2 (aq) + NaOH (aq) → H2O (l) + NaC2H3O2 (aq) 1. Weigh precisely (on an electronic balance) ~ 1.5 grams of pure potassium hydrogen phthalate (KHC8H4O4) into a 50 mL beaker. 2. Dissolve the acid in ~50 mL of water and transfer carefully into a 100 mL volumetric flask. 3. Rinse the beaker several times with small portions of water to ensure quantitative transfer. 4. Make the volume up to the mark with distilled water, cap the flask and mix thoroughly. This solution will be used to standardize a solution of sodium hydroxide. Standardization of the Sodium Hydroxide Solution Potassium hydrogen phthalate, the primary standard, reacts with sodium hydroxide as shown below: 1. Select a clean 50 mL burette, rinse it with a small portion of the sodium hydroxide solution, and fill it to just below the zero mark. 2. Read and record the initial volume to the nearest 0.01 mL. 3. Rinse a 250 mL Erlenmeyer flask with distilled water to make sure it is clean. 30 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 4. Pipette exactly 25.00 mL of the potassium hydrogen phthalate solution into the flask. 5. Add three drops of phenolphthalein indicator and titrate with the sodium hydroxide solution until a permanent colour change is first detected. The palest pink colour denotes the end point of this reaction. The colour should persist throughout the entire solution when swirled for at least 10 seconds. The colour will fade slowly on standing. 6. Repeat the titration on a second 25.00 mL aliquot of the primary standard solution. If the titration volumes do not agree within ±0.1 mL continue to repeat the titration. 7. Report your two best titrations. The Determination of Acetic Acid in Vinegar The acetic acid (CH3COOH) concentration in commercial vinegar may be easily determined by titrating a suitable sample of the vinegar with the standardized sodium hydroxide solution. 1. Pipette exactly 10.00 mL of the commercial vinegar sample into a 250 mL Erlenmeyer flask and add ~5 mL of distilled water. 2. Using three drops of phenolphthalein indicator, titrate the acetic acid with the standard base to a pale pink equivalence point. Record the burette readings. 3. Repeat the titration at least once more using a fresh aliquot of vinegar. Results should agree within ±0.2 mL or additional titrations are required. 4. Report your two best titrations. 31 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Data and Calculation Part A: Standardization of sodium hydroxide solution Titration 1 2 Mass of beaker (g) Mass of beaker + KHP (g) Mass of KHP (g) Volume of NaOH to neutralize the KHP solution (mL) Part B: Molarity of acetic acid and percent of vinegar No Volume of NaOH used (mL) Titration 1 Titration 2 Calculations 1. Determine the number of moles of sodium hydroxide required to titrate the vinegar for each titration from the known molarity and the titration volume (V = V2 - V1) of sodium hydroxide. Be sure that the volume of the sodium hydroxide has been converted from milliliters to liters (1 L = 1000 mL). MNaOH = ௠௢௟௘௦ே௔ைு ௏௢௟௨௠௘ே௔ைு so moles NaOH= (VNaOH) x (MNaOH) 2. The moles of acetic acid are equal to the moles of sodium hydroxide at the equivalence point. The equivalence point is close to the endpoint so we can use the endpoint value. The endpoint is when the phenolphthalein changes color. moles acetic acid = moles sodium hydroxide 3. Determine the mass of acetic acid present in each titration from the molar mass (sometimes called molecular weight) of acetic acid and moles of acetic acid. Molar mass = ࢓ࢇ࢙࢙ ࢓࢕࢒ࢋ࢙ so mass of acetic acid = molar mass x moles 4. The mass of the vinegar for each titration is found from the measurements using the 32 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 analytical balance (m2 - m1). 5. The percentage mass of acetic acid in the vinegar is found from the mass of acetic acid and the mass of vinegar. % mass = ࢓ࢇ࢙࢙࢕ࢌࢇࢉࢋ࢚࢏ࢉࢇࢉ࢏ࢊ ࢓ࢇ࢙࢙࢕ࢌ࢜࢏࢔ࢋࢍࢇ࢘ x 100 Questions 1. How accurately does the 50 mL of water used to dissolve the KHP in the standardization of the NaOH solution need to be measured? Explain. 2. When transferring the KHP in Part A, if some of the KHP missed the opening to the Erlenmeyer flask and fell onto the weighing pan and stayed there, how would the calculated molarity of the NaOH solution compare to the actual value (i.e., is the calculated concentration more, less or the same as the actual value)? Explain. 3. During the titration of KHP in part A of this experiment, you obtain a dark pink endpoint (instead of a pale pink endpoint). Will this result in the calculated molarity of the NaOH solution being higher, lower or the same as the actual molarity? Explain your answer 4. How does obtaining a dark pink endpoint (instead of a pale pink endpoint) in the titration in Part B, affect the calculated mass % of acetic acid in vinegar compared to the actual value (what it should be)? That is, is the calculated mass % greater than, less than or equal to the actual value? Explain. 33 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 EXPERIMENT-5 Determination of Fluoride Ion Using an Ion Selective Electrode Objective: To determine the F- ion concentration in toothpaste Introduction The primary purpose of brushing the teeth with dentifrice is to clean the accessible tooth surface of dental plaque, stains and food debris. Fluoride (F-) is an important anion present in various environments, clinical and food samples. In many countries, fluoride is purposely added to the water supply (water fluoridation) as sodium fluoride (NaF) and to toothpastes in 0.1% concentration as sodium monofluorophosphate, Tin difluoride or sodium fluoride {Na2 3POF, SnF2, NaF. In topical fluoride agents are the main dental products used in caries prevention. Though a small amount of fluoride is beneficial, and has been used to treat osteoporosis, fluoride causes mottled teeth and bone damage at about 5mg L-1 when it is present in water. Studies have shown that bone cancer in male children and uterine cancer deaths are linked to water fluoridation due to fluoride’s gradual build up in the bones thereby causing adverse changes to the bone structure. Recent independent research has shown that fluoride build up in the brain of animals when exposed to moderate levels of fluoride. Two new epidemiological studies have also confirmed fluorides’ neurotoxic effects on the brain, as children exposed to higher levels of fluoride had lower IQs., showed that rats drinking 1ppm fluoride (NaF) in water had histologic lessions in their brain similar to Alzheimer’s disease and dementia. Fluoride has also been reported to cause birth defects and perinatal deaths, impaired immune system, acute adverse reactions, severe skeletal fluorosis at high levels, osteo- arthritis, acute poisoning and contributes to the development of repetitive stress injury. The determination of fluoride concentration in the various samples requires very sensitive methods. Electro analysis, spectra analysis, chromatography and miscellaneous methods with various adaptations have been employed in the analysis of fluoride. In many recent applications, ion-selective electrode (ISE) methods are replacing existing time consuming and expensive 34 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 analytical methods with resulting increases in efficiency and simplicity of measurement. They are cost-effective, and sufficiently sensitive, selective, accurate and precise. The fluoride selective electrode is a solid-state type electrode consisting of a lanthanum fluoride crystal sealed over the end of an inert plastic tube which contains an internal electrode and filling solution usually, 0.1M NaCl and 0.1M NaF. A potential arises because of the difference in fluoride activity on either side of the crystal. The ionic strength and the pH of sample and standard solutions should be matched when determining F- concentration using F- ISE. Ion-selective electrode (ISE) is a type of membrane electrode incorporates a special ion-sensitive membrane which may be glass, a crystalline inorganic material or an organic ion-exchanger. The membrane interacts specifically with the ion of choice, in our case fluoride, allowing the electrical potential of the half cell to be controlled predominantly by the F- concentration. The potential of the ISE is measured against a suitable reference electrode using an electrometer or pH meter. The electrode potential is related to the logarithm of the concentration of the measured ion by the Nernst equation. If the measurements are made with very little current flowing in the cell, the reference electrode potentials are fixed, and if the sample solution is essentially the same matrix for all measurements the junction potentials are also unchanged. Then the measured cell potential can be expressed as ‫ܧ‬௠௘௔௦ ൌ ‫ ܭ‬െ ͲǤͲͷͻͳ͸ ݈‫݃݋‬ ௔೔೚೙೔೙೙೐ೝ ௔೔೚೙೚ೠ೟೐ೝ Where K is a constant and ‘’a’’ is the activity of the analyte ion. The ISE filling solution contains a large concentration (activity) of the analyte ion and is essentially unchanged during operation of the electrode (aion inner is fixed). Thus, at 25 Ԩ ‫ܧ‬௠௘௔௦ ൌ ‫ ܭ‬൅ ͲǤͲͷͻͳ͸ ݈‫ܽ ݃݋‬௜௢௡௢௨௧௘௥ For fluoride ion solutions at 25oC and constant ionic strength, E meas K  0.05916 log[ F  ] Thus, for an ideal fluoride ISE, the cell potential is linearly related to the logarithm of the fluoride ion con centration and should increase 59.16 mV for every 10-fold decrease in the [F-]. When the ionic strength of all standards and samples is constant, the response of a real fluoride ISE is described by a similar relationship E meas K  E (0.05916 ) log[ F  ] where β is the electromotive efficiency and typically has a value very close to unity (> 0.98) 35 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Direct Potentiometric Measurement To check if the electrode is working properly, you will measure the cell potential of three fluoride standards prepared in a Total Ionic Strength Adjustment Buffer (TISAB). The TISAB contains an acetic acid/acetate buffer that fixes the pH of the solution at about 5. At this pH the formation of HF is negligible and the concentration of OH-, the only other anion that the electrode responds to is insignificant. It also contains NaCl to establish a high and constant ionic strength, and a complexing agent that removes cations that could interfere by forming complexes with fluoride. From a linear least-squares fit to a plot of Emeas versus log [F-] you can obtain the slope [S = β(0.05916)]. Typically S equals 56 ± 2 mV. Method of Standard Addition The method of variable volume standard addition will be used to determine the fluoride content of an unknown solution. In this approach, a solution containing fluoride will be mixed with the TISAB and the potential will be measured. Then successive amounts of a fluoride standard solution will be added and the potential will be measured after each addition. The following describes how the unknown fluoride concentration can be obtained from these measurements. The measured potential (E) can be represented by E K  S log C Where K is a constant; S: is the slope of the calibration curve and equals β (0.05916) and C: is the analyte ion (F-) concentration The equation can be rearranged to give C 10 E K S The analyte ion concentration after any addition of the standard is given by C CoVo  C std Vstd Vo V std Where C0 is the analyte concentration before any standard is added; V0 : is the volume of the solution before any standard is added; Cstd: is the concentration of the standard solution; Vstd: is the volume of standard solution that is added. 36 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Substituting this expression for C in the previous equation gives CoVo  Cstd Vstd Vo V std 10 E / S 10 K / S This equation can be rearranged to give 10 E / S (Vo  Vstd ) 10 K / S CoVo 10 K / S C std Vstd A plot of 10E/S (Vo + Vstd) versus CstdVstd will give a linear plot with an x-intercept (y = 0) equal to the negative of the amount (μg) of analyte in the solution before addition of the standard. The analyte concentration (μg/mL) in the original unknown solution (Cunk) can then be determined by dividing by the volume of the unknown fluoride solution (Cunk). Procedure Preparation of Fluoride Standard Solutions By serial dilution of the 1000 μg/mL fluoride standard solution, prepare 50 mL each of 200, 20 and 2 μg/mL fluoride standards in 50-mL volumetric flasks. After thorough mixing, transfer each diluted standard solution to a labeled plastic reagent bottle for storage. Calculate the concentration of each diluted standard using the exact concentration of the stock solution. If you do not have fluoride standard solution in your lab, you can prepare it from solid NaF dried at 100 Ԩ for hour . Calibration of Electrode 1. Carefully pipette 25.0 mL of the most dilute fluoride standard into a 50-mL volumetric flask and dilute to the mark with the TISAB. Stopper the flask and thoroughly mix the solution. 2. Transfer this solution to a 100 mL plastic beaker. Place the beaker on a stirring plate, add a magnetic stirring bar and begin stirring at a constant rate. 3. Connect the fluoride ISE to a pH meter and set the meter to the mV mode. Rinse the electrode with deionized water and blot dry. 4. Lower the electrode into the standard solution and when the reading is stable record the mV value. 37 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 5. Repeat steps 1-4 for each of the remaining fluoride standards. 6. Estimate of the slope (S) from the difference in the mV readings for each factor of 10 of increases in the fluoride ion concentration. If your value is outside the expected range, consult your lab instructor. Analysis of Unknown 1. Accurately weigh about 0.2 g of toothpaste into a 100 mL beaker. Add 10 mL of 1N KCl and about 40 mL of water to the beaker. 2. Boil the mixture gently for 3-5 minutes, breaking up the toothpaste with a stirring rod if necessary. 3. Cool the solution, quantitatively transfer the liquid to a 100 mL volumetric flask and dilute to volume with KCl 4. Prepare a 500 μg/mL fluoride standard by pipeting 5.0 mL of the 1000 μg/mL fluoride standard solution into a 10 mL volumetric flask and diluting to the mark with the TISAB. 5. Carefully pipette 50.0 mL of prepared toothpaste which contains the TISAB at the same concentration as used for the standard calibrations into a 100 mL plastic beaker. Place the beaker on a stirring plate, add a magnetic stirring bar and begin stirring at a constant rate. 6. Rinse the ISE with deionized water and blot dry. 7. Lower the electrode into the unknown solution; when the reading is stable record the mV value. 8. Pipet 1.0 mL of the 500 μg/mL F- standard solution into the unknown solution and record the mV value when the reading is stable. 9. Make three additional 1.0 mL additions of the standard solution and record the mV reading after each addition as before. 10. When finished, rinse the ISE with deionized water and place it in the storage container. Calculations a. Determination of Calibration Slope 1. Using EXCEL, plot the mV reading for the diluted fluoride standards versus the log of the actual fluoride ion concentration. 2. Fit the data points with a linear least-squares line and from the equation for the line obtain the slope (S). 38 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 b. Determination of Unknown Concentration by Standard Addition 1. Using the slope determined in ‘’a’’, plot 10E/S (V0+Vstd) versus CstdVstd. Remember to include the initial reading with no added standard. 2. Fit the data points with a linear least-squares line and obtain the equation for the line. 3. Use the equation for the line to determine the x-intercept and from this calculate the fluoride ion concentration in the unknown solution. Report the fluoride ion concentration (μg/mL) in the unknown solution. Questions 1. Why is the calibration plotted in log concentration? 2. Explain how to determine the concentration of fluoride by ion selective electrode? 3. What is the importance of TISAB in this analysis? Comment on your results and the technique in general? 39 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 EXPERIMENT-6 Analysis of Turbidity, Colour, pH, and Alkalinity of Water Objective: To perform alkalinity, pH, turbidity and colour analysis on a given set of water samples Introduction Turbidity is caused by suspended materials which absorb and scatter light. These colloidal and finely dispersed turbidity-causing materials do not settle under quiescent conditions and are difficult to remove by sedimentation. Turbidity is a key parameter in water supply engineering, because turbidity will both cause water to be aesthetically unpleasant and cause problems in water treatment processes, such as filtration and disinfection. Turbidity is also often used as indicative evidence of the possibility of bacteria being present. Turbidity measurements performed using proprietary nephelometric instruments are expressed as Nephelometric Turbidity Units (NTU). The nephelometric apparatus is designed to measure o forward scattering of light at 90 to the path of an incandescent light beam. Suspended particles present in a water sample reflect a portion of the incident light off the particle surface. The light o reflected at 90 is measured by a photoelectric detector and is compared against light reflected by a reference standard. Many surface waters are coloured, due primarily to decomposition of organics, metallic salts or coloured clays. This colour is considered as "apparent colour" as it is seen in the presence of suspended matter, whereas "true colour" is derived only from dissolved inorganic and organic matters. Samples can be centrifuged and/or filtered to remove turbidity in order to measure true colour. Waters which obtain their colour from natural organic matter usually pose no health hazard. However, because of the yellowish brown appearance of such waters, the consumers may not find the water aesthetically acceptable. Consumers of highly coloured but already properly treated water may not believe the water is in fact properly treated. Many processing industries 40 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 require low coloured water. PUB requires drinking water to meet the "highest desirable" World Health Organisation (WHO) standards of less than 5 colour units. One standard colour unit is defined as a 1 mg/L concentration of platinum in the form of potassium chloroplatinate K PtCl . Measurement of colour is by comparison of the sample with standard 2 6 colour solutions using a spectrophotometer. A straight line calibration curve is initially developed by plotting absorbance versus platinum-cobalt colour standard. In practice, the absorbance of a sample is determined and corresponding concentration is read off the calibrated curve. When measuring true colour, pre-treatment has to be carried out to remove turbidity. Unfortunately, either centrifugation or filtration has some effect on true colour. So when reporting the true colour value, specify the details of the pre-treatment method and its operating conditions. Likewise, the colour value of water is extremely pH dependent, too, and invariably increases as the pH of the water is raised. When reporting a colour value, specify the pH at which colour is determined. pH is a way of expressing the hydrogen-ion concentration of a solution. As acids and bases in + - solution dissociate to yield hydrogen ions [H ] and hydroxyl ions [OH ] respectively, pH is used to indicate the intensity of the acidic or alkaline condition of a solution. Alkalinity is a measure of the acid-neutralizing capacity of dissolved substances in water and equals the amount of strong acid required to lower the solution from initial pH to about 4.5. Many materials may contribute to the alkalinity of water. For most practical purposes, it is due primarily to presence of salts of weak acids (mainly bicarbonate and carbonate) and hydroxide (at high pH). pH and alkalinity are key water quality parameters in environmental engineering practice. In the water supply and treatment fields, these parameters have great influence on the chemical coagulation, disinfection and softening processes, and corrosion control for water distribution pipe networks. Effective chemical coagulation of water, for instance, occurs only within a specific pH range. Chemicals used for coagulation release, as a by-product of their reactions with water to form insoluble hydroxide precipitates, hydrogen ions (acid-causing). If unchecked, these hydrogen ions could lower the pH of the water sufficiently to render the coagulants ineffective. The presence of sufficient amount of alkalinity in the water can react and remove the hydrogen ions released by the coagulants, thus buffering the water in the pH range where the coagulant can be effective. In 41 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 pure water, water molecules dissociate into equal amounts of hydrogen and hydroxyl ions (10 -7 moles/L). From the law of mass action, it can be shown that, for pure water at about 25°C: [OH-] [H+] = Kw = 10 -14 The pH value of a solution has been defined to be the negative log of the hydrogen ion concentration: pH = log [H+] The pH scale runs from 0 to 14, with pH 7 representing neutrality. Acid conditions increase as pH values decrease, and alkaline (base) conditions increase as the pH values increase. Measurement of the hydrogen ion concentration is made by pH meters via a glass electrode and a calomel reference electrode. The alkalinity of water is its quantitative capacity to neutralize acids. The three major forms of alkalinity ranked in order of their association with high pH values are - (1) hydroxide alkalinity, [OH ], 2- (2) carbonate alkalinity, [CO ], and 3 - (3) bicarbonate alkalinity [HCO ] 3 + Their ability to react with H ends at pH 4.5 when both have turned into carbonic acid (H CO ). In 2 3 nature, bicarbonates are the major form of alkalinity because they result from the reactions of CO2 on calcium and magnesium rocks. Some CaCO3 (up to about 20 mg/L) may also go into solution 2+ 2- as Ca and CO3 ions. For all practical purposes, alkalinity due to other sources in natural waters may be ignored. Alkalinity of waters is measured by means of titration with a standard solution of a strong acid (usually H2SO4) to designated pHs, and is reported in terms of equivalent CaCO3. Alkalinity depends on the end-point pH or indicator used. Either titration curve technique or colour indicators can be used for the determination. The alkalinity measurement is based on the titration curve for a hydroxide-carbonate-bicarbonate mixture, as shown in figure below. 42 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Titration curve for a hydroxide-carbonate-bicarbonate mixture For samples whose initial pH is above 8.3, the titration is made in two steps. In the first step, the titration is conducted until the phenolphthalein indicator end-point is reached (i.e. pH of about 8.3) with a colour change from pink to colourless. During this first phase, the acid added to the sample - 2- reacts with [OH ] alkalinity, if present, and [CO ] alkalinity as follows: 3 OH- + H+ → H2O CO32- + H+ → HCO3In the second phase, the titration is continued until the methyl orange indicator end-point is reached with a colour change from yellow to red (i.e. pH of about 4.5). During this phase, the addition of - acid changes the HCO ions, initially present as well as those produced by Reaction (1-4), into 3 carbonic acid; HCO3- + H+ → H2CO3 43 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 In the above titration, the result of first step is known as "phenolphthalein alkalinity" and the overall titration is known as "total alkalinity" (the amount of acid required to react with all the hydroxide, carbonates and bicarbonates in the sample) respectively. When the pH of a sample is less than 8.3, a single titration is made to the methyl orange end point. Also for routine work, it is common that only the total alkalinity is determined. It is possible to determine the various components of alkalinity (i.e. hydroxide, carbonate and bicarbonate fractions) from a combination of titration, pH measurements and chemical equilibrium equations. An understanding of the buffering capacity of alkalinity can be derived from an evaluation of figure above. At the inflection points of pH 8.3 (phenolphthalein alkalinity) and pH 4.5 (total alkalinity), the carbonate system will react with a considerable pH change when only a small fraction of titrant is added. However, at the points where only half of the initial carbonate has been converted to bicarbonate and only half of the resultant bicarbonate has been converted to carbonic acid, considerably more titrant is required to effect a pH change. It is during these conditions that the buffering capacity is exhibited. Quantifying the alkalinity to the inflection points is a measure of this buffering capacity. Material and Instrument ¾ Turbidimeter ¾ UV visible spectroscopy ¾ Beaker ¾ pH meter ¾ Magnetic stirrer ¾ Erlenmeyer flask Chemical and Reagent ¾ Methyl orange indicator ¾ Sulphuric acid Sampling Surface water and Tap water collect from around your campus. 44 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Determination of Turbidity 1. Select the operating range at ”AUTO” mode of the Turbidimeter. 2. Fill a clean sample cell to the mark with the test sample and place it in the cell holder. The sample cell must be clean, dry and free of fingerprints. Wipe the outside of the cell with a lens tissue and align the dot on the sample cell with the raised mark on the spill ring around the cell holder opening. Be sure the cell is kept down completely and held in place by the spring clip. Cover the sample with the light shield. 3. The digital readout is in Nephelometric Turbidity Units (NTU). Determination of Colour 1. Place the cell containing the blank (distilled water) in the sample compartment with the transparent sides facing the light source of UV visible spectroscopy. Close the sample compartment lid. (Note: Do not touch the transparent sides of the sample cell and keep it clean). 2. Press “AUTOZERO” key to set the zero absorbance. 3. Discard the distilled water and place the cell containing the sample in the measuring position. Close the sample compartment lid. 4. Press “START” key to measure colour. 5. Record the “Conc” as colour units for the sample Determination of pH 1. Calibrate the pH meter according to instructions supplied by the Lab instructor. 2. Pour sample into a clean beaker. 3. Rinse the probe thoroughly with distilled water to prevent any carry-over. Switch to pH mode. 4. Immerse the probe in the sample. 5. Establish equilibrium between probe and sample by stirring to insure homogeneity. Gently drop a stirring bar into the sample and place the beaker on a magnetic stirrer. Start the magnetic stirrer and adjust the speed to give thorough but gentle mixing. 6. Read and record the pH. 7. Rinse the electrode thoroughly with distilled water. 8. When not in use, the electrode should be replaced in the beaker containing water. 45 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Determination of Alkalinity (Total Alkalinity) 1. For each sample, place 100 mL of sample in an Erlenmeyer flask. 2. Add 3 drops of methyl orange indicator solution to the flask. 3. Titrate sample with 0.02 N H2SO4 (sulphuric acid), constantly swirling the flask content above a white surface until just after the colour of the flask content change from yellow to red. 4. Record the volume of titrant used. 5. Calculate Total Alkalinity as follows: Total alkalinity as mg/L CaCO3 = ‫ۯ‬ൈ‫ۼ‬ൈ૚૙૙૙ൈ૞૙ ‫܍ܔܘܕ܉ܛ܎ܗۺܕ‬ where: A = volume of 0.02 N H SO used for methyl-orange end point. 2 4 N = Normality of H SO , 0.02 N. 2 4 Data and Calculation Sample Turbidity Apparent (NTU) colour True colour pH Alkalinity (mg/L CaCO3) Tap water Surface water Calculate the total alkalinity of the tap and surface water sample? Questions 1. Why you rinse the electrode in the glass beaker? 2. From your results, are there any observable relationships between turbidity and apparent colour and between turbidity and true colour? 3. What form of alkalinity would you expect to predominate in Tap and Surface waters? 46 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 EXPERIMENT-7 Determination of Permanent Hardness due to Ca2+ and Mg2+ in Tap Water by EDTA Method Objective: To determine the level of permanent hardness of tap water by EDTA Method Introduction Hard waters are generally considered to be those waters that require considerable amounts of soap to produce foam and that also produce scale in water pipes, heaters, boilers and other units in which the temperature of water is increased. Hard water is appropriate for human consumption similar to that as soft waters, however it produces adverse actions with soap and thus their use for cleaning purposes is unsatisfactory and thus their removal from water is required. Hardness of waters varies from place to place. In general, surface waters are softer than ground waters. Waters are commonly classified based on degree of hardness: Classification of hardness types Hardness (mg/L) Degree of hardness 0-75 Soft 75-100 Moderately hard 150-300 Hard >300 Very hard Hardness: Hardness is caused by polyvalent metallic cations, though the divalent cations, such as calcium and magnesium cations are usually the predominant cause of hardness. In addition, hardness is also caused by Ca2+ and Mg2+ ions. For example, when hard water is heated, Ca2+ ions react with bicarbonate (HCO3-) ions to form insoluble calcium carbonate (CaCO3) (Eq. 1). This precipitate, known as scale, coats the vessels in which the water is heated, producing the mineral deposits on your cooking dishes. Equation 2 presents magnesium hardness. Ca2+ (aq) + 2HCO3-(aq) → CaCO3(s) +H2O +CO2 Mg2+(aq) + 2OH-(aq) → Mg(OH)2 (s) 1a 1b 47 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Total hardness is defined as the sum of the calcium and magnesium concentrations, both expressed as calcium carbonate in mg/L. When hardness (numerically) is greater than the sum of carbonate and bicarbonate alkalinity, amount of hardness equivalent to the total alkalinity is called “Carbonate hardness”. Carbonate hardness (mg/L) = Alkalinity (2a) When alkalinity > Total hardness: Carbonate hardness (mg/L) = Total hardness (2b) The amount of hardness in excess of this is called “Non-carbonate hardness (NCH)”. These are associated with sulfate chloride, and nitrate ions. Temporary hardness is due to the presence of bicarbonates of calcium and magnesium ions. It can be easily removed by boiling. When water is boiled, temporary hardness producing substances (bicarbonates) are precipitated as insoluble carbonates or hydroxides. This precipitate can be removed by filtration. However, Permanent hardness is due to the presence of chlorides and sulphates of calcium and magnesium ions. This type of hardness cannot be removed by boiling. The filtrate obtained contains permanent hardness producing substances and is estimated against EDTA using EBT indicator. The estimation of hardness is based on complexometric titration, is used to find the total calcium and magnesium content of milk, sea water and various solid materials. It can also be used to determine the total hardness of fresh water provided the solutions used are diluted. The combined concentration of calcium and magnesium ions is considered to be the measure of water hardness. The method uses a very large molecule called EDTA which forms a complex with calcium and magnesium ions. EDTA is short for ethylenediaminetetraacetic acid. A blue dye called Eriochrome Black T (ErioT) is used as the indicator. This blue dye also forms a complex with the calcium and magnesium ions, changing colour from blue to pink in the process. The dye–metal ion complex is less stable than the EDTA–metal ion complex. For the titration, the sample solution containing the calcium and magnesium ions is reacted with an excess of EDTA. The indicator is added and remains blue as all the Ca2+ and Mg2+ ions present are complexed with the EDTA. 48 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 A back titration is carried out using a solution of magnesium chloride. This forms a complex with the excess EDTA molecules until the end-point, when all the excess EDTA has been complexed. The remaining magnesium ions of the magnesium chloride solution then start to complex with ErioT indicator, immediately changing its colour from blue to pink. Estimation of hardness by EDTA method is based on the principle that EDTA forms complexes with hardness causing metal ions in water. The complexes are stable within pH range of 8 to 10. Thus, to maintain the pH range buffer solution (NH4Cl and NH4OH mixture) is used. Eriochrome Black-T (EBT) indicator is used to indicate the completion of complexation reaction. ଶ ‫ܶܤܧܽܥ‬ ‫ ܽܥ‬ା ൨ ܿ‫ݔ݈݁݌݉݋‬ ቈ ଶ ቉ ൅ ‫ ܶܤܧ‬՜ ൤‫݃ܯ‬ ‫݃ܯ‬ା unstable complex When this solution is titrated against EDTA, it replaces the indicator from the indicator complex. When all the hardness causing ions are complexed by EDTA, the indicator is set free and end point is marked by color change from purple red to blue. The total hardness is thus determined. ªCa EBT º ªCa EDTA º « Mg » complex  EDTA o « Mg »  EBT ¬ ¼ ¬ ¼ The formed complex is blue in color. The temporary hardness is removed by boiling and then precipitate formed is removed by filtration and the permanent hardness in filtrate is determined by titration with EDTA. Temporary hardness = total hardness - permanent hardness The most common multivalent metal ions in natural waters are Ca2+ and Mg2+. In this experiment, we will find the total concentration of metal ions that can react with EDTA and we will assume that this equals the concentration of Ca2+ and Mg2+. In a second experiment, Ca2+ is analyzed separately after precipitating Mg (OH)2 with strong base NaOH. Material and Instrument ¾ Desiccator ¾ Volumetric flask ¾ Burette ¾ Pipette 49 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Reagents Required ¾ EDTA: Na2H2EDTA. 2H2O ¾ Buffer (pH 10): Add 142 mL of 28 % aqueous NH3 to 17.5 g of NH4Cl and dilute to 250 mL with distilled water. ¾ Eriochrome black T indicator: Dissolve 0.2 g of the solid indicator in 15 mL of triethanolamine plus 5 mL of absolute ethanol. (Alternatively, Calmagite could be used by dissolving 0.05 g in 100 mL of water. The color changes are the same for both indicators) ¾ Hydroxynaphthol blue indicator ¾ 50 % (w/w) NaOH: Dissolve 100 g of NaOH in 100 g of H2O in a 250-mL plastic bottle. Store tightly capped. When you remove solution with a pipette, try not to disturb the solid Na2CO3 precipitate. ¾ Unknowns: Collect water from streams or lakes. To minimize bacterial growth, plastic jugs should be filled to the top and tightly sealed. Refrigeration is recommended Procedure 1. Dry Na2H2EDTA .2H2O at 80 Ԩ for 1 hour and cool in the desiccator. Accurately weigh out ~0.6 g and dissolve it with heating in 400 mL of water in a 500-mL volumetric flask. Cool to room temperature, dilute to the mark, and mix well. 2. Pipet a 50.00-mL sample of tap water into a 250-mL flask. 3. To each sample, add 3 mL of pH 10 buffer and 6 drops of Eriochrome black T indicator 4. Titrate with EDTA from a 50-mL burette and note when the color changes from wine red to blue 5. Repeat the titration with three samples to find an accurate value of the total Ca2+ + Mg2+ concentration. 6. Perform a blank titration with 50 mL of distilled water and subtract the value of the blank from each result. ¾ Let V1 mL volume of EDTA consumed during titration and let V be the volume of tap water taken. Thus, total hardness of the sample = V 1 x 1000 ppm of CaCO3 equivalent V ¾ For the determination of permanent hardness due to Ca2+, pipette out the same volume of unknown sample as previous into clean flasks 50 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 7. Add 30 drops of 50% (w/v) NaOH to each solution and swirl for 2 minutes to precipitate Mg(OH)2 (which may not be visible). Add~0.1 g of solid hydroxynaphthol blue to each flask. This indicator is used because it remains blue at higher pH than does Eriochrome black T. 8. Collect the filtrate into volumetric flask and titrate in the same way as above. After reaching the blue end point, allow the sample to stand for 5 min with occasional swirling so that any Ca(OH)2 precipitate may redissolve. Then titrate back to the blue end point if the blue color turns to red upon standing. 9. Perform a blank titration with 50 mL of distilled water. Calculate the permanent hardness of water due to calcium as = ௏ൈேൈହ଴ൈଵ଴଴଴଴ ௩௢௟௨௠௘௢௙௦௔௠௣௟௘௧௔௞௘௡ CaCO3 equivalent. Where V = volume of EDTA consumed during the titration N= normality of EDTA Permanent hardness due to Mg+2 = Total hardness - permanent hardness due to Ca+2 N.B. In this experiment temporary hardness is assumed to be negligent Calculation Calculate the total and permanent hardness of the water sample in ppm of CaCO3? Questions 1. Why is hardness of water expressed in terms of calcium carbonate equivalent? 2. Mention the disadvantages of hard water for industrial purpose. 3. Why is the colour of solution wine red before titration and blue colour at the end of titration? 4. State the salts responsible for temporary and permanent hardness of water? 5. Why is ammonium hydroxide-ammonium chloride buffer added during the determination of hardness of water? 51 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 EXPERIMENT-8 Determination of Chemical Oxygen Demand (COD) of Wastewater Using Open Reflux Method Objective: To determine the COD of Wastewater Introduction Water pollution and its impacts on the environment are serious issues for present world. To limit the water pollution and improve the water quality, advanced wastewater treatment technologies are invented. These technologies are implemented by removing physical, chemical and biological contaminants from wastewater and producing an environmentally safe fluid waste stream (treated effluent) and a solid waste (treated sludge). It may then even be possible to reuse sewage effluent for drinking water with the help of more advanced technologies. If untreated wastewater containing contamination enters into the surface and ground water resources, it leads to a serious environmental and human health risk. To minimize the potential risks from untreated wastewater entering freshwater resources, industrial wastewater plants go through a water quality assessment by monitoring some parameters. Water quality professionals assess water quality by measuring the concentrations of these parameters and comparing with their standards. Some of the unique analytical parameters of the water pollution control industry are biochemical oxygen demand, chemical oxygen demand, taste, odor, color, chlorine demand, hardness, alkalinity and biodegradability tests. Finding excessive levels of one or more of these parameters can serve as an early warning of potential pollution problems. One of these parameters are COD and BOD that indicate the amount of organic pollution and water degradation. COD is defined as the amount of oxygen equivalents consumed in oxidizing the organic compounds of samples by strong oxidizing agents such as dichromate or permanganate. It is expressed in milligrams per liter (mg/L) that indicates the mass of oxygen consumed per liter of solution. The higher the chemical oxygen demand, the higher the amount of pollution in the water sample. COD is considered one of the most important quality control parameters of an effluent in wastewater treatment facility. COD values are used to monitor wastewaters before (influent) and after (effluent) treatment, and, therefore, their reliability is important to protect the environment 52 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 and to guarantee the economical sustainability of the treatment facility. COD measurements are commonly made on samples of wastewater treatment facility or of natural waters contaminated by domestic and industrial wastes. COD is measured as a standardized laboratory assay in which a closed water sample is incubated with a strong chemical oxidant under specific conditions of temperature and for a particular time. A commonly used oxidant in COD assays is potassium dichromate (K2Cr2O7) which is used in combination with boiling sulfuric acid (H2SO4). Chemical Oxygen Demand (COD) is rapidly measured parameters as a means of measuring organic strength for streams and polluted water bodies. The test can be related empirically to BOD, organic carbon or organic matter in samples from a specific source taking into account its limitations. The test is useful in studying performance evaluation of wastewater treatment plants and monitoring relatively polluted water bodies. COD determination has advantage over BOD determination. COD results can be obtained in 3-4 hrs as compared to 3-5 days required for BOD test. Further, the test is relatively easy, precise, and is unaffected by interferences as in the BOD test. The intrinsic limitation of the test lies in its inability to differentiate between the biologically oxidizable and biologically inert material and to find out the system rate constant of aerobic biological stabilization. The open reflux method is suitable for a wide range of wastes where a large sample size is preferred. The closed reflux methods are more economical in the use of metallic salt reagents and generate smaller quantities of hazardous waste, but require homogenization of samples containing suspended solids to obtain reproducible results. The dichromate reflux method is preferred over procedures using other oxidants (e.g. potassium permanganate) because of its superior oxidizing ability, applicability to a wide variety of samples and ease of manipulation. Oxidation of most organic compounds is up to 95-100% of the theoretical value. The organic matter gets oxidized completely by potassium dichromate (K2Cr2O7) with silver sulphate as catalyst in the presence of concentrated H SO to produce CO and H O. The excess K Cr O remaining after the reaction is 2 4 2 2 2 2 7 titrated with ferrous ammonium sulphate [Fe (NH ) (SO ) ]. The dichromate consumed gives the 4 2 4 2 oxygen (O ) required for oxidation of the organic matter. The chemical reactions involved in the 2 method are as under: 53 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 2K2Cr2O7 + 8 H2SO4 →2 K2 SO4 + 2Cr2 (SO4)3 + 8 H2O + 3O2 C H O + 6O → 6CO + 6H O 6 12 6 -2 2 2 +2 + 2 +3 3+ Cr2O7 + 6Fe + 14H → 6Fe + 2Cr + 7H2O Interferences Oxidation of most organic compounds is 95 to 100% of the theoretical value. Pyridine and related compounds resist oxidation and volatile organic compounds will react in proportion to their contact with the oxidant. Straight-chain aliphatic compounds are oxidized more effectively in the presence of a silver sulfate catalyst. The most common interferent is the chloride ion. Chloride reacts with silver ion to precipitate silver chloride, and thus inhibits the catalytic activity of silver. Bromide, iodide, and any other reagent that inactivates the silver ion can interfere similarly. Such interferences are negative in that they tend to restrict the oxidizing action of the dichromate ion itself. However, under the rigorous digestion procedures for COD analyses, chloride, bromide, or iodide can react with dichromate to produce the elemental form of the halogen and the chromic ion. Results then are in error on the high side. The difficulties caused by the presence of the chloride can be overcome largely, though not completely, by complexing with mercuric sulfate (HgSO4) before the refluxing procedure. Although 1 g HgSO4 is specified for 50 mL sample, a lesser amount may be used where sample chloride concentration is known to be less than 2000 mg/L, as long as a 10:1 weight ratio of HgSO4: Cl- is maintained. Do not use the test for samples containing more than 2000 mg Cl-/L. Halide interferences may be removed by precipitation with silver ion and filtration before digestion. This approach may introduce substantial errors due to the occlusion and carry down of COD matter from heterogenous samples. Ammonia and its derivatives, in the waste or generated from nitrogen-containing organic matter, are not oxidized. However, elemental chlorine reacts with these compounds. Hence, corrections for chloride interferences are difficult. Nitrite (NO2-) exerts a COD of 1.1 mg O2/mg NO2--N. Because concentrations of NO2- in waters rarely exceed 1 or 2 mg NO2--N/L, the interference is considered insignificant and usually is ignored. To eliminate a significant interference due to NO2-, add 10 mg sulfamic acid for each mg NO2--N present in the 54 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 sample volume used; add the same amount of sulfamic acid to the reflux vessel containing the distilled water blank. Reduced inorganic species such as ferrous iron, sulfide, manganous manganese, etc., are oxidized quantitatively under the test conditions. For samples containing significant levels of these species, stoichiometric oxidation can be assumed from known initial concentration of the interfering species and corrections can be made to the COD value obtained. Apparatus and equipment ¾ 250 or 500 mL Erlenmeyer flask with standard (24/40) tapered glass joints ¾ Friedrich’s reflux condenser (12 inch) with standard (24/40) tapered glass joints ¾ Electric hot plate ¾ Volumetric pipettes (10, 25, and 50 mL capacity) ¾ Burette, 50 mL with 0.1 mL accuracy ¾ Analytical balance, accuracy 0.001g ¾ Volumetric flasks (1000 mL capacity) ¾ Boiling beads ¾ Magnetic stirrer and stirring bars. Reagents and standards a. Standard potassium dichromate solution, 0.25 N (0.04167 M): Dissolve 12.259g K Cr O dried at 2 2 7 103 °C for 24 h in distilled water and dilute to 1000 mL. Add about 120 mg sulphamic acids to take care of 6 mg/L NO2-N. b. Sulphuric acid reagent: Add 10 g of Ag SO to 1000 mL concentrated H SO and let stand for one 2 4 2 4 to two days for complete dissolution. c. Standard ferrous ammonium sulphate approx. 0.25 N (0.25 M): Dissolve 98 g Fe (NH4)2(SO4)2.6H2O in about 400 mL distilled water. Add 20 mL concentrated H SO and dilute to 1000 mL. 2 4 d. Ferroin indicator: Dissolve 1.485 g 1, 10-phenanthroline monohydrate and 695 mg FeSO .7H O in 4 2 distilled water and dilute to 100 mL. 55 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 e. Mercuric sulphates: HgSO4 crystals analytical grade f. Potassium hydrogen phthalate (KHP) Standard: Dissolve 425 mg lightly crushed dried potassium hydrogen phthalate (HOOC. C6H4.COOK) in distilled water and dilute to 1000 mL. This solution has a theoretical COD of 500 μg O2/mL. This solution is stable when refrigerated, up to 3 months in the absence of visible biological growth. Sample collection, preservation and Sample preparation Preferably collect wastewater/tap water in glass bottles. Remove settleable solids by sedimentation or decantation. If there is delay between collection and analysis, preserve sample by acidification to pH≤2 using concentrated H2SO4. Samples can be preserved for maximum 7 days. All samples high in solids should be blended for 2 minutes at high speed and stirred when an aliquot is taken for analysis. Select the appropriate volume of sample based on expected COD range, e.g. for COD range of 50-500 mg/L take 25-50 mL of sample. Sample volume less than 25 mL should not be pipetted directly, but serially diluted and then a portion of the diluted sample taken. Dilution factor should be incorporated in calculations. a) 500 mL of sample diluted to 1000 mL = 0.5 mL sample/mL of diluent, 50 mL = 25 mL of sample. b) 100 mL of sample diluted to 1,000 mL = 0.1 mL sample/mL diluent, 50 mL of diluent = 5 mL of sample Calibration Since the procedure involves chemical of organic matter by potassium dichromate as oxidizing agent which is a primary standard, calibration is not applicable. For standardization of ferrous ammonium sulphate, dilute 10 mL standard K Cr O to about 100 mL. Add 10 mL concentration 2 2 7 of H SO and allow it to cool. Titrate with ferrous ammonium sulphate (FAS) to be standardized 2 4 using 2-3 drops of ferroin indicator. Calculate normally Normality of FAS = mL of K 2 CrO7 mL of FAS required The deterioration of FAS can be decreased if it is stored in a dark bottle. 56 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Procedure 1. Place 0.4 g HgSO4 in a 250 mL reflux sample 2. Add 20 mL sample or an aliquot of sample diluted to 20 mL with distilled water. Mix well 3. Add clean pumic stones or glass beads. 4. Add 10 mL 0.25 N (0.04167M) K2Cr2O7 solution and mix. 5. Add slowly 30 mL concentrated H SO containing Ag SO mixing thoroughly. This slow 2 4 2 4 addition along with swirling prevents fatty acids to escape due to generation of high temperature. Alternatively attach flask to condenser with water flowing and then add H SO slowly through condenser to avoid escape of volatile organic substance due to 2 4 generation of heat 6. Mix well. If the color turns green, either take fresh sample with lesser aliquot or add more potassium dichromate and acid. 7. Connect the flask to condenser. Mix the contents before heating. Improper mixing will result in bumping and blow out of flask content. 8. Reflux for a minimum of 2 hours. Cool and then wash down condenser with distilled water. 9. Disconnect reflux condenser and dilute the mixture to about twice its volume with distilled water. Cool to room temperature and titrate excess K2Cr2O7 with0.1M FAS using 2-3 drops of ferroin indicator. The sharp color change from blue green to reddish brown indicates end-point or completion of the titration. After a small time, gap, the blue-green color may reappear. Use the same quantity of ferroin indicator for all titrations. 10. Reflux blank in the same manner using distilled water instead of sample. Alternate procedure for low COD samples less than 50 mg/L: Follow similar procedure with two exceptions (use standard 0.025 N (0.004167 M) K2Cr2O7 and titrate with standardize 0.025 M FAS. The sample volume should be 5 mL. Exercise extreme care with this procedure because even a trace of organic matter on the glassware or from the atmosphere may cause gross errors. Compute amount of HgSO4 to be added based on chloride concentrations. Carry blank reagent through the same procedure. 57 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Calculations COD as mg/L = Where ሺࢇെ࢈ሻൈࡺൈૡ૙૙૙ ࢂ࢕࢒࢛࢓ࢋ࢕ࢌ࢙ࢇ࢓࢖࢒ࢋሺ࢓ࡸሻ a = Volume of FAS used for blank b = Volume FAS used for sample N = normality of FAS 8000 = Milieq. Wt. of O x 1000 2 Questions 1. What is the significant of COD? 2. What is the colour change at the end point in the determination of COD? 3. Why is the blank titre value higher than sample titre value? 4. Why dil. H2SO4 is used to dissolve FAS crystals while preparing standard solution? 58 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 EXPERIMENT-9 Soil Sample Collection and Preparation for Heavy Metal Analysis Objectives: To Familiarize Students with Soil Sample Collection and Preparation for Further Analysis Introduction THE SOIL SYSTEM Soil is defined as “the unconsolidated mineral material on the immediate surface of the earth that has been subjected to and influenced by genetic and environmental factors. The true soil component can also be defined as all mineral and naturally occurring organic materials with a particle size less than 2 mm. The physical and chemical characteristics of the soil system influence the transformation, retention, and movement of pollutants through the soil. Clay content, organic matter content, texture, permeability, pH and cation exchange capacity will influence the rate of migration and form of the chemical found in leachate migrating from the waste. Elevated concentrations of heavy metals in soils are of potential long term environmental and health concerns because of their persistence and cumulative tendency in the environment, and their associated toxicity to biological organisms. These factors must be considered by the investigator when designing a soil sampling plan. Furthermore, restricted use of contaminated lands and the costs of soil remediation also pose liabilities and financial burdens on landowners and other stakeholders. As a consequence, environmental assessment of lands with respect to heavy metal contamination, and identification its environmental and health implications have become increasingly important in environmental research. For a reliable and cost-effective investigation of heavy metal contamination of soils, a well-planned sampling strategy, appropriate selection of analytical methods, and careful interpretation of results are of vital importance. The soils are contaminated with heavy metals, analysis of the heavy metal concentrations of the soils will be adequate, and sampling of the soils will be relatively simple. However, if knowledge of the spatial distribution of heavy metals in soils is also sought, a systematic sampling approach will be required. 59 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Sampling Strategy Sampling is the process of obtaining representative sample which reliably represents the population under question both in composition and size. The sampling process must ensure that the items chosen are representative of the bulk of material or population. Sampling is inherent to any research program in science because the measurement of properties of the total population is impossible or difficult for any realistic study. However, it is clear that the larger the sample size the more closely your sample data will match the entire population. The goal of sampling is thus to produce a sample that is representative of the target population. The following sampling techniques are commonly in soil sampling. RANDOM SAMPLING The basis of most sampling plans in environmental sampling is the concept of random or probabilistic selection of the sample to be collected and the subsample that is to be analyzed. The random sampling strategy is the simplest methods, where soil samples are collected randomly and stochastically independently across the site of interest. It can be used as a quick sampling program of a pilot study. In random sampling of a site, each sample point within the site must have an equal probability of being selected. The same can be said for the selection of particles within a sample. Each and every particle within the sample must have an equal chance of being selected. Each particle that is not in the sample should have a zero probability of being selected. Properly designed sampling plans based upon the laws of probability provide a means of making decisions that have a sound basis and are not likely to be biased. On contrary, there are nonrandom samples collected for a particular reason. They are based solely on the choice of collector. Such samples are called purposive samples. A major disadvantage of this sampling strategy is that soil samples may not represent the whole study site. Therefore, this sampling strategy is usually employed in relatively homogenous sites and applicable to investigations where the major objective is to determine whether heavy metal concentrations of the soils are elevated above background and/or legislative standards. 60 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 STRATIFIED SAMPLING One of the tools of sampling that can be used to reduce the variability of the sample is stratified sampling. Strata are identified as regions of the site that are expected to be uniform in character. In the soil environment, strata are often associated with soil types or as areas of known pollution versus areas where pollutants are not expected to be present. Sampling points within the strata are selected systematically or by some random process. The stratified sampling method is based on the fact that the entire population might embrace certain number of distinct samples which would be divided into subpopulations. The subpopulations are called strata and the sums of strata constitute the total population. For stratified random sampling, each stratum is sampled independently and each individual in the subpopulation is randomly selected, i.e., a simple random sample is taken in each stratum. For this method, each subpopulation can be considered as independent and thus each stratum and thus one is able to get inferences from each of them. Correctly applied stratified sampling is likely to give a better result than simple random sampling, but four main requirements should be met before it is chosen: 1. Population must be stratified in advance of the sampling. 2. Classes must be exhaustive and mutually exclusive (i.e., all elements of the population must fall into exactly one class). 3. Classes must differ in the attribute or property under study; otherwise there is no gain in precision over simple random sampling. 4. Selection of items to represent each class (i.e., the sample drawn from each class) must be random SYSTEMATIC SAMPLING When the population can be ordered in some scheme and the samples are drawn at regular intervals through an ordered list, then the sampling method is called systematic sampling. In this method, the first sample is randomly selected and then the next samples drawn at regular fixed intervals till the end of the list. The systematic sampling plan is an attempt to provide better coverage of the soil study area than could be provided with the simple random sample or the stratified random sampling plan. The systematic sampling design is in reality stratification based upon spatial 61 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 distribution over the site. Systematic sampling collects samples in a regular pattern (usually a grid or line transect) over the areas under investigation. In a systematic sampling, soil samples are collected in a systematic manner, such as at a regular distance from each other across the study area, and some of the fixed sampling grids, including the bottle rack grid and the rectangular grid. The systematic sampling strategy is often employed in the geochemical mapping of heavy metals, since it enables detailed characterization of the spatial distribution of heavy metals in a large region Factor affecting Soil Sampling Factors that should also be considered during soil sampling include sampling density, sampling depth and the use of composite soil samples. In an ideal situation, the larger the number of soil samples collected, the better the sample population can reflect the conditions of the site. However, in reality, sampling density is often a compromise between representativeness of the site and the availability of resources. Sampling depth is determined based upon the purpose of the investigation and/or the specific requirements of a regulatory guideline. Also, in cases where heavy metal contamination of subsurface soils is suspected or groundwater contamination is a concern, sampling of soil profiles or subsurface soils may be necessary. The two common approaches are metric (depth-related) sampling and soil –horizon-related sampling. In general, the metric sampling approach is used for the purposes of screening analysis of potentially contaminated land. In more detailed environmental assessments, a horizon-related sampling approach is recommended. The use of composite soil samples offers the advantage of increased accuracy/representativeness through the use of large numbers of sampling units per sample. A composite soil sample is formed by combining equal portions of individual sub-samples. It is based on the fundamental assumption that analysis of the composite sample yields a valid estimate of the mean, which is obtained by averaging the results of analysis from each of the sampling units contributing to the composite. Ultimately, a suitable sampling strategy should maximize the representativeness of the study area with a minimal number of soil samples and resources to be utilized, while meeting the requirements of the investigation. It is suggested that, in a preliminary investigation, surface soil samples may be collected randomly or systematically at a low sampling 62 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 density. If contamination of subsurface soils is suspected, soil profiles may also be obtained. Analytical results of this preliminary assessment can provide an initial confirmation of the contamination. If signs of contamination are found, a more comprehensive systematic and strategical sampling program can be employed for the next stage of the study. In general, a sampling strategy should be tailored specifically to the intended study area to reflect the objectives and site conditions. Chemicals and Materials Materials ¾ Nylon sieve (0.2mm) ¾ glass bottles ¾ mortar and pestle ¾ hot plate ¾ refrigerator ¾ Whatman filter No.42 ¾ volumetric flask and spade Chemicals 20 % HNO3 (v/v), distilled and deionized water, HCl Procedure for Soil Sample Collection 1. Select a site in your campus or outside your campus and form hexagonal soil sampling grids of 2m edge length from a total of 8m2 of the sampling site. 2. Remove leaves, grass or any other solid material from each edge of the hexagon. 3. Then collect soil samples from each edge of the hexagon at depth of 0 cm, 5 cm, 10 cm and 20 cm using auger or spade. 4. Collect the soil sample from an edge of the hexagon into polyethylene bag and label it using tape 5. Don’t mix the samples from different edges rather collect them into different polyethylene bags 6. Bring the labeled samples to your laboratory and allow them to air dry 63 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 In this figure the arrows indicate the sampling point on the hexagonal grid Procedure for Soil Sample Preparation 1. Dry the collected soil sample at 60°C; sieve it through a 2.0-mm Nylon sieve to remove sand, gravel, and plant debris. 2. Store the sieved sample in glass bottles at room temperature. 3. Homogenously mix all the samples 4. Grind about 20 g of dried soil sample to fine powder by mortar and pestle 5. Sieve the powdered soil sample through a 0.2 mm Nylon sieve. 6. Take 0.5 g of the sample obtained in the above step and place it a reaction vessel 7. Moist the sample with 1mL of deionized water add a dilute mixture of 3.0 mL of HNO 3 and 9.0 HCl mL to the moistened soil sample 8. Heat the content of the vessel (glass beakers covered with watch glass) on hotplate to a maximum of 110 Ԩ for about 3 hours. Start heating from 50 Ԩ and continue by 10 Ԩ difference until the maximum is attained. Wait for ten minutes before proceeding to the next temperature. This gradual rise of temperature is required to avoid excessive release of due possible formation exothermic reaction. 64 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 9. Dilute the sample with 3 mL of 20 % nitric acid (v/v) after heating near to dryness and filter it with Whatman filter No. 42 if there is any residue 10. Transfer it to 100 mL volumetric flask and dilute with distilled and deionized water 11. Keep your sample in a refrigerator until you analyze it using FAAS Questions 1. Which sample strategy is best to get accurate result? 2. What is the most crucial steps of soil sample preparation for analysis? 65 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 EXPERIMENT-10 Determination of Manganese in Soil Sample Using FAAS Objective: To determine manganese from soil sample Introduction Manganese is regarded as essential for human nutrition because it is an activator and constituent of many enzymes present in humans but in small amount. It is an essential component of numerous enzymes involved in bone formation and in the metabolism of amino acids, lipids, and carbohydrates. Its deficiency in humans appears to be rare because manganese is present in much common food stuff; however, if it occurs in excess it can cause poor reproductive performance, growth retardation, abnormal formation of bone and cartilage, and an impaired glucose tolerance. Different symptoms and diseases such as headache, drowsiness, psychotic behavior and lesions are associated with the excessive intake of manganese. This metal is known to be present at trace levels in different environmental samples including water and soil bodies. This element participates in different oxido-reduction reactions along with other elements such as nitrogen, oxygen, sulfur and iron. Manganese (II) ion is the dissolved form of manganese found in water samples and mainly associated with water pollution. Thus, determination of manganese levels in soil and water sample has paramount importance. Therefore, the determination of manganese in environmental samples requires very sensitive techniques. A pre-concentration method is often required to remove matrix interferences, and enhance the sensitivity of instruments to allow determination of Mn (II) at trace concentrations. Different enrichment methods such as co-precipitation, liquid-liquid extraction, ultrasound assisted-dispersive liquid-liquid microextraction with the use of natural deep eutectic solvent, solid-phase extraction, In-syringe solvent-assisted dispersive solid phase extraction and cloud point extraction have been mentioned in the literature for the determination of trace elements. Unfortunately, these methods are sometimes unsatisfactory because of time limitation, high chemical consumption, waste generation, low pre-concentration factor and limited applicability. 66 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 However, heavy metals in soil, water or biological samples can be determined by means of atomic absorption spectrophotometry. This technique involves the study of the absorption of radiant energy in the UV and visible regions by neutral atoms in the gaseous state. In an atomic absorption analysis, the element being determined must be converted to the elemental state through a process called atomization; vaporized, and imposed in the beam of radiation from the light source. This process is most frequently accomplished by drawing a solution of the sample as a fine mist, into a suitable flame. The flame in flame atomic absorption spectroscopy provides energy for both excitation and atomization of the metal under investigation. The absorption is measured at a selected wavelength which is characteristic for each individual element. Flame atomic absorption spectrophotometry (FAAS) is a simple, rapid, accurate, and highly specific technique, with a high degree of freedom from interferences. The absorbance measured is proportional to the concentration of the analyte. A flame atomic absorption spectrophotometer consists essentially of the following components: ¾ A stable light source, emitting the sharp resonance line of the element to be determined. ¾ A flame system into which the sample solution may be aspirated at a steady rate, and which is of sufficient temperature to produce an atomic vapor of the required species from the compounds present in the solution. ¾ A monochromator to isolate the resonance line and focus it upon a photomultiplier. ¾ A photomultiplier to detect the intensity of light energy falling upon it, which is followed by facilities for simplification and read out. Schematic diagram of Double beam AAS 67 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Preparation of Standard Solutions Standard solutions of known metal concentrations should be prepared in deionized water with a matrix similar to that of the sample. Standards which bracket the expected sample concentration and are within the working range of the method should be employed. It is preferable to prepare stock solutions of standards in concentrations above 500 mg/L and store them in a refrigerator diluting as required. For samples containing high and variable concentrations of matrix materials, the diluted standard solutions should contain similar concentrations of the major ions in the sample. If the sample matrix is complex and components cannot be matched accurately with standards, the method of standard additions should be used to correct for matrix interference effects. Procedure for Preparation of Standard Solutions Pipette out 100 ml from 1000 mg/L standard stock solutions of manganese and transfer it to a one liter standard volumetric flask and dilute to the solution to the mark with deionized distilled water. This solution contains and 100 mg/L of Mn. From this solution prepare the following working standards using serial dilution. 0.04ppm, 0.08ppm, 0.16ppm, 0.55ppm, 1.00ppm, 2.00pppm, 3.00ppm Procedure for Instrument Operation Because of difference among different models of atomic absorption spectrophotometers, it is not possible to formulate instructions applicable to every instrument. The manufacturer's instructions for each particular instrument should be followed. In general, however, the following procedures applicable for FAAS with air-acetylene flame: (a) Install the hollow cathode lamp for the manganese and set wavelength to 279.5 nm (b) Set the slit width and lamp current at the values suggested in the manufacturer's instructions, and allow the instrument to warm up until the energy source becomes stable (10-20 minutes). Readjust the current as necessary after warm up. 68 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 (c) Optimize wavelength by adjusting wavelength dial until optimum energy gain is obtained. Align lamp in accordance with manufacturer's instructions, ask your technical assistant for the instrument manufacturer’s instructions. (d) Install air-acetylene burner and adjust burner head position. Turn on air and adjust flow rate to that specified by manufacturer to give maximum sensitivity for the metal being measured. Turn on acetylene, adjust flow rate to value specified, and ignite flame. (e) Aspirate each of the standard solution of the manganese and adjust aspiration rate of the nebulizer (if variable) and record the absorbance readings for all the standard solutions. (f) Aspirate your triplicate sample solutions into the instrument and note the absorbance values for all replicate sample solutions (g) On completion of the analysis, extinguish flame by turning off first acetylene and then air. Calculation Calculate the concentration of manganese ion from calibration curves obtained by plotting concentrations of the standard solutions versus the corresponding absorbance readings. Questions 1. What is the purpose of light source and flame in flame atomic absorption spectroscopy (FAAS) to analyze manganese metal? 2. Briefly discuss the principle of flame atomic absorption spectroscopy for heavy metal analysis? 69 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 EXPERIMENT-11 Determination of Soil Organic Matter by Walkley and Black Method Objective: To determine organic matter of soils Introduction Soil contains a large variety of organic materials ranging from simple sugars and carbohydrates to the more complex proteins, fats, waxes, and organic acids. Important characteristics of the organic matter include their ability to form water-soluble and water insoluble complexes with metal ions and hydrous oxides; interact with clay minerals and bind particles together; sorb and desorb both naturally-occurring and anthropogenically-introduced organic compounds; absorb and release plant nutrients; and hold water in the soil environment. As a result of these characteristics, the determination of total organic carbon is an essential part of any site characterization since its presence or absence can markedly influence how chemicals will react in the soil or sediment. Soil’s total organic carbon (TOC) determinations are typically requested with contaminant analyses as part of an ecological risk assessment data package. Soil is the largest pool of terrestrial organic carbon in the biosphere, storing more C than is contained in plants and the atmosphere combined. The abundance of organic C in the soil affects and is affected by plant production, and its role as a key control of soil fertility and agricultural production has been recognized for more than a century. The patterns and controls of soil organic carbon (SOC) storage are critical for our understanding of the biosphere, given the importance of SOC for ecosystem processes and the feedback of this pool to atmospheric composition and the rate of climate change. Our capacity to predict and ameliorate the consequences of climate and land cover change depends in part on a clear description of SOC distributions and the controls of SOC inputs and outputs. Soil organic matter (SOM) is the product of decayed plant and animal tissues, forest floor development, living and dead microbial tissues, microbially synthesized compounds, and derivatives of these materials, produced as the result of microbial decay. Decomposition processes play an important role in soil fertility in terms of nutrient cycling and the formation of soil organic 70 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 matter. Organic matter is an important constituent of every soil which increases the cation exchange capacity of the soil thus, the base saturation increases, the relative amount of acid cations decreases. For plant growth the nutrient storage capacity (high CEC) of the organic matter is important, especially in clay poor soils. In clay rich soils it enables the formation of aggregates with large pores and improves therefore water and air availability. Besides being essential in soil studies, the soil organic matter parameter is the most important one in several soil properties such as structure, coloring, water retention, and cation exchange capacity; as a result, it becomes important in guiding soil fertilizing and irrigation. Several methods have been used to determine soil organic carbon, and each of the methods has its own advantages and limitations. The most common analytical method for soil organic carbon determination is Walkley and Black (1934) method. This method is routine, relatively accurate, and popular method for the determination of soil organic matter in most soil laboratories. However, it requires a lot of reagents and chemicals including potassium dichromate which is toxic especially when it is in a hexavalent form. Nowadays, determination of soil organic carbon needs initial high cost investment. Recently, the cost of chemicals increases continuously. It is very important to be able to reduce the amount of chemicals used during the analysis of organic carbon and it is also very important to reduce environmental pollution since the chemicals released to the environment become reduced. Although it is a metal that occurs naturally in the environment in the trivalent state (Cr3+), considered essential to living things, when it takes the hexavalent form (Cr6+), it is considered toxic to humans, and may cause ulceration, irritation, and inflammation; it is also associated with the risk of cancer. The Walkley and Black technique uses a strong oxidizing agent, potassium dichromate, to react with the organic matter in the soil. Chromium (VI) is converted to chromium (III) and unreduced chromium is back titrated with ferrous ammonium sulfate which provides an indication of the amount of organic matter present. The determination of soil organic carbon is based on the Walkley-Black chromic acid wet oxidation method. Oxidizable matter in the soil is oxidized by 1 N K2Cr2O7 solution. The reaction is assisted by the heat generated when two volumes of H2SO4 are mixed with one volume of the dichromate. The remaining dichromate is titrated with ferrous sulphate. The titre is inversely related to the amount of C present in the soil sample. Organic carbon is oxidized with potassium 71 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 dichromate in the presence of concentrated sulphuric acid. Potassium dichromate produces nascent oxygen which combines with the carbon of organic matter to produce CO2. The excess volume of K2Cr2O7 is titrated against the standard solution of ferrous ammonium sulphate in presence of H3PO4 using ferroin to detect the first appearance of unoxidized ferrous iron and thus volume of K2Cr2O7 can be found out which is actually required to oxidize organic carbon. 2K2Cr2O7 + 8H2SO4 → 2K2SO4 + 2Cr2 (SO4)3 + 8H2O + 6O2 3C + 6O2 → 3CO2 (Mol. wt. of K2Cr2O7 = 294.212, Eq. wt. of K2Cr2O7 = 294.212/6 = 49.03) 2 K2Cr2O7 = 3C, K2Cr2O7 = 3C/2 OR 49.03 g K2Cr2O7 = 12C/4 = 3.0 g C As 1000 cc (N) K2Cr2O7= 3.0 g C 1 cc (N) K2Cr2O7 = 3 g C / 1000 = 0.003 g C Apparatus ¾ Conical flask (500 mL) ¾ Pipettes (2 mL, 10 mL, & 20 mL) ¾ Burette ¾ Volumetric measuring flask ¾ Reagent bottles ¾ Asbestos sheet Reagents a) 1 N potassium dichromate: Dissolve 49.04 AR grade K2Cr2O7 (dry) in distilled water and make up the volume to one liter. b) Concentrated sulphuric acid (Sp. Gravity 1.84, 96 %): If the soil contains chloride, then 1.25 % silver sulphate may be added in H2SO4. c) Ortho-phosphoric acid (Sp. Gravity 1.75, 85 %) d) Sodium Fluoride (chemically pure) 72 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 e) 0.5 N Ferrous ammonium sulphate- Dissolve 196.0 gm of AR grade ferrous ammonium sulphate in distilled water, add 20 mL of concentrated H2SO4 and make volume to one litre. The ferrous ammonium sulphate should be from a fresh lot and light green in color. f) Ferroin indicator Procedure for Sample Collection and Preparation ¾ Remove coarse sand, stone and undecomposed grass from outer surface of your sampling site, not remove decomposed litter or grass. ¾ Make a hexagonal area of 10 m2 using meter and dig down to depth of 20 cm below the upper surface of the soil using auger or spade. ¾ Take about 1g of soil at 0 cm, 5 cm, 10 cm, 15 cm and 20 cm below the upper surface of the soil at each corner of the hexagonal area you made on the ground. ¾ Avoid direct contact of the collected soil samples with sun. ¾ Make a composite sample by homogeneously mixing samples collected at each corner ¾ Air dry the soil, sieve through 1mm-mesh size sieve and pulverize it. ¾ Store the sample near 4 Ԩ until analysis. Procedure for Analysis 1. Weigh 1 gm. of 0.5 mm sieved soil into dry 500 mL conical flask. Add 10 mL of K2Cr2O7 into the flask with pipette and swirl. 2. Add rapidly with a burette 20 mL conc. H2SO4 and swirl gently until soil and reagents are mixed then more vigorously for one minute. 3. Allow the reaction to proceed for 30 min on asbestos sheet to avoid burning of table due to release of intense heat due to reaction of sulphuric acid. 4. Add slowly 200 mL of distilled water, 10 ml of concentrated ortho-phosphoric acid and add about 0.2 gm NaF (one small teaspoon) and allow the sample to stand for 1.5 hours. The titration end point is clear in a cooled solution 5. Just before titration add 1 mL ferroin indicator into the conical flask. Titrate the excess K2Cr2O7 with 0.5 N ferrous ammonium sulphate till the color flashes from yellowish green to greenish and finally brownish red at the end point. 6. Simultaneously blank test is run without soil 73 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Data and Calculation Black reading Burette reading Difference (B- % Organic % Organic (B) (S) Carbon matter % Organic Carbon = (B-S) ×N× 0.003 × S) ૚૙૙ ࢃ࢚Ǥ࢕ࢌ࢙࢕࢏࢒ሺ࢕࢜ࢋ࢔ࢊ࢘࢟ሻ Where, B = mL of std. 0.5 N ferrous ammonium sulphate required for blank. S = mL of std. 0.5 N ferrous ammonium sulphate required for soil sample. N = Normality of std. ferrous ammonium sulphate (0.5N) Soil organic matter contains (58%) of organic carbon, the percentage of organic carbon multiplied by 100/58 = 1.724 which gives the percentage of organic matter i.e. Organic matter = Organic Carbon x1.724 Questions 1. Why add K2Cr2O7 first in soil sample? 2. What is the colour change at the end point in the determination of SOC? 3. Discuss the different between soil organic carbon and soil organic matter? 4. What is the outcome of determine soil organic carbon of the soils by Walkley and Black method? 74 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 EXPERIMENT-12 Determination of Extraction Efficiency of Some Organic Solvents Using Soxhlet Extractor Objective: Acquainting students with plant sample collection and extraction Introduction Soxhlet Extraction Natural medicines were formed and developed in the daily life of ancient people and in the process of their fight against diseases over thousands of years, and they have produced a positive impact on the progress of human civilization. Today, natural medicines not only provide the primary health-care needs for the majority of the population in developing countries but have attracted more and more attention in developed countries due to soaring health-care costs and universal financial austerity. In our country, most of the population has tried natural medicines for the prevention and treatment of diseases. Chemicals known to have medicinal benefits are considered to be “active ingredients” or “active principles” of natural medicines. Natural products have provided the primary sources for new drug development. Natural products offer more drug-like features to molecules from combinatorial chemistry in terms of functional groups, chirality, and structural complexity. The amounts of active ingredients in natural medicines are always fairly low. The lab-intensive and time-consuming extraction and isolation process has been the bottle neck of the application of natural products in drug development. There is an urgent need to develop effective and selective methods for the extraction and isolation of bioactive natural products. Extraction as the term pharmaceutically used, can be defined as the technique used for separation of therapeutically desired active constituent(s) and elimination of unwanted insoluble material by treatment with selective solvents. Screening the crude plant extracts for the desired bioactivity is among the most important operations in medicinal plant research, and extraction is the first crucial step of the process. To have a complete idea of bioactivity of crude extracts, it becomes necessary to optimize the extraction methodology, so as to achieve maximum possible extraction efficiency. Obtaining better quality and high efficiency of extraction from medicinal plant being significant, one has to optimize the extraction methods for better extraction efficiency. 75 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Extraction methods include solvent extraction, distillation method, pressing and sublimation according to the extraction principle. Soxhlet extraction is a typical example of an exhaustive solid–liquid extraction technique and its principle of operation is based on the transfer of the target analyte(s) from the sample matrix (solid) to appropriate organic solvent(s) by ensuring that the extraction solvent (liquid) maintains continuous contact with the sample during the heat-aided extraction process. As shown in Figure below, the conventional Soxhlet extraction apparatus generally consists of a distillation flask, sample holder (thimble), siphon, and condenser. In practice, a preweighed and sufficiently dried solid sample is packed in filter paper and placed inside the thimble. Solvent vapors generated from the heated flask containing the extraction solvent(s) pass through the thimble and become liquefied in the condenser. The extraction process begins the moment the liquefied solvent maintains contact with the sample in the thimble. The level of the condensed solvent, therefore, continues to rise until it reaches the overflow level. At this point, an instant siphon effect rapidly makes the extract (solute + solvent) to fall back into the distillation flask. Within the flask, only the fresh solvent evaporates resulting in a continuous cyclical movement of the extraction solvent. A complete cyclical movement of fresh solvent from the distillation flask until it returns back to the flask as an extract is referred to as a “cycle.” The process is repeated until complete extraction is achieved. The cyclical movement of evaporated solvent from the flask that is subsequently condensed facilitates the mass transfer of the target analyte in every cycle completed during the extraction. Generally, the more the number of cycles completed, the faster the rate of extraction of the target analyte from the sample matrix. The advantage of this method compared to previously described methods is that large amounts of drug can be extracted but it is time taking process compared to the modern extraction techniques. 76 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 5 4 7 3 6 2 1 8 Soxhlet Extractor Material and Chemical Powdered plant material, Soxhlet extractor, chloroform, ethanol and acetone Procedure Find any plant which is claimed to have medicinal values in your locality in which the active principle is believed to in leaves of the plant. Identify the local and scientific name of the plant and write them on your laboratory note book. 77 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Procedure Leaf Sample Collection and Preparation 1. Identify at least three nearby sites where the plant is available 2. Collect healthy and unaffected leaves from four mature plants per site starting from the bottom of braches of the plant to tips of the same braches. 3. Collect all the leaf samples from a site into polyethylene bag and bring each sample from the three sites to your laboratory. 4. Wash the sample with tap water and then with distilled water so as to remove dust and other particulates. 5. Dry the cleaned samples in air in dark room which has a well circulation of air. 6. Homogenously mix the dried plant materials from the three sites while grinding to fine powder using electric grinder or local iron mill. You can alternatively use mortar and pestle if your sample size is small enough. Solvents for Extraction Successful determination of biologically active compounds from plant material is largely dependent on the type of solvent used in the extraction procedure as different organic solvents have different polarities and extraction efficiencies. However, in this experiment we are limited only to chloroform (bp 61-62Ԩ), acetone (bp 56 Ԩ) or ethanol (bp 78 Ԩ) to extract the crude extract from leaves of the plant using Soxhlet extraction techniques. Extraction Procedure 1. Weigh 15-20 g of the powdered plant material 2. Place the weighed powdered plant material in paper thimble. 3. Measure the mass of the paper thimble with pant material. Let the mass be W1 4. Inset the paper thimble containing the weighed plant material into in Soxhlet extractor chamber. 5. Connect the Soxhlet extractor chamber containing the paper thimble with plant material to the condenser of the Soxhlet 78 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 6. Place sufficient amount of chloroform (about 300 mL) in 500 mL round bottom flask. Add 1-2 boiling chips into the flask containing chloroform. 7. Place the flask containing chloroform with boiling chips in heating mantle which has temperature scale. 8. Connect the flask placed in mantle with the condenser previously made ready 9. Heat the setting made so far now at boiling point of chloroform and continue the extraction until the paper thimble becomes color less. In case the paper is not previously colored, you expected to continue the extraction at least for six hours. 10. Take out the thimble from the extractor and dry it in air. Make sure that there is no solvent is remaining in it 11. Measure the dry paper thimble with plant residue and let the mass be W2 12. Follow the same procedures (1-10) for the other acetone or ethanol Calculation The extraction efficiency of the solvent = Amount extracted = W1-W2 ୟ୫୭୳୬୲ୣ୶୲୰ୟୡ୲ୣୢ ୟ୫୭୳୬୲୲ୟ୩ୣ୬ x100 You can also measure the plant residue without the paper thimble after quantitative transference of the residue on a tared clean paper. The difference between the mass of the residue and the mass taken gives the amount extracted plant material. Questions 1. What is active ingredients in natural medicine? 2. Explain the extraction principle of solvent/Soxhlet/ extraction method? 3. List some common solvent that used to extract the crude extract from medicinal plant? 4. What is the advantage of Soxhlet extraction method over the other extraction method? 79 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 EXPERIMENT-13 Determination of the Lipid Content of Snack Food Using Soxhlet Extraction Method Objective: To determine the lipid contents of various snack foods by the Soxhlet extraction Introduction The term “lipid” refers to a group of compounds that are sparingly soluble in water but show variable solubility in a number of organic solvents including ethyl ether, petroleum ether, acetone, benzene and others. There are two types of lipids, free and “bound,” which together constitute total lipid. Free lipid components often occur in storage tissue and are easily extracted, if the sample is dry and well ground to enable solvent penetration. Free lipid may be determined by a simple extraction with a nonpolar solvent (e.g. hexane), whereas total lipid may involve a combination of solvents (e.g. nonpolar and polar) or sample hydrolysis/digestion to extract bound lipid. Although fat derived from meat potentially contains some polar lipids, bound lipids are generally associated with cereal grains. Whether free or bound lipids are extracted, the extract will almost certainly contain components that do not fit the definition of fat. Consumers, industrial food processors, and governmental agencies all have an intense interest in fat, although for very different reasons. Consumers are concerned with the reduction in the intake of total fat, saturated fat, and cholesterol for improving human health. Fat control is necessary for food processors who must strive to meet consumer demands for products containing less fat (i.e. “fat-free” or “low-fat” foods) as well as to maintain costs and to comply with labeling requirements. Governmental agencies must have suitable methods for fat determination to assure accurate labeling of food products. Although the determination of fat content is one of the most common analyses performed in a food stuffs laboratory, the quantitative extraction and analysis of fat is far from straightforward. With the ever-increasing range of processed, composite, and novel foods available, the analyst faces an increasingly difficult task of selecting an appropriate method for fat determination. 80 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 The fat content of a food determined by extraction with one solvent may be quite different from the fat content as determined with another solvent of different polarity. Fat content is determined often by solvent extraction methods like Soxhlet extraction, but it also can be determined by instrumental methods that rely on the physical and chemical properties of lipids (e.g., infrared, density, X-ray absorption). The accelerated solvent extraction technique also provides many advantages over the traditional Soxhlet extraction methods, and is an automated extraction technique that significantly reduces extraction time (<0.5 h per sample) and solvent consumption (<25 mL per sample). This technique enables extraction of analytes from solid and semi-solid samples using common solvents (e.g. hexane, water, and isopropanol) at elevated pressures and temperatures to increase extraction efficiency. The accelerated solvent extraction technique is a superior extraction technique to Soxhlet for the determination of fat in food products. In this experiment however, we will focus on determination of fat content from snack foods using Soxhlet extraction. However, other appropriate foods could be substituted and results compared between methods. Also, the experiment specifies the use of petroleum ether as the solvent for the Soxhlet. However, anhydrous ethyl ether could be used for both methods but appropriate precautions must be taken. SOXHLET METHOD Fat is extracted, semi continuously, with an organic solvent. Solvent is heated and volatilized, then is condensed above the sample. Solvent drips onto the sample and soaks it to extract the fat. At 1520 minutes intervals, the solvent is siphoned to the heating flask to start the process again. Fat content is measured by weight loss of sample or weight of fat removed. Chemicals ¾ Petroleum ether ¾ Ethyl ether N.B: Petroleum ether and ethyl ether are fire hazards; avoid open flames, breathing vapors, and contact with skin. Ether is extremely flammable, hygroscopic, and may form explosive peroxides. Thus, adhere to normal laboratory safety procedures. Petroleum ether and ether liquid wastes must be disposed of in designated hazardous waste receptacles. 81 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Apparatus and Equipment Analytical balance, Soxhlet extractor, with glassware, vacuum oven Chemicals ¾ Three Al weighing pans, pre-dried in 70 Ԩ vacuum oven for 24 hours, beaker (250 mL), cellulose extraction thimbles, pre-dried in 70 Ԩvacuum oven for 24 hours, desiccator, and glass boiling beads, glass wool, pre-dried in 70 Ԩ vacuum oven for 24 hours graduated cylinder (500 mL), mortar and pestle, plastic gloves, spatula, tape and tongs. ¾ Snack foods (need to be fairly dry and able to be ground with a mortar and pestle) Procedure 1. Record the fat content of your snack food product as reported on the package label and record serving size so you can calculate g fat/100 g product. 2. Slightly grind ~30 g sample with mortar and pestle (excessive grinding will lead to greater loss of fat in mortar). 3. Wearing plastic gloves, remove three pre-dried cellulose extraction thimbles from the desiccator. Label the thimbles on the outside with your initials and a number (use a lead pencil), then weigh accurately on an analytical balance. 4. Place ~2–3 g of sample in the thimble and reweigh. Place a small plug of dried glass wool in each thimble. Reweigh. 5. Place the three samples in a Soxhlet extractor. Put ~350 mL petroleum ether in the flask, add several glass boiling beads, and extract for 6 h or longer. Place a 250-mL beaker labeled with your name below your samples on the Soxhlet extraction unit. Samples in thimbles will be placed in the beaker after extraction and before drying. 6. Remove thimbles from the Soxhlet extractor using tongs, air dry overnight in a hood and then dry in a vacuum oven at 70 Ԩ for 24 hours. Cool dried samples in a desiccator then reweigh. 7. Correct for moisture content of product as follows: 82 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 (a). Using the remainder of the ground sample and three dried, labeled, and weighed aluminum sample pans, prepare triplicate 2-3 g samples for moisture analysis (b). Dry sample at 70 Ԩ for 24 hours in a vacuum oven. (c). Reweigh after drying, and calculate moisture content of the sample Data and Calculations Using the weights recorded in the tables below; calculate the percent fat (w/w) on a wet weight basis as determined by the Soxhlet extraction method. If the fat content of the food you analyzed was given on the label, report this theoretical value. Name of Snack Food: Label g fat/serving: Label serving size (g): Label g fat/100 g product: Data from Soxhlet extraction: Mass of Mass of wet Wet sample , thimble and Mass of Dry sample, thimble and glass thimble sample and glass wool wet wool thimble sample 1. 2. 3. Data from moisture analysis: Pan (g) pan + wet sample (g) pan + dried sample (g) % moisture 1. 2. 3. Average = _______________ SD = _______________ 83 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL % Moisture = Chem3118 ( wt of wet sample  pan)  ( wt of dried sample  pan) x100 ( wt of wet sample  pan)  ( wt of pan) % (fat+ moisture) = (initial wt of sample  thimble  glass wool )  ( Final wt of thimble  glasswool ) ( wt of wet sample thimble)  ( wt ofthimble) % fat =% (fat+ moisture) -% Moisture 84 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 REFERENCES American society for testing and materials. 1995. Standard test methods for chemical oxygen demand (dichromate oxygen demand) of water. D1252-95, ASTM Annual Book of Standards. American Soc. Testing & Materials, Philadelphia, Pa. AOAC, Offifficial Method 983.23. Fat in foods. Chloroform-methanol extraction method, in: Offifficial Methods of Analysis of AOAC International, 19th edition., AOAC International, Gaithersburg, MD, USA, 2012. Arya, S.P., Mahajan, M. and Jain, P. 2000. Non-spectrophotometric methods for the determination of Vitamin C. Analytica Chimica Acta, 417(1):1-14. ASTM International. 2006. Standard Test Methods for Acidity or Alkalinity of Water Annual Book of ASTM Standards, ASTM D 1067-06, American Society for Testing and Materials, Philadelphia, PA. Carpenter, C.E. and Ward, R.E., 2017. Iron determination by Ferrozine method. In Food Analysis Laboratory Manual (pp. 157-159). Springer, Cham. Doreen Amponsah. 2014. Determination of the amount of caffeine and benzoic acid in selected soft drink. International Journal of scientific and engineering research, 5: 6. Kendüzler, E., Türker, A.R. and Yalçınkaya, Ö. 2006. Separation and preconcentration of trace manganese from various samples with Amberlyst 36 column and determination by flame atomic absorption spectrometry. Talanta, 69(4): 835-840. Nour Violeta Trandafir I. and Ionica Mira Elena. 2008. Quantitative determination of caffeine in carbonated beverages by an HPLC method. Journal of Agroalimentary Processes Technology, 14:123-127. Petersen, R.G., and Calvin, L.D. 1996. “Sampling.” Methods of Soil Analysis Part 3 – Chemical Methods, Soil Science Society of America, Wisconsin, 1-18. Sato, J.H., Figueiredo, C.C.D., Marchão, R.L., Madari, B.E., Benedito, L.E.C., Busato, J.G. and Souza, D.M.D. 2014. Methods of soil organic carbon determination in Brazilian savannah soils. Scientia Agricola, 71(4): 302-308. Sawyer, C.N., McCarty, P.L., and Parkin, G.F. 2000. Chemistry for Environmental Engineering, 4th Edition. Tata McGraw-Hill Publishing Company Limited. 85 | P a g e By: Abdu H. (MSc) ANALYSIS OF REAL SAMPLE, LABORATORY MANUAL Chem3118 Sleutel, S., De Neve, S., Singier, B. and Hofman, G. 2007. Quantification of organic carbon in soils: a comparison of methodologies and assessment of the carbon content of organic matter. Communications in Soil Science and Plant Analysis, 38(19-20): 2647-2657. Titration.info .2017. Determination of acetic acid in vinegar by titration, from http://www.titrations.info/acid-base-titration-acetic-acid-in-vinegar Tokalioglu, S., Kartal, S. and Sahin, U. 2004. Determination of fluoride in various samples and some infusions using a fluoride selective electrode. Turkish Journal of Chemistry, 28: 203211. United Nations Industrial Development Organization, Handa, S.S., Khanuja, S.P.S., Longo, G. and Rakesh, D.D. 2008. Extraction technologies for medicinal and aromatic plants. Earth, Environmental and Marine Sciences and Technologies. THE END!!! 86 | P a g e By: Abdu H. (MSc) Buy your books fast and straightforward online - at one of world’s fastest growing online book stores! Environmentally sound due to Print-on-Demand technologies. 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