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Prepared for Dept. Of Civil Engineering by Susan C.M. Harper September 2007
Table of Contents
Course Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Station/Drawer Supply List (if supplies are missing, please report) . . . . . 2 First Session: Laboratory Safety and Orientation . . . . . . . . . . . . . . . . . . . . 3
L ABORATORY R EPORT W RITE U P G UIDELINES . . . . . . . . . . . 5
Laboratory Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Laboratory 1: Solids Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Laboratory 2: COD, DO and BOD 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Laboratory 3: Bacteriological Examination of Sewage . . . . . . . . . . . . . . . 17 Laboratory 4: Chloride Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Laboratory 5: Chlorine and Chlorine Demand . . . . . . . . . . . . . . . . . . . . . 34 Laboratory 6: Alkalinity, Acidity, pH, Hardness, Colour, Turbidity . . . . 39 Laboratory 7a: Coagulation and 7b: Coagulation & Softening . . . . . . . . . 47
Appendices see: CIVL407Manual_CourseNotes.pdf . . . . . . . . . . . . . . . . . 54
(Environmental Laboratory Analysis - 1-3-0, 0-0-0) Testing procedures used in water quality studies and in the operation of water and wastewater treatment plants. Prerequisite Chem 151. Sc h e d u le Lecture CEME1204: Laboratory CEME1301: Wednesday 1:00 pm 1) Tuesday (time to be arranged by consensus) 2) Wednesday 2:00 to 5:00 pm 3) Thursday (time to be arranged by consensus) 4) Friday (time to be arranged by consensus) (Note: Lab Session must have at least 4 students to run)
Subject No Lab No Lab Laboratory Safety & Orientation (~1 hr) #1 Solids determination #2 Dissolved oxygen, COD, BOD No lab - Thanksgiving week - catch up #3 Bacteriological Examination of waste water #4 Chloride #5 Chlorine demand #6 Acidity, alkalinity, hardness, colour, turbidity No lab - Remembrance day - catch up #7a Coagulation #7b Coagulation and Softening
Date 2007 Sep. 4 - 7 Sep. 11 - 14 Sep. 19 Sep. 25 - 28 Oct 2 - 5 Oct 9 - 12 Oct. 16 - 19 Oct. 23 - 26 Oct. 30 - Nov.2 Nov. 6 - 9 Nov. 13 - 16 Nov. 20- 23 Nov. 27 - 30
Lecturer: Lab instructor: T.A.s: Lab: Marker:
Victor Lo, email: Susan Harper, email: Margaret Parsotan, email: Kazi Parvez Fattah, email:
email@example.com firstname.lastname@example.org email@example.com firstname.lastname@example.org
(604) 822-4880 (604) 822-4397
September 2007 CIVL 407 Lab M anual
Sta tio n /D ra w e r Su p p ly Lis t (if s u p p lie s a re m is s in g , p le a s e re p o rt)
The following items will be maintained in each stations supply drawer or at station on bench when required: 2 pair safety glasses 2 permanent marker pens 2 stir bars and a bar retriever 1 stir plate 2 buret funnels 4 30 ml beakers 4 50 ml beakers 2 100 ml beakers 2 150 ml beakers 2 250 ml beakers 2 1 L plastic beakers 2 600 ml plastic beakers 2 400 ml plastic beakers 2 10 ml grad cylinders 2 25 ml grad cylinders 2 50 ml grad cylinders 2 100 ml grad cylinders 1 buret stand 1 double buret clamp 2 50 ml burets 2 25 ml burets 2 10 ml burets 0-14 pH strips Thermometer 2 Stir stick or spatula Filter funnel (Nalgene magnetic) Tweezers (Millipore or Gelman for membrane filters)) On benches each row: Latex gloves- small, medium and large Non-latex gloves (keep on reserve for latex sensitive persons only) Filter racks with funnels #4 Whatman filter paper or equivalent Vacuum flasks and hoses 8 1 L grad cylinders 8 3 L plastic beakers
September 2007 CIVL 407 Lab M anual
Putting pens in your mouth that may have been on the bench or handled with gloves could be hazardous. if there is room. Spills: Wipe up all spills immediately.2 First Session: Laboratory Safety and Orientation 1) 2) 3) 4) NO Food or drink.up to 10. Hygiene: At times you will be working with sewage. Do not put lids and small items onto racks that may slip through to the filthy drip tray. You must be informed of the hazards of a substance before using it. packs. They are available in 0. Safety Equipment: Eye Wash Station.. gloves as required. Please take your labcoat away in a plastic bag. No sandals or open-toe or heel shoes. with the tip against the side of the receiving vessel.0. 2. etc. MSDS: is a supplier information sheet that must be available for each product and describe the hazards and necessary handling requisites. 50 and 100 ml. Most pipettes are calibrated “to deliver” or TD and should never be blown out. calculators.5. 25. Remember to empty and rinse burets as well. These should be used whenever standard solutions are measured or when upmost accuracy is required. 20. Safety Shower. Clean-up: You must leave your work station as you found it . WHMIS: Workplace Hazardous Materials Information System is a government regulation.0 ml. but if you do please ask for assistance. with contaminated gloves will contaminate those items. wash down and dry the bench. Pipettes must be placed with tips pointing up into the pipet basket supplied.0. Broken Glass: Be careful not to break glass. 15. Be aware that handling your pens. Pipettes/ graduated cylinders/ beakers/ flasks: 5) 6) 7) 8) 9) 10) Volumetric pipettes are the most accurate way to measure volume. Some pipettes may be “to 3 September 2007 CIVL 407 Lab M anual . They are calibrated by weighing the volume of distilled water that will flow from them by gravity. 1. Wearing lab coats out of the lab is forbidden. disinfectant for bench surfaces. DO NOT PUT BROKEN GLASS INTO THE REGULAR GARBAGE CONTAINER.clean and dry. If you cut yourself. please get attendant help. no matter how minor. Please ask for assistance if chemicals are spilled. All glassware must be rinsed well (at least 5 times with tap water) and put on paper towels at your station or on drying racks.. A small amount of liquid always remains in the tip and must not be blown out. eye protection. Personal Protective Equipment: Lab coat (long). Sanitation: antibiotic soap for hand washing.
Do not squish bulbs in the direction of another person. YOU WILL USUALLY NEED THE INFORMATION GIVEN TO COMPLETE YOUR LAB WRITE-UPS. 12) Cup sinks: Please do not use these sinks for the disposal or delivery of liquids. Ie If you want to measure 20 mls. Chemical residues can be splashed in you face. Chemicals will be put out as required for each lab and should be used sparingly. 1. some are not meant to drain completely . 14) Glassware/supplies: Each station has a drawer where some glassware and other useful tools will be kept. not wastefully as quantities are limited.deliver with Blow-Out”. Additional supplies are available in the supply room (around the corner). LISTEN AND MAKE NOTE OF VERBAL AND CHALK BOARD INSTRUCTIONS. 11) Gas/air/vacuum taps: Do not touch these unless instructed to do so. Pipette bulbs or pumps must be used and great care must be taken not to contaminate bulbs. Graduated cylinders: Not as accurate as pipettes. They are useful for measuring samples 20 mls . 100 mls. They can be unpredictable in force and are close to eye level.1000 mls. Cylinders are available: 5 mls.. 13) Stir bars: Please remove from containers before pouring contents down the drain. Even the force of air from the air taps can be dangerous because it can contain grit or water. chemicals or for titrations. 50 mls.merely approximations.pay attention to the markings. 10 mls. If liquid should enter a bulb or pump. Make sure that you label beakers and flasks to avoid confusion and waste. 25 mls. please rinse out immediately and set aside to dry. 4 September 2007 CIVL 407 Lab M anual . Some are blow-out. Undetected gas leaks can be deadly. 250 mls. They are usually identified by a double etched or coloured band at the top of the pipette. They are calibrated the same way as TD except that the remaining drop is blown into the receiving vessel. Flasks: Vo lu m e tric flasks are very accurate and should be used when diluting standards (together with volumetric pipettes). Use the size of cylinder CLOSEST TO THE VOLUME YOU WISH to measure for best accuracy. Erle n m e y e r or conical flask markings are not accurate. Graduated or “measuring” pipettes are not as accurate as volumetric (bulb type) pipettes and the one having the maximum capacity closest to the volume required is the most accurate one to select. 500 mls. especially at smaller volumes. A demonstration will be given. markings are not accurate at all . 2 & 4 L Beakers: are cylindrical in shape with straight sides and flat bottoms. They are containers best suited for “swirling” liquid ie to mix reagents with samples in a colourimetric determination (Lab 4 or 6). Pipettes are in drawers indicated. NEVER mouth pipette. Used to hold/transfer approximate volumes of samples. do not use a 1 L graduated cylinder.
Total Mark L ONG R EPORT L AB 7 Required Required Required Include a brief description of the procedure. Please note that the marking outline is intended only as a guideline to assist in report write-up and that marks would also be allocated for overall presentation and clarity of the report. R EPORT E LEMENTS Title page Objective Materials and Method S HORT R EPORT L ABS 1 TO 6 Required Required Details are not required. Sufficient detail should be provided so that what was done could be understood and the experiment could be repeated from the procedure reported.L ABORATORY R EPORT W RITE U P G UIDELINES Guidelines for laboratory report write up are also given in the CIVL407 Manual Course Notes and summarized here. The short report is intended to be a concise yet complete laboratory report whereas the long report is more elaborate. The mark allocation for some sections for both reporting format is indicated in parenthesis in the table below. Required  Required  Brief discussion required  Required Required  Required Observations and Results Sample Calculations Discussion Including sources of error Conclusions Questions and Answers Other Students’ Data and Results References Presented in an acceptable format. Required  Required  Required  Required  Required  Required  Required   5 September 2007 CIVL 407 Lab M anual . State that the procedures were in accordance with CIVL 407 Lab Manual and note any deviations to the manual’s procedure.
Do not include any surface floating material as settled solids. 2. If large pockets of liquid form between the particles of settled matter. Settle for 45 minutes. OBJECT: -to become familiar with a number of the most common sewage treatment plant tests and to illustrate some of the difficulties in performing these tests. sludge. gently dislodge any solids that have clung to the sides using a stirring rod. Alternatively a place will be provided in the lab to store your coat. SETTLEABLE SOLIDS 1. 6 September 2007 CIVL 407 Lab M anual . Do not wear lab coats outside of lab and do not take away from lab unless stored in a plastic bag. and record the volume of settleable solids. balance. evaporating dishes. if necessary.. to measure sewage treatment plant efficiency in removing residue. wipe up all spills and wash with disinfectant. keep hands and pencils away from your mouth and wash hands frequently. oven muffle furnace. A 1 L graduated cylinder may be used in place of the Imhoff cone for the sludge sample. tin dishes. graduated cylinders. Use pipette bulbs.3 Laboratory Exercises La b o ra to ry 1: So lid s D e te rm in a tio n WARNING You will be handling sewage samples which may contain pathogenic bacteria. samples of raw sewage. MATERIALS: -Imhoff cones. A. Mix the samples thoroughly and fill individually marked Imhoff cones to the 1 litre mark. settle for 15 minutes longer. subtract an estimated volume from the measured volume of matter. desiccators. final effluent. distilled water bottles.
The furnace will then be allowed to cool significantly before dishes are removed to a desiccator. 4. 6. Assemble filtering apparatus. 3.] Rinse the graduated cylinder with small amounts of distilled water and add to filter. The dish containing the dried total solids can now be placed in a muffle furnace or. immediately after stirring the beaker. the solids remaining represent the fixed solids.done for you. Obtain the tare weights of three aluminum dishes each containing a glass fibre filter (already pre-washed. Calculate the mg/L volatile and fixed solids. After drying overnight. TOTAL SOLIDS and TOTAL VOLATILE AND FIXED SOLIDS AT 550o C 1. Record the total volume filtered. Subtract the tare weight to obtain the total solids weight and calculate the mg/L value. [Hint: pour out small portions (say 25 mls at a time for Effl. Conversely. dried. This is a different test to the one we are doing using Imhoff cones and should not be confused.]. rinse the cylinder. C. rapidly pour sample into a 50 ml graduated cylinder to approximately equal 35-40 mls (if using 50 ml dishes). Obtain the gross weight of the dish [Note: it should be room temperature before weighing. Do not write on crucibles as high temperatures during the next steps will burn writing off. 2. prior to session).[Note: it may be harder to wash out solids if you allow them to settle before pouring into the dish . 3. The loss in weight represents the volatile solids lost from the originally measured sample volume. [Notes: to allow for rinsing and to avoid spillage when transferring dish to oven don't pour more than 40 mls]. the dish will be transferred to a desiccator. stir the beaker contents and then rapidly (so that it doesn't settle) measure a volume of sample into the appropriate graduated cylinder. Using the sample poured out for Part B (for consistency). when filtering slows significantly do not add more.so be quick. 2. SUSPENDED SOLIDS . Once dishes are at room temperature they can be re-weighed. Wet filter with a small volume of distilled water to seat it. Quickly record the exact volume and pour the sample into the dish.TOTAL AND VOLATILE 1. Mix the sample in the carboy thoroughly. SVI= settled sludge volume (ml/L) x 1000/suspended solids (mg/L)] B. position the filter and begin suction (vacuum tap). dried and fired). pour about 500 mls (for Part B & Part C) into a beaker and then. 25-50 mls of raw sewage and 5-10 ml of sludge u s in g g ra d u a te d c y lin d e rs to measure . adding the rinsings to the dish. Suggested portions: 100-200 ml of final effluent. Carefully remove filter from filtration apparatus and transfer it back to the aluminum dish. tare weight. pre-fired and desiccated . sample source and sample volume.not beakers.[Note: Sludge Volume Index is the volume occupied by 1 gram of sludge after 30 minutes of settling. on a cart in preparation for staff to do so when all are ready. Make sure that you have recorded all the pertinent details: Dish #. 10 mls for Raw & 5 mls for sludge) of well-mixed sample and filter entire portion before pouring another portion. Place the aluminum dish into the 103°C oven to dry for at least one hour (or leave drying 7 September 2007 CIVL 407 Lab M anual 4. 5. if instructed. Put the dish in the 103-105 °C oven for drying.] Using very small amounts of distilled water. Pinch sides of dish in a bit to protect the filter from oven drafts. The samples will be fired at 550° C for one hour. . Obtain the tare weight of a properly prepared porcelain evaporating dish (ie one that has been washed.
% reduction in settleable matter _________ % reduction in total solids _________ % reduction in volatile solids _________ % reduction in fixed solids _________ % reduction in suspended solids _________ % reduction volatile susp. What major types of solids are removed in primary treatment? in secondary treatment? 2. suspended. DO NOT DISCARD! The gain in weight represents the total suspended solids of the sample. where the volatile solids are reduced from 65% to 40%. what is the percentage reduction in solids. What. if any. 6. If sludge is used in Part A. overnight). volatile. Calculate the mg/L total volatile solids and total fixed suspended solids. Calculate as the mg/L total suspended solids. 8. After firing the dishes will be moved to a desiccator. affect would there be to the TSS and TVSS results if your bare hands were used to handle a tin dish a) prior to obtaining the initial tare weight and b) after obtaining the initial tare weight? 5. From the above determinations it will now be possible to obtain a value for the dissolved solids present in the sample. A raw sewage sludge goes through an anaerobic digestion process. They must be allowed to cool completely before re-weighing them to determine the loss on ignition or volatile suspended solids. The aluminum dish and filter (from #4) must now be fired in a muffle furnace at 550°C for at least 30 minutes (or until a constant weight is achieved). compute the sludge volume index (SVI). Transfer dish to a desiccator. Your hands may have moisture and natural and/or applied oil/lotion on them. Discuss the meaning and significance of this index. solids _________ % reduction fixed suspended solids _________ % reduction dissolved solids _________ Lab 1 Questions 1. Classify each of the following into its proper solids category(s) i. Please return tomorrow to obtain final weight. Lab personnel will perform the timed-firing after the class. If all of the volatile solids reduced is given off as gas and if the specific gravity of the volatile solids is 1.5.) a) a bacterium b) sodium chloride c) sugar d) clay 8 September 2007 CIVL 407 Lab M anual . Enter all data in the tables on following page. 3. place the dishes on a cart designated by the instructor. What two techniques can be used to overcome or correct for the loss of material from filter pads when firing at 550 oC? 4. Calculate the percent solids reduction through the treatment plant.e. dissolved.3 and fixed solids 2. For the time being. 7.50. 6. fixed (use more than one adjective to describe each substance. cool and weigh.
Total Solids Dish marking (do not use ink . Settleable Matter After 1 hour (mL/L) B.fired (G) Loss on ignition (G) Volatile solids (mg/L) Fixed solids (mg/L) Percentage fixed residue C.oven dry (G) Dish tare weight (G) Total solids (G) Total solids (mg/L) Gross weight .oven dry (G) Tin dish with filter tare weight (G) Total suspended solids (G) Total suspended solids (mg/L) Gross weight .use embossed mark) Sample volume (ml) Gross weight . Suspended Solids Tin dish marking (do not use ink .fired (G) Loss on ignition (G) Volatile suspended solids (mg/L) Fixed suspended solids (mg/L) Dissolved solids (mg/L) 9 September 2007 CIVL 407 Lab M anual Final Effluent .use permanent mark on dish) Sample volume (mL) Gross weight .DATA AND RESULTS Raw Sewage Aerobic Sludge A.
vials with pre-measured reagents. otherwise reagents will run to the bulb end and make pipetting difficult and contamination likely. spectrophotometer. Always use and store pipettes with the top higher than the tip. Materials: Raw sewage and final effluent samples. etc out of your mouth. pencils. D O a n d B O D 5 WARNING You will be using concentrated acid and other corrosive and toxic chemicals. 10 September 2007 CIVL 407 Lab M anual . Colourimetric Method NOTE: Open reflux method is the most accurate means of determining COD because a large sample size is used (20 mls). and to demonstrate the use of dissolved oxygen probes. Wash hands frequently with soap. Wipe up all spills immediately. Keep fingers. NEVER pour water into acid. 400.Closed Reflux. The closed reflux method is more economical but because a small sample volume (2 mls) is used. wash/rinse bench tops with water/disinfectant. COD . as well as handling samples probably containing pathogenic bacteria. Hach block digester. OBJECT: -to familiarize you with the commonest tests run for assessing treatment plant efficiency and pollution loading to receiving waters.La b o ra to ry 2: CO D . to illustrate the shortcomings of these tests. potassium hydrogen phthalate standards: 800. 100 and 50 mg/L as COD. etc. 200. homogenization of samples containing suspended solids may be necessary in order to get a representative sample and to improve reproducibility. I. pipettes. Digests from either method can be titrated or read colourimetrically.
Remove the vials and allow them to cool to room temperature (use cold water to speed up if necessary). 100.the azide modification of the iodometric method) and a dissolved oxygen probe and meter. 11 September 2007 CIVL 407 Lab M anual . a volumetric correction must be made to the value obtained from the curve. 5. 2. If sample is known to be outside the standard range (above 800 mg/L). You will use a chemical titration (Winkler . into the appropriate tubes and screw cap on snugly. DISSOLVED OXYGEN (DO) Materials: Raw sewage and final effluent samples. before the lab. alkaline-iodide-azide reagent. raw sewage and final effluent. Put identifying labels on each tube ie your initials. [Note: it should look homogenous and be careful it will be very hot!] 3. a smaller aliquot (making volume up to 2 mls with distilled water) or 2 mls of a pre-diluted sample can be used. dilution water. Standards have been digested for you. 10-ml graduated pipettes. except without mercuric sulfate). aerated dilution water. Allow samples to digest for 30 minutes (normally this would be 2 hours). 200. thermometers. in which case.Procedure: 1. II. concentrated sulphuric acid. and are at the spectrophotometer. At your station you will find four vials with pre-measured solutions containing potassium dichromate and sulfuric acid (prepared according to standard methods. Prepare calibration curve. 4. manganous sulfate. The four liquids are cold tap water. starch indicator. Pipette 2 mls of sample (using volumetric pipettes unless particulate is present. DO NOT ADJUST THE SETTINGS ON THE METER. 400. 6. The vials should be clean and dry on the outside. absorbance versus concentration and calculate the COD in samples. Note: You are going to measure DO on four different liquids using two different methods. burette stand with burettes. Take vials to fumehood and place in block digester . well-mixed samples in the 25-position digester. which is set at 600 nm to read absorbance of light by the sample. Raw Sewage (Infl) and/or Effluent (Effl) in the upper 1/4 of the tube using permanent markers. The instructor will demonstrate the use of the spectrophotometer. Make sure sample is well mixed with the acid contents of the vial. 250-ml graduated cylinder. 50 and 0 COD as mg O 2 /L.025 N sodium thiosulphate. 500-ml Erlenmeyer flasks. BOD bottles.noting the location of your well-labelled. Read your samples and the set of COD standards: 800. 0. use wide mouth graduated pipettes) in duplicate. The probe has been previously calibrated. If less than two mls of sample or diluted sample was used.
(Note: DO meter temperature is usually not accurate. Use the specially marked volumetric flask to dispense the 201 ml. right to the top and place stopper on bottle.think about it!) Complete all four titrations. as demonstrated. Fill four BOD bottles with the four samples (cold tap water.) You can save these bottles for B. Determine DO. 6. invert several times again and allow to settle again. Tip out the liquid from the area around the stopper and invert several times to mix thoroughly. Take each stoppered bottle. Neglect any reappearance of the blue colour. . until the first complete disappearance of the blue colour. aerated dilution water. B. 1. Record the volume of titre. 5.A. Allow the floc to settle to about 1/3 the bottle volume. The chemical floc should completely dissolve. When the floc has settled the second time to about 1/3 bottle volume. Note: if solution already seems pale yellow add starch right away. (Note: biological solids originally present in the sample will not dissolve. taking care not to entrain additional air. raw sewage or influent and final effluent). again quickly replacing the lid. Fill a BOD bottle with each of the four samples (or use ones saved from A ). for each of the four samples. Titrate this volume with 0. Stopper the bottles without trapping any air bubbles. (Fill carefully. add 1 ml of concentrated H 2 SO 4 and quickly restopper. 4.) Transfer 201 ml of each bottle (do one at a time) to a 500 ml conical flask. 9. Instructions on the use of the probe will be given in the lab. add 1 ml of alkaline-iodide-azide solution the same way. using the DO probe and meter. 8. Determine the percent saturation for each sample. remove the stopper. 2. Set back in sink. tip the liquid out of the space around the stopper (caution -it may be corrosive) and invert several times. For the cold tap water sample. DISSOLVED OXYGEN PROBE NOTE: DO NOT add any reagents to the samples used for the probe method. 2. Do not mix droppers and do not allow tap water into the reagent bottles. To the same bottles. 12 September 2007 CIVL 407 Lab M anual 3.025 N Na 2 S2 O 3 until only a faint yellow or "straw" colour remains. 7. to thoroughly mix contents. submerge the hose in the bottle and allow the water to overflow for 15-30 seconds). Record the temperature of each sample using the DO meter or a thermometer. add 1 (one) ml of manganous sulfate reagent using plastic dropper provided (keeping the dropper tip just below the surface) and quickly replace the lid. Add enough starch solution to give a strong blue colour (one dropper full) and continue to titrate. (Hint: if you measured very low DO level with the meter. then the same sample will require little or no titre and may not turn blue when starch is added . The DO concentration in mg/L is equal to the number of ml of titre as long as 201 ml is titrated. WINKLER TITRATION (Azide modification) NOTE: Use only the droppers attached to the reagent bottles for dispensing into samples. dropwise. Place the bottles in the sink and one at a time remove the stopper. 1.
(Probe) Temp (oC) (thermometer) Saturation concentration Percent saturation DETERMINING PERCENT SATURATION THE "QUICK AND EASY" METHOD For a quick and easy determination of the percent saturation value for dissolved oxygen at a given temperature. use the saturation chart above.Dissolved Oxygen Data Sample/Data Bottle # Sample Volume (mls) Initial Buret reading Final Buret reading Net Titre (mls) DO conc. Note that this nomogram assumes that the water is at sea level The saturation value can also vary slightly depending on barometric pressure with lower values expected when a storm front moves through as compared to bright and sunny "high pressure" days. (mg/L) DO conc. The percent saturation is the value where the line intercepts the saturation scale. Draw a straight line between the water temperature and the mg/l of dissolved oxygen. Pair up the mg/l of dissolved oxygen you measured and the temperature of the water in degrees C. Raw Sewage Final Effluent Tap Water Dil’n Water 13 September 2007 CIVL 407 Lab M anual .
All bottles should have liquid in the well around the stopper . carefully measure the waste water samples into BOD bottles. Calculate the 5-day (or 6-day if unavoidable) BOD. Note: Each group should have 9 BOD bottles in the incubator. groups). 14 September 2007 CIVL 407 Lab M anual . Two dilutions will be made for each sample and each dilution will be in duplicate for a total of eight bottles (plus one bottle for the blank mentioned in Step 4). The blank is used to check that the dilution water has negligible BOD. After five (or six)days. dilution water. 4. remove bottles and measure the DO.III. ie Rinse the filling tube if it has been in the sample. Because you want to be sure that the dilution water has no BOD. fill one BOD bottle with DILUTION WATER alone. Stopper the BOD bottles. it may indicate that probe may not have been calibrated properly or the dilution water had been contaminated. graduated cylinders. but if there is a significant BOD then it will have to be subtracted before the dilution factor is applied in the calculation. There should be negligible BOD in the Dilution water. BIOCHEMICAL OXYGEN DEMAND Note: Because of the logistics of organization some groups may have 6 day BOD 5 instead of the usual 5 day. Place all bottle sets in the 20 o C incubator for five days (or 6 for Tues. to bring the levels above the ground glass neck of the bottle. Dissolved oxygen meter and probe. Measure the Initial DO of the blank using the calibrated probe and meter. These caps are designed to prevent the evaporation of the liquid around the stopper. preferably using the same meter and probe used to get IDO reading. 1. Place plastic caps over all bottles. 10-ml graduated pipettes. showing sample calculations. In addition. Materials: Raw sewage and final effluent samples. If the dilution water BOD is greater than 1 ppm. Using the volumes and samples provided by the instructor. Be sure to record the bottle numbers and sample volumes on the following table .do not write on the bottles and do not use the numbers on the lids. thereby maintaining a water seal for each bottle. be careful not to contaminate it with sample. Measure the Initial DO using a calibrated probe and meter. being sure to exclude any air bubbles that might be trapped under the stopper by adding dilution water. Be sure to note this in your lab write-up. 5. Try not to aerate the contents by keeping the filling tube below the surface of the liquid in the bottle or carefully running the water down the side of the bottle.if not add a some dilution water (or distilled water) to the well. BOD bottles. 3. 6. Carefully top up the BOD bottles with DILUTION WATER.
DO of diluted sample immediately after preparation. 15 September 2007 CIVL 407 Lab M anual . and ratio of seed water in diluted sample to seed in seed control= (%seed in diluted sample) / (%seed in seed control). = = = = decimal volumetric fraction of sample used DO of seed control before incubation. mg/L DO of seed control after incubation.BIOCHEMICAL OXYGEN DEMAND (BOD) DATA Sample source: ________________________________ Raw Sewage Bottle number (not lid #) Sample volume (ml) Initial DO (mg/L) Final DO (mg/L) Final Effluent Bottle number (not lid #) Sample volume (ml) Initial DO (mg/L) Final DO (mg/L) Dilution water Bottle number (not lid #) Sample volume (ml) Initial DO (mg/L) Final DO (mg/L) _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ BOD Calculation When dilution water is not seeded (our water is not seeded for this exercise): When seeded water is used: where: D 1 = D2 P B1 B2 f mg/L. mg/L. mg/L = DO of diluted sample after 5 d incubation at 20 oC.
Diln. Both wastes have equal BOD 5 temperature and volume. open reflux. 2) 3) 4) Show on a graph the approximate dissolved oxygen sag curves in a river receiving two wastes. the other 0.Summary Raw Sewage. Diln.17. if at all possible. what would you expect its I) BOD 5 and ii) BOD L to be. Diln. One waste has a K value of 0. If an industrial waste has a COD of 450 mg/L. 1 Final Effluent. 2 ________ ________ ________ ________ ________ ________ ________ ________ Average ________ Average ___________ ________ Percentage BOD reduction through treatment plant Lab 2 Questions 1) At what relative times should influent and effluent samples be taken from a sewage treatment plant in order to determine true % removal efficiency? Give two reasons why samples for dissolved oxygen should be “fixed” in the field.) Why is your COD result different from the BOD result? 5. titrimetric method. Calculate theoretical oxygen demand for 300 mg/L of methyl alcohol. 6) 7) 8) 16 September 2007 CIVL 407 Lab M anual . 2 Final Effluent. What interference does the Azide Modification of the Winkler Dissolved Oxygen method overcome and why is it the most common method used for domestic sewage? Why is a blank titrated in the COD test? (Refer to standard methods. Diln.25. 1 Raw Sewage.
lauryl tryptose broth tubes. Keep hands. plates.. Do not wear lab coat out of lab . Wipe lab benches with disinfectant before and after each lab. Students will do only sections marked ? MATERIALS: Multiple tube method: dilution tubes. T h e re f o re . sterile dilution water. DO NOT MOUTH PIPETTE. Membrane filter method: dilution tubes. etc. Get raw data from another group and include in your report.La b o ra to ry 3: B a c te rio lo g ic a l Exa m in a tio n o f Se w a g e WARNING As you are handling and pipetting samples probably containing pathogenic organisms please observe the following rules: Wear gloves. 35 oC incubator. EC tubes and/or Hach A1 tubes. Brilliant Green Lactose Broth tubes. F iv e d if f e re n t p ro c e d u re s w ill b e s tu d ie d . agar tubes. Demonstration only. Heterotrophic plate count: dilution tubes. Demonstration only SAMPLE: You will do either a raw sewage sample or a final effluent sample. not both. sterile pipets. y o u w ill d o th o s e m a rke d b y ? only 17 September 2007 CIVL 407 Lab M anual . Wash hands with soap before leaving. OBJECT: -to demonstrate different methods for the determination of the coliform group of organisms and to acquaint you with bacteriological procedures. LES Endo agar plates. HACH P/A Broth with MUG.leave it here or take away in a plastic bag to launder. wipe up all spills and disinfect the area of the spill.b e c a u s e o f lo g is tic s it is n o t p o s s ib le f o r y o u to p e rf o rm th e la b a s p re v io u s ly w ritte n . eye protection. Quebec colony counter. 35oC incubator. sterile pipettes. pencils. filtering apparatus. lab coats. membrane filters. away from your mouth.
2. The number of colonies counted directly. Consider positive EC broths incubated at the elevated temperature (44. Completed Phase LES Endo agar Plates: A p la te w ill b e a v a ila b le to lo o k a t. All tubes have been examined for gas production.7oC for 24 hours (confirmed test for faecal coliforms). is USEPA-accepted for testing non-potable waters. using a ready-to-use P/A Broth with MUG. Calculate the MPN value from the number of positives using the Table 1. Demonstration. Heterotrophic Plate Count . Multiple-tube Fermentation or Most Probable Number: Serial dilutions of sample are incubated in lauryl tryptose broth at 35oC for 48 hours (swirl after 24 hrs).5 oC) as a positive completed test response for both Total and faecal coliform. Note: these methods will be demonstrated but data must be recorded.A. EC or A-1 tubes positives: 1. Brilliant green lactose bile (BGLB) tubes are inoculated and incubated at 35oC for 48 hours (confirmed test for total coliforms) and EC tubes are inoculated and incubated at 44. as demonstrated. Bacterial numbers are calculated from the numbers of positive tubes. MUG produces a fluorogenic product when hydrolyzed by an enzyme specific to E. Gas-positive Hach A-1 tubes indicate the presence of faecal coliform. ? C. Presence/Absence Testing: HACH technique to simultaneously screen for total coliform and E. Parallel positive BGLB broth cultures with negative EC broth cultures indicate the presence of nonfaecal coliforms. D. Gas formation indicates the possible presence of coliform organisms. 3. 1.COLI in 4 to 24 hours.Pour Plate Method: Serial dilutions of samples mixed with agar and incubated for 48 hrs at 35oC. It is used as a faster alternative to the LTB/EC procedure for enumerating faecal coliforms in water. Using aseptic technique. Hach’s Most Probable Number Method 8368. E. (Single colonies are obtained by streaking LES Endo agar plates with an inoculum from positive BGLB or EC broth tubes and incubating for 24 hours. streak one LES Endo agar plate from each tube of BGLB broth showing gas from the highest dilution and the second highest dilution. Microscope Slides (Completed Phase of Multiple-Tube Fermentation): Single colonies arising from single cells are prepared and stained for microscope examination. A-1 Medium. Express as Total coliforms. Another series of tubes are inoculated from these positive lauryl tryptose tubes. Results will be provided for the demonstration set. Formation of gas in any amount in the inverted vial of the BGLB broth fermentation tube at any time within 48 hr ±3 hr constitutes a positive confirmed phase.coli. ? B.coli. Streak plates in a manner to insure presence of some discrete colonies. Membrane Filter Technique: Serial dilutions of sample are collected on membrane filters and placed on M-Endo media. Colonies that are pink to dark red with a golden green metallic sheen are counted after incubation 20-22 hrs at 35oC. The broth will turn yellow indicating total coliform and fluoresce in the presence of E.) Methods: ? Recording BGLB. 18 September 2007 CIVL 407 Lab M anual . Use Table 2 on page 27.
Heterotrophic Plate Count . open the valve slowly and allow all the liquid to be pulled through. 4. 3. If you haven’t already. choose appropriate dilutions for counting. or colourless and are considered to be non-coliforms. Membrane Filter Technique -Total Coliform 1. Prepare one set of six dilution tubes as described in Section A. ?C. Do not push down on the filter except on the outside edge and use only sterile forceps . 5. red. 2. Prepare Gram Stain slides for examination under a microscope. Gram staining technique can be also be used. from one edge. The sheen area may vary in size from a small pinhead to complete coverage of the colony surface. Close valve and rinse the funnel with three aliquots (portions) of sterile dilution water opening and closing valve after each aliquot. therefore the results table should specify #coliform colonies/100 ml. After 20-22 hr. Inoculating. Using sterile forceps. Be sure dishes are labelled with your name and dilutions. Place the filter on a small LES Endo agar plate carefully..1. -Note: “typical” colonies count as coliforms in this procedure. with a motion that excludes air as it comes in contact with the agar. Filtering. prepare one set of six dilution tubes following the instructions given under Multiple Tube Fermentation. Incubate inverted Petrie dishes for 20-22 hr at 35 oC. Verification is usually done by a test for lactose fermentation. Colonies that lack sheen may be pink. Flame loop between second and third quadrants to improve colony isolation.applying gentle pressure. Dilution tubes.Pour Plate Method 1. Verification of both types of colonies is advisable as some typical sheen colonies may be noncoliform and some atypical may be coliform. Pipette about 10 ml of sterile dilution water over the surface of the filter with the suction valve still closed. A high count of non-coliform colonies may interfere with the maximum development of coliforms. 19 September 2007 CIVL 407 Lab M anual .2. place a sterile membrane filter (grid side up) onto the filtration apparatus. The sheen area may vary in size from a small pinhead to complete coverage of the colony surface. In general. Use dishes with 20 to 80 coliform colonies and not more than 200 of all types. then immediately replace the cover. Using a sterile pipette. remove filter using flamesterilized forceps (allow forceps to cool or they will damage the filter). Pour the full volume of dilution tube # 6 onto the filter. coliform bacteria are Gram-negative (pink) rods (sometimes coccus-like). Repeat for the rest of the dilution tubes to # 2. The typical coliform colony has a pink to dark-red colour with a golden green metallic surface sheen. transfer 1 ml of sample from dilution tube # 6 (most dilute for Raw sewage) or #5 (for Effluent) to an empty (large) Petrie dish. Mount apparatus on suction flask with valve sideways to block suction. When filtering is complete and the valve is closed. Atypical coliform colonies can be dark red or nucleated without sheen. The total count of colonies (coliform and noncoliform) on Endo-type medium has no consistent relationship to the total number of bacteria present in the original sample. Dilution tubes. The filtration apparatus has been sterilized by autoclaving. Counting. white. removing the cover just enough to insert pipette tip and dispense sample. ?B. 2. Incubate plates (inverted) at 35 oC for 24 ± 2 hr.
Flood the slide with fresh alcohol-acetone solution to act for 30 seconds.too hot for a bacterium. Let them cool down from the 60C that they are kept at but not enough to solidify. refer to Standard Methods for more instruction. e. on the bench. if fewer than 10/cm 2. use the plate(s) that will give from 30 to 300 colonies/plate. D. Microscope Slides: 1. A hand counter is also available. Allow iodine to act for 30 seconds. Wash off stain with water and then with iodine solution (Lugol’s). Work quickly or the agar will solidify in the tube before you get a chance to pour it.d e m o n s tra tio n -s ta rte d a h e a d o f la b 1. c. Test temperature on your wrist . Slide staining. Drain off excess iodine and wash freely with water and then alcohol-acetone decolourising solution. to mix the sample with the agar. place 1 loopful of sterile dilution water in the centre of a slide. a. Use the Quebec colony counter to aid in counting and a grease pencil to mark off colonies counted.coli). Instead. Multiply the number by 5 to compute estimated colonies per plate (66 cm 2 ). Allow the agar to solidify in the dish before inverting. Spread the colony in the drop of water so that you get a uniform dispersion over an area about the size of a dime. Label the slide using a grease pencil (T or A). Air dry the slide high above the Bunsen burner and fix by passing slide briefly through a flame. Incubate at 35 oC for 24-48 hours. Record results of previously prepared test. f. average and multiply by 57 (for plastic Petrie dish). count four representative squares.3. Ideally. select the appropriate dilution to count. ?3. Do not report as “too numerous to count” if all plates exceed 300 colonies. ?4. 2. Repeat for the rest of the dilutions. marking (on edge of dish so as not to impede counting) and incubating for 48 hr at 35 oC. Preferably select seven consecutive squares horizontally and six consecutive squares vertically. Counting. Flood slide with crystal violet solution and allow to act for 30 seconds. b. The aim of using several dilutions is to have at least one dilution giving colony counts between these limits. If more than 10/cm 2. pour and replace cover. 6. E.too hot for a baby . Compute bacterial density and report as “colony-forming units” (CFU) per ml. Add 100 mls of sample directly to pre-measured medium provided. 20 September 2007 CIVL 407 Lab M anual . 2. 5. Presence/Absence Testing . d. After 48 hr incubation. Take 5 tubes of melted agar from the incubator. Slide the cover of a Petrie dish back just far enough to pour the agar. If necessary. Gently swirl the dish. Examine for yellow colour (total coliforms) and fluorescence (E. Wash slide in tap water. Resterilise the loop and remove one colony from the surface of the Les Endo streaked plate (prepared previously). Do this for a typical and an atypical colony. 4. Slide preparation: With a flame sterilized inoculating loop. count colonies in 13 squares (of the colony counter) of the most dilute plate having representative colony distribution. Repeat with dilutions 5 to 2 (for Raw) and 4-1 (for Effluent).
. cocci or rods. Note: in the staining process: before decolourisation all organisms appear Gram positive (blue). BGLB and EC and AI tubes . After application of the counterstain. . . odour. blot and then dry in air high above the Bunsen burner (so as not to burn away your efforts). concave). plump rods.do not turn knobs except the positioning ones. colour. nonsporing. after decolourisation Gram negative organisms are no longer visible. or in short chains. gram positive or negative. rough.Membrane filter prepared dilutions.3 µm in size.Record Presence/absence testing results of Hach Prepared Media bottles. c. Description of bacteria: Describe what you see on the plates: colonial morphology of each type of colony seen ie surface (shiny. . h. pairs chains). and on the slide: typical or atypical. Wash in water.Come in and count Heterotrophic Plate colonies after 48 hours. Apply fuchsin or safranin (counter)stain for 30 seconds. 21 September 2007 CIVL 407 Lab M anual .g.5 . .Record positives in all Multiple Tube Fermentation inoculations: LTB. Gram negative organisms are visualized (pink). in pairs. Cells occur singly. sometimes coccus-like and 0. cell groupings (single. Examine the prepared slides . . In general. place on LES Endo agar dishes and incubate.counts will be provided on the board. . b. dull.Examine prepared slides. ?3. Schedule Summary Day 1. transparency. coliform are Gram-negative (pink). smooth. . short. Day 2. metallic. Day 3.Prepare dilutions for Heterotrophic Plate Count and Membrane Filter technique. a. length to width ratio. Microscopic examination. usually motile.Prepare and incubate Heterotrophic (Pour) Plates using dilutions above. Plates ready on a weekend will be put in cold room until Monday.Come in and count colonies on LES Endo Membrane Filter dishes.
pour plate method. 1/ 10 and 1/100 are used. 35 oC/48 hr Tube # 1 2 3 Mls of inoculum Infl Effl Plate count Infl Effl *CFU/ml Infl Effl *CFU= colony-forming units Membrane Filter Technique Tube # Dilution (mls) Infl Effl Coliform Colonies Infl Effl #Colonies/100 mls Infl Effl 1 2 3 4 5 6 4 5 6 22 September 2007 CIVL 407 Lab M anual . 1 2 3 4 5 6 Heterotrophic Plate Count . Multiply the MPN index by the dilution factor from the series used when dilutions other than 1/1.Bacteriological Examination Data Multiple Tube Fermentation : indicate the number of positives for each dilution Tube # Mls of sample/diln tube LTB 24 hr (+ or -) BGLB 48 hr (+ or -) EC 24 hr (+ or -) AI 24 hr (+ or -) Infl Effl Infl Effl Infl Effl Infl Effl Infl Effl From MPN Index table find MPN Index per 100 ml..
coli because it produces a fluorogenic product when hydrolyzed by glucuronidase. To select the three dilutions to be used in determining the MPN index. that in the denominator. E. With MUG reagent added to P/A Broth. Both media meet USEPA guidelines for testing of total coliforms in drinking water. MF results. A long-wave UV light is required to detect fluorescence. you can simultaneously detect total coliforms and E.coli. In the examples given below. They are in concentrated form and merely require the addition of 100 ml of sample (or diluted sample) to the media and incubation. MUG (4methylumbelliferyl-$-D-glucuronide) can be used to detect E. choose the highest dilution that gives positive results in all five portions tested (no lower dilution giving any negative results) and the two next succeeding higher dilutions. it is not necessary to enumerate. Use the results at these three volumes in computing the MPN index. The number in the numerator represents positive tubes.coli will fluoresce as early as 4-24 hours. the total tubes planted. If zero total coliform is the maximum contamination goal.1 ml 5/5 4/5 1/5 0. Do they agree? Why/why not? -compare MPN/MF with Heterotrophic Plate Count.Discussion: -sources of error? -compare MPN. the significant dilution results are in boldface.Most Probably Number (MPN) When more than three dilutions are used in a decimal series of dilutions.001 ml 0/5 0/5 0/5 Combination of positives 5-2-0 5-4-2 0-1-0 MPN Index /100 ml 5000 2200 20 23 September 2007 CIVL 407 Lab M anual . the combination of positives simply represents the total number of positive tubes per dilution: Example a b c 1 ml 5/5 5/5 0/5 0. an enzyme specific to E. Estimation of Bacterial Density . use the results from only three of these in computing the MPN.coli.01 ml 2/5 2/5 0/5 0. Do they agree? Why/why not? What is expected? What is a heterotroph? -were the reductions through the sewage treatment plant as expected? Additional notes Presence/Absence (P/A) Testing: Media chosen may be Presence/Absence broth (P/A) or Lauryl Tryptose broth with Bromcresol Purple broth (LT/BCP) to indicate acid formation.
0. Of Positives MPN Index/ 100 ml 22 26 27 33 34 23 30 40 30 50 60 50 70 90 80 110 140 170 130 170 220 280 350 240 300 500 900 1600 95% Confidence Limits Upper Lower <2 2 2 4 2 4 4 6 6 4 7 9 9 12 8 11 11 14 14 17 13 17 17 21 26 1 10 13 11 15 15 18 18 17 20 24 25 29 24 29 29 35 35 40 38 45 46 55 63 4-2-0 4-2-1 4-3-0 4-3-1 4-4-0 5-0-0 5-0-1 5-0-2 5-1-0 5-1-1 5-1-2 5-2-0 5-2-1 5-2-2 5-3-0 5-3-1 5-3-2 5-3-3 5-4-0 5-4-1 5-4-2 5-4-3 5-4-4 5-5-0 5-5-1 5-5-2 5-5-3 5-5-4 5-5-5 9 12 12 15 16 9 10 20 10 20 30 20 30 40 30 40 60 80 50 70 100 120 160 100 100 200 300 600 -— 56 65 67 77 80 86 110 140 120 150 180 170 210 250 250 300 360 410 390 480 580 690 820 940 1300 2000 2900 5300 ---- $1600 24 September 2007 CIVL 407 Lab M anual .1 mL) Comb. 1.Table 1 . Of Positiv es 0-0-0 0-0-1 0-1-0 0-2-0 1-0-0 1-0-1 1-1-0 1-1-1 1-2-0 2-0-0 2-0-1 2-1-1 2-2-0 2-3-0 3-0-0 3-0-1 3-1-0 3-1-1 3-2-0 3-2-1 4-0-0 4-0-1 4-1-0 4-1-1 4-1-2 MPN Index/ 100 ml 95% Confidence Limits Lower 1 1 1 1 1 1 2 2 1 2 3 3 5 3 4 4 6 6 7 5 7 7 9 12 Upper Comb.0 mL.MPN Index and 95% Confidence Limits for Various Combinations of Positive Results when 5 Tubes are used per Dilution (10 mL.
Hach .MPN Table 2 25 September 2007 CIVL 407 Lab M anual .
D. -The Use of Indicator Organisms to Assess Public Water Safety . 2) 3) 26 September 2007 CIVL 407 Lab M anual . What are the important pathogens transmitted by water? How is quality control achieved in microbiological analysis.htm Lab 5 Questions: 1) Compare the advantages and disadvantages of the multiple tube fermentation and membrane filter techniques. Mara: -Bacterial Indicators/ Health Hazards Associated with Water -Bacteriology for Sanitary Engineers Microbiology 321: -Manual of Microbiological Techniques Website: http://www.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/keen/Gram stainkeen.hach.http://www.com/ ¸Information Central/Learning Library/Microbiological Testing ASTM: (STP635) D.Further information can be found in the laboratory library (do not remove without permission): Standard Methods: -Part 9000 Microbiological Examination HACH publications:-Catalogue listing testing methods with descriptions.
Note and consider carefully: Different methods of analysis have different optimum ranges.La b o ra to ry 4: Ch lo rid e D e te rm in a tio n WARNING You will be using concentrated acid and other very dangerous chemicals. beakers. graduated cylinders. volumetric pipettes. That means you will probably have to dilute the sample by the most accurate means available. immediately. burettes. goggles or glasses and lab coats. standard Hg(NO 3)2. indicator. buffer. standard AgNO 3 solution. OBJECT: -to acquaint you with the use of specific ion electrodes. You will be told the approximate concentration in the sample and it is up to you to make sure that the sample will fit within the specified ranges. beakers. magnetic stirrer. MATERIALS: -millivolt meter. reference electrode (double junction). 125 ml Erlenmeyer flasks. spectrophotometer. pH meter with double-junction electrode and silver billet electrode. to illustrate the differences in operation and accuracy between instrumental methods and wet chemical methods. standards. Use personal protective equipment: gloves.5 mg/L). conc. known addition and calibration techniques and colourimetric and potentiometric titrations. standard chloride solution (0. semi-log graph paper. sample. 27 September 2007 CIVL 407 Lab M anual . Hint: optimum ranges will coincide with the standards provided or will be indicated within the pertinent section. indicator-acidifier reagent. NaHCO 3. If chemicals are spilled alert instructors. Mercuric thiocyanate is very toxic. chloride specific ion electrode. HNO 3.
labelled beakers. 4.= ____________________ 28 September 2007 CIVL 407 Lab M anual . 2. Standards of 1000. 3. As probe tends to drift for some time it may be expedient to select a “standard time to read” (i. Unless already done by a previous group. 2 minutes) and use it for all standards and samples. added 2 mls of ISA.SPECIFIC ION ELECTRODE Part One: Preparation and reading standards and samples 1. Calculate the chloride concentration. Determine the concentration of chloride in the sample.added to the sample "slope" = change in potential over 10 fold change in concentration of standard (use data from Step 2).e.in sample = mg/L Cl. place the lowest standard on the stirrer and lower the electrodes into it. stir and obtain a reading for it. 5. 100 and 10 ppm (mg/L) have been prepared for you and are at the meter. Using semi-log paper or software equivalent. CHLORIDE . where: E1 = E2 = E = Vo = Vs = A = S = potential of sample (mV) potential of sample plus stock addition potential change = E 2 . pour out 100 mls of each standard into separate. Unless already done by a previous group (values written on the board). Turn on stirrer and while stirring record the millivolt (mv) reading when the reading appears to be stable. 6. Method of Standard Addition :Add 5 mls of the stock (4 mg/ml) chloride solution (V s) to the sample measured in Step 3 (E 1) and take a new reading after value is stable (E 2 ). Measure 100 mls of sample. Repeat for rest of standards. rinsing and blotting dry the electrodes between each.E 1 volume of sample volume of stock solution added mg Cl. Add 2 mls of IONIC STRENGTH ADJUSTMENT (ISA) buffer to each using the plastic dropper (filled to the mark). plot the millivolt readings (linear axis) against the concentration of the standards (log axis) to produce a calibration curve. mg Cl.I.
II. After all Cl. and the sample (diluted if necessary) into separate and labelled 125 ml Erlenmeyer flasks (5 in total). 5.0.] After 10 minutes has passed (since the combined reagent was added to the blank). the three standards (2. CHLORIDE . discard and refill at least twice to rinse cuvette. III. 3. insert in the spectrometer and record the absorbance displayed on the meter. 6.. zero instrument on distilled water and obtain an absorbance value for the blank. add 5 ml of the “combined reagent” (containing ferric alum and mercuric thiocyanate solutions) to the blank first. pure blue.0 and 8. use a plastic dropper to transfer blank into a spectrophotometer cuvette (do not fill more than 3/4 ).5 ± 0.BY COLOURIMETRIC METHOD The principle of this method is based on the following formula: 2Cl. add the reagent to the second standard and so on for the rest of the standards and sample (which may need to have been diluted).+ Hg(SCN)2 º HgCl2 + 2SCN SCN . (Use extreme caution as this is a corrosive & toxic chemical. (Alternatively.0 ppm). Wait two or three minutes and then add the reagent to the first (lowest) standard. the excess mercuric ions combine with diphenylcarbazone (DPC) to produce a violet colour (the end-point).0. which then must be subtracted from the standards and sample.º HgCl2 1. Determine the chloride concentration in the sample from the calibration curve. 2.) Every two or three minutes thereafter you will rinse the cuvette with the next standard or sample.8. Add 1 ml (use pipette to measure accurately) of indicator-acidifier reagent to sample.ions in the sample combine with added mercuric ions forming a soluble but virtually undissociate-able complex. it should be rerun from the start after diluting. Marking the time (T=0). Insert cuvette into the spectrophotometer (wavelength must be set at 463 nm) and press “zero” or “ref set” (depending on model) to make the meter read zero. (No further adjustment is required after the blank or distilled water has been set to zero. the solution should be green-blue and will turn to pure blue (no green) within a few 29 September 2007 CIVL 407 Lab M anual .ions have been bound. measure 25 mls of “zero” standard or “blank” (halide-free distilled water). (Light green indicates a pH of less than 2. Pour 100 ml of sample (or a portion diluted such that the concentration is less than 100 mg/L) into a 250 ml beaker. For most samples. fill. after a few more minutes.+ Fe+++ º Fe(SCN) ++ (Reddish brown) 1. If sample is above the highest standard. Using a 25 ml graduated cylinder. 4. the 1 ml of the indicator-acidifier reagent will adjust the pH to 2. Hg ++ + 2Cl.they can’t all be at 10 minutes at once. Near the end-point. 2. [Note: this delay between additions is to allow you time to measure absorbance of each at the 10 minute time required . 4. and Hg ++ (excess) + DPC º violet colour 3.) Prepare a calibration curve by plotting the absorbance readings versus the concentration of chloride in the standards.1. The colour of the solution should be green-blue at this point.) Mix thoroughly by gently swirling the flask. a pH of greater than 3. using the dispenser provided. MERCURIC NITRATE METHOD FOR CHLORIDE DETERMINATION The principle of this method is as follows: Cl. Titrate with 0.014N Hg(NO 3) 2 to a definite purple end-point.
Immerse stir bar. 4. Start the stirrer and set the millivolt reading to zero or record initial mv reading [Note: Some meters can not be set to zero]. Begin titrating. Take care that stirrer is not hitting the electrodes. where: A = ml titrant for sample B = ml titrant for blank N = normality of titrant IV. Plot a differential titration curve to determine the exact end-point. The end-point occurs at the greatest mV change per unit addition of AgNO 3. recording the volume and millivolt reading for each increment. place beaker on stir plate and lower electrodes into the solution. and add 2.if one is necessary.4. Part one: Standard 1.2 ml or drop-wise) should be added. with standard AgNO 3. The first group will do the standard (part one) in duplicate or triplicate depending on the size of the group and the other will do the sample (part two) in duplicate (or triplicate). as the end-point of the reaction is approached (ª mV/ml increases). then.0 ml (use volumetric pipette) of standard chloride solution in a 250 ml beaker. Calculate the chloride concentration: 5. 2.1 to 0. Place 10. until past the endpoint when larger increments can be used again (ª mV/ml decreases). large increments (1-2 mls) may be added. 5. A reference set of data will be provided for comparison. CHLORIDE BY POTENTIOMETRIC TITRATION Note: For this part form two groups comprised of 1 member from each station. Determine the blank by titrating 100 ml of distilled water containing 10 mg of NaHCO 3 which serves to provide some alkalinity to the blank (to make it similar to the sample) and provides a correction for alkalinity and reagents . Calculate the Normality of the AgNO 3 using the following equation: 30 September 2007 CIVL 407 Lab M anual . The titre from this step must be subtracted from the titre for the sample. Continue the titration about 5 ml past the end-point.. 6. drops of the purple end-point. in increments. At the start. Rotate the work within the group so that different pairs are doing the titrating and recording of numbers. 3.0 ml of concentrated HNO 3 (use plastic dropper). Don’t forget to add the indicator.. Nitric acid is in the sink and is very corrosive. dilute to about 100 ml with distilled water. smaller increments (0. This way each station will have data for each part.
Add 2 ml of HNO 3 and proceed as described in Part One. Where: A = ml AgNO 3 B = ml Blank N = normality of titrant (AgNO 3) D = ml of sample ml From graph a b c mg/L Cl- S))))))))Q S))))))))Q S))))))))Q S))))))))Q S))))))))Q S))))))))Q 31 September 2007 CIVL 407 Lab M anual .Part Two: Sample 1. Measure 100 ml of sample or portion made up to 100 ml. Calculate the chloride concentration in the sample using the following equation: 3.) Plot three curves for this titration: a. Millivolts per ml versus ml of titrant. It is not. if dilution is necessary into a 250 ml beaker (use a graduated cylinder to measure). Millivolts per ml per ml versus ml of titrant. 2. which are merely first and second derivatives. be careful to use the average value of ml of titrant on the abscissa. 4. c. in this case. In plotting b and c. (If pH of the sample were quite high it would be necessary to first adjust the pH to about 4 and then add 2 ml HNO 3. Millivolt reading versus ml of titrant. b.
Chloride . 32 September 2007 CIVL 407 Lab M anual .Potentiometric Titration RAW DATA A Volume AgNO 3 Added (Vol) (ml) B Meter Reading (E) (mV) C FIRST DERIVATIVE D SECOND DERIVATIVE F G H ª (Vol) (ml) ª (E) (mV) E ave (AgNO 3 ) Volume (ml) ª (E)/ ª (Vol) (mV/ml) ª ( ª E/ ª Vol) (mV/ml) ª [Ave(Vol)] (ml) I Ave of Ave(Vol) (ml) J ª( ª E/ ª Vol) /ml (mV/ml/ml ) Extra pages are at the back of this lab manual in appendix.
hydrazine. # Environmental samples usually require complex pre-treatment. Also. From your chloride results. in itself. # Chromate. ferric. strongly reducing solutions may form a surface layer of silver. thiosulphate. specific ion electrodes are rarely usable in environmental samples. 3. iodide. In the above tests. Each sample type requires development of an appropriate method which. timeconsuming process requiring a sound knowledge of chemistry and extensive experience working with environmental samples. PRETREATMENT TECHNIQUES # There are no "standard" pretreatment techniques. cyanide. In the Mohr method for chlorides (described in Sawyer and McCarty) what would the equilibrium concentration of silver ions be (in mg/L) when the chloride concentration has been reduced to 0.INTERFERENCES IN CHLORIDE DETERMINATIONS POTENTIOMETRIC METHOD # Iodide. 2.3 mg/L? Outline the advantages and disadvantages of all the methods used in this lab to measure chloride concentration. # In practice. chromate. # Colour may obscure or interfere with end-point determination. MERCURIC NITRATE TITRATION METHOD # Iodide. # Other interferences: ferricyanide. can you see any advantage in suing the first or second derivative curve? Discuss. and nitrite interfere. 33 September 2007 CIVL 407 Lab M anual . This is a complex. # High levels of ions which form very insoluble salts of silver may deposit a layer of salt on the electrode membrane. fluoride and bromide titrate as chloride. known addition cannot correct for any ions that test as chloride ion. Lab 3 Questions 1. SPECIFIC ION ELECTRODE # All halides interfere (the electrode is a halide electrode). # Sample pH may need adjustment beyond the capacity of indicator-acidifier reagent. dichromate. It can only correct for substances that enhance or suppress the test response proportional to the amount of chloride ion present. # Environmental samples usually require pretreatment. COLOURIMETRIC METHOD # Bromide. KNOWN (STANDARD) ADDITION TECHNIQUE # The known addition techniques will only correct for certain types of interferences. requires knowledge of the compounds in the sample which are likely to interfere. and sulfite ions interfere when concentration greater than 10 mg/L. # Pretreatment of environmental samples usually required. fluoride. # Mercury must be absent from samples. ferric ion. causing electrode malfunction. # Colour in the sample may interfere in the absorbance measurement. fluoride and bromide titrate as chloride.
025 N sodium thiosulphate. Bleach/chlorine solutions are strong oxidants. starch indicator. 0. Wipe up all liquid spills immediately. pipettes. about 5 ml of glacial acetic acid (use a graduated cylinder). allow to mix again. to illustrate the concepts of free and combined chlorine residuals. phosphate buffer solution. burettes and stands.N-diethyl-p-phenylenediamine (DPD) indicator solution. pages 4-56 to 4-57 or older/newer editions. (concentrated) glacial acetic acid. Mix well (on stir plate with stir bar). When weighing chemicals at the balance. Prepare a stock solution of strong chlorine water by adding 5 mls of bleach solution (sodium hypochlorite) to about 200 mls of water in a 250 ml volumetric flask. Take a 250 ml Erlenmeyer flask and add about 1 gram of potassium iodide. 100 ml graduated cylinder. 250 ml Erlenmeyer flasks. Make up to the 250 ml and invert flask several times to mix. potassium iodide (KI) crystals. 1995. bleach or hypochlorite solution. 250 ml volumetric flasks. Titrate rapidly with the stock chlorine solution (in burette) until the appearance of a constant 34 September 2007 CIVL 407 Lab M anual . gloves and lab coats. chlorine free water. 3. and exactly 10 ml of 0. standard ferrous ammonium sulphate (FAS) solution. Rinse a burette and fill it with this stock chlorine solution.. N. PREPARATION OF STOCK CHLORINE SOLUTION (Iodometric method . Materials: -600 ml beakers. clean up all spills with a brush into a container and wash crystals down the drain. REFERENCES: Standard Methods 19th ed. about 50 ml of chlorine-free water (distilled water will do). OBJECT: -to acquaint you with disinfection of water supplies and to demonstrate techniques for the determination of chlorine residuals.La b o ra to ry 5: Ch lo rin e a n d Ch lo rin e D e m a n d WARNING Glacial acetic acid is very corrosive. 2.025 N sodium thiosulphate solution (use a volumetric pipette). add 1 ml of starch solution. I. chlorine demand and break-point chlorination. Personal protective equipment required at all times when working in the lab are: eye protection.total chlorine residual) 1.
as there is lots to do . Record the burette reading (mls) = Reading A. add full amount of KI at the start (ie 2 drops KI solution and 1 g of KI crystals) to a fresh sample. c) Dichloramine: Add about 1 g KI (to the same sample) and mix to dissolve.Breakpoint Chlorination using Free and Combined Chlorine Residuals Values 1. Place 5 ml of phosphate buffer solution and 5 ml of DPD solution in a 250 ml conical (Erlenmeyer) flask and mix. Mix usual volumes of buffer reagent and DPD indicator solution with distilled water b e fo re adding sufficient sample to bring total volume to 100 ml or the test will not work. For dichloramine concentrations greater than 1 mg/L. pp 4-43. These 6 flasks can be prepared at once to make them ready for the next step. a) Free chlorine: titrate rapidly with standard FAS (Ferrous ammonium sulfate) titrant until the red colour is discharged. Method 4500-Cl F. It is likely that the highest dose will require dilution. 2. Record the burette reading (Reading C). 3. b) Monochloramine: Add two drops of KI solution (to the same sample) and mix. please refer to Standard Methods. Go to c.) Free and combined chlorine or total chlorine: To obtain total chlorine in one reading.). if colour drifts back (indicating an incomplete reaction). Determination of Free and Combined Chlorine by the DPD Method 1. Sequentially add the necessary volumes of chlorine stock solution to each beaker at the appropriate time spacing to provide the applied dosages given by the instructor (see board). (*NOTE: If total chlorine exceeds 5 mg/L use a smaller sample and dilute to a total volume of 100 mls with chlorine-free water. Add 100 ml of sample or diluted* sample using a 100 ml graduated cylinder and mix again. Allow at least 5 minutes of time to elapse between applications of chlorine doses (use the buret containing diluted chlorine solution from Part I above) to successive beakers in order to allow enough time for titrating the free and combined chlorine residuals. Work in groups of three or four for this part. 4. mix to dissolve. d) Alternatively: (Not to be done in this lab. After 15 minutes of contact time. let stand for two minutes and titrate until the red colour (if present) is discharged. Total chlorine (Free plus Combined) = Reading C Free residual chlorine = Reading A Combined residual chlorine (dichloramine plus monochloramine) = Reading C .A Monochloramine= B-A Dichloramine= C-B For interferences. refilling burette. Let stir for 2 min and then continue titrating until red colour (if any) is discharged (Reading C).purplish-blue colour. Set up a numbered row of six 600 ml beakers each containing 500 mls of the sample of water provided (it has been prepared using Ammonium chloride and Glutamic acid). 2. Let stand 2 minutes more. calculate the chlorine concentration in the stock solution (See Data and Results section for formula). Continue titrating until red colour (if any) is discharged once again (Reading B). From the amount of chlorine solution used. IIb. 18th edition. etc. and again after 1 hour of contact time determine the free and combined chlorine (see below) in 100 ml samples taken from each of the beakers at the appropriate time spacing (15 minutes and 60 minutes after the addition of the chlorine dose to each beaker). let stand 2 min more if colour drifts back indicating incomplete reaction. titrate to colourless again. 35 September 2007 CIVL 407 Lab M anual .COORDINATE! IIa.
Guaranteed analysis is normally printed on the container and is usually 5-6%. Make a separate graph for the 15 minute and 1 hour contact time. c. Discuss your results as a function of the residual forms of chlorine present at different chlorine dosages and at different contact times. If this is true. d. b. II. How would you expect the forms of residual chlorine and their speed of formation to change as a function of 1) temperature and 2) pH? Lab 5 Questions 1. free and combined residual chlorine for each applied chlorine dose. Given: HOCl W H + + OClK eqv = 2.DATA AND RESULTS I. 250) then convert mg/L to g/100 mls = %. a. FAS titrant concentration is 1 ml = 100 µg Cl as Cl2 a. what percentage of bacteria would be killed in 10 minutes at a chlorine residual of 0. Under what conditions is the practice of break point chlorination necessary? 4. Make a plot of the three residual chlorine components against the applied chlorine dosage. If a sewage sample contains 10 x 10 6 coliforms/100 ml. Standardization of chlorine stock solution: Volume of sodium thiosulphate used Normality of sodium thiosulphate used Volume of stock chlorine solution Cl concentration of stock solution = = = = ______ mls (A) ______ (N) ______ mls (B) = _________mg/L Cl as Cl2* *To determine bleach concentration as percent you must multiply first by your dilution (i. Why is it important to determine chlorine residuals in domestic water supplies? 2. The rate of kill of bacteria by chlorination follows first-order reaction kinetics.5 mg/L if 50% are killed in 1½ minutes at this concentration? b.7 x 10-8 at 25 o C % HOCl = 27 What is the pH of the solution? 3. how many coliforms would remain after a chlorine contact time of 10 minutes? 20 minutes? 36 September 2007 CIVL 407 Lab M anual . Determine the concentration of total.e.
(mls) (Stock=_____mg/L Cl as Cl2) Applied Cl dosage (mg/L) Volume of FAS to endpoint A Volume of FAS to endpoint B (includes A) Volume of FAS to endpoint C (includes A & B) Free chlorine (mg/L) (A) Monochloramine (mg/L) (B-A) Dichloramine (mg/L) (C-B) Combined residual chlorine (mg/L) (C-A) Total chlorine (mg/L) © Time of Cl addition Time of Residual measurement 1 2 3 4 5 6 37 September 2007 CIVL 407 Lab M anual . of Cl Soln.15 MINUTE CONTACT TIME BEAKER NUMBER Vol.
of Cl Soln. of FAS to endpoint C (mls) Free chlorine (mg/L) (A) Monochloramine (mg/L) (B-A) Dichloramine (mg/L) (C-B) Combined residual chlorine (mg/L) (C-A) Total chlorine (mg/L) © Time of Cl addition Time of residual measurement 1 2 3 4 5 6 (includes A+B) 38 September 2007 CIVL 407 Lab M anual .60 MINUTE CONTACT TIME BEAKER NUMBER Vol. (mls) (Stock=_____mg/L Cl as Cl2) Applied Cl Dosage (mg/L) Vol. of FAS to endpoint B (mls) (includes A) Vol. of FAS to endpoint A (mls) Vol.
pH test paper and other water quality assessment equipment. p H. DETERMINATION OF pH 1. Ac id ity . 4. metres and calibrated glassware are available in limited quantities and are very expensive to replace. Probes. rinse & dry probe. beakers. Check the pH of a small aliquot of the sample by wetting a pH test paper and comparing the colour combinations to those shown on the case. turbidimeters. magnetic stirrer. Rinse probe when you are finished and leave immersed in pH 7 buffer for storage. Always wear personal protective equipment. Use care when using lab equipment. Calibrate the pH meter by following the procedure outlined on the instruction sheet beside the meter unless told otherwise. standard base. bromphenol blue. metacresol purple. EDTA. if above 7. turn on stirrer and obtain a stable reading. MATERIALS: -pH meter.(Do not allow the stir bar to hit the probe. Be sure that buffers are being stirred when you perform the calibration and that when you change buffers you thoroughly rinse the probe into a waste beaker and blot dry so that you do not contaminate other buffers or your samples. 3. After calibration is complete. 1 N NaOH. then use buffers 4 and 7 to calibrate. lower probe. then use buffers 7 and 10. I. OBJECT: -to familiarize you with the most common water treatment tests. flasks. graduated cylinders. phenolphthalein.) Record value in following table. Normally. colour comparator. hardness indicators. 39 September 2007 CIVL 407 Lab M anual . T u rb id ity WARNING Please use caution when handling all chemicals. particularly those labelled with specific hazards. bromcresol green. Hard n e s s . buffer solution. the two buffers that are chosen for calibration will bracket your sample ie if sample is less than 7. water samples. to acquaint you with the use of pH metres. Co lo u r. standard acid. (Take care not to contaminate paper in case.Lab o rato ry 6: Alkalin ity .) 2. pH test paper. burettes and stand. place a beaker containing the sample and stir-bar on the stir-plate.
2. Add the last few drops at 3-5 second intervals so that you do not "overshoot" the end-point. 2.EDTA TITRIMETRIC METHOD 1.. Record the burette reading in the following table. 2. add 1 drop of 0. titrate until just colourless. Add 1 drop of sodium thiosulphate solution. 4.. A pH much above 10 promotes CaCO 3 precipitate. Rinse and fill a burette with standard EDTA solution and record the initial burette reading. 3.1 N sodium thiosulphate to the sample.think what that means!) 4.. Complete the titration within 5 minutes to minimize the tendency toward CaCO 3 precipitation. Measure 100 ml of sample (with minimum agitation or exposure to air) using a graduated cylinder and transfer carefully to an Erlenmeyer flask. Apply dilution factor to obtain final result. 5. Measure out 25 mls of sample using a graduated cylinder and dilute to 50 ml with distilled water. Pour into a 250 ml Erlenmeyer flask. Titrate slowly with EDTA. 3.II. Titrate to the first uniform pink colour (colour stays). DETERMINATION OF ALKALINITY 1.?) 4. Add stir-bar.. Measure 100 ml of water sample into a clean. TOTAL HARDNESS DETERMINATION . (If solution is clear after addition of P. Measure out a new sample and add 1 drop of sodium thiosulphate and a full dropper of phenolphthalein. White paper placed under the flask will facilitate this determination. (If the solution is already blue after indicator. DETERMINATION OF ACIDITY 1. until the reddish tinge disappears from the solution and a pure blue remains. add 90% of the titrant to the sample before adjusting the pH with buffer. III. Add a dropper full of bromcresol green (BG= MO) to this same water sample and continue the titration until the colour changes from blue to yellow (at pH 4. The buffer elevates the pH to 10. while stirring. IV. Rinse and fill a labelled burette with standard acid solution. In order to remove any free residual chlorine (which interferes with the indicator colour response). if the approximate hardness is known (by unsuccessful attempts). Alternatively. Add a full dropper of bromphenol blue indicator and titrate until the colour changes from yellow to blue ("MO" Acidity). If a ppt does form within the 5 minutes. 3. (BG or MO) 6. sample-rinsed graduated cylinder and transfer to an Erlenmeyer flask.5). If sample is highly alkaline (titration goes over one burette full) dilute the sample and titrate again. 40 September 2007 CIVL 407 Lab M anual .. Add a full dropper of phenolphthalein (P) indicator and if the solution stays pink or red. Record the volume of titrant required in the following table. Add 1-2 ml of buffer solution using dropper bottle (3 full droppers) and one aliquot of Total Hardness Indicator using the special dispenser on the chemical bottle. Record burette reading (P). it may be necessary to repeat the titration with the sample diluted 1 to 1 (again) with distilled water. Rinse and fill another burette with standard base solution and label it. Add a stir-bar.
Insert the sample cell in the instrument cell compartment so the diamond or orientation mark aligns with the raised orientation mark in front of the cell compartment. 4.HACH Spectrophotometer. with continuous stirring to a pure blue end-point. The display will show SIG AVG when the instrument is using signal averaging. Record all colour data in the following table. Cap the cell.. Values are reported as APHA Colour Units (eqv. 2. Add one aliquot of the Calcium Hardness Indicator and titrate with EDTA slowly.EDTA TITRIMETRIC METHOD 1. Measure out 50 ml of sample into an Erlenmeyer flask and add 2. The instrument may suggest diluting if over range. VII.TRUE AND APPARENT PART A: TRUE COLOUR . Refill the burette with EDTA titrant and note the initial reading. Demonstration of Jackson Candle and Hellige PART A: Hach Turbidimeter 1. The display will show AUTO RNG when the instrument is in automatic range. Place the cell containing sample in it and press Read. 2.BY HACH 2100P Turbidity Meter. TURBIDITY .Enter the stored program number for true colour=120 1. 3. 3.. Wipe the cell with a soft. to determine the apparent colour (measures the influence of turbidity on the reading). CALCIUM HARDNESS DETERMINATION . Record the final burette reading in the following table. Record the final burette reading in the following table.. Add a stir-bar and stir to mix. The reading might be higher so if you had to dilute in Part A dilute for Part B. taking care to handle the sample cell by the top. Select automatic range by pressing the RANGE key. Use the technique outlined in Part A. COLOUR . Filter about 20 mls of sample using a filter funnel and stand and the pre-fluted filter paper provided 2. 5. 4. to mg/L platinum as chloroplatinate) PART B: APPARENT COLOUR 1. V. Press: I/O. Pour the supernatant into one of the cells (to the etched mark) and fill another with distilled water. Select signal averaging mode by pressing the SIGNAL AVERAGE key. Close the lid. Place the cell containing water into the spectrophotometer and press Zero. or a volume sufficient to produce a pH of 13-14 (designed to precipitate the magnesium as a hydroxide). . Fill a sample cell to the line (about 15 mL). 2. 4.5.0 ml of 1 N NaOH solution (use 3 droppers full). VI. lint-free cloth to remove water spots and fingerprints. The instrument will turn on but will turn off automatically after a short time so you should be ready. 3. Use signal average mode if 41 September 2007 CIVL 407 Lab M anual . except on an unfiltered sample.
. record the number and refer to the appropriate graph to determine the turbidity as mg/L SiO 2. that the filter selector shows "none" and that bulb B is in use. DO NOT PLACE AN EMPTY TUBE IN THE TUBE HOLDER WITH THE CANDLE LIT . Record the turbidity after the lamp symbol turns off. T = Total alkalinity. page 272-273.NTU. 3. Try to be quick and not burn the candles more than a few minutes at a time. 2.T.demonstration only.. Note that the rectangular door mirror is closed. Result of Titration Hydroxide Alkalinity as CaCO 3 Carbonate Alkalinity as CaCO 3 Bicarbonate Concentration as CaCO 3 T T-2P 0 0 0 P=0 0 0 P<1/2T 0 2P P=1/2T 0 2P P>1/2T 2P-T 2(T-P) P=T T 0 Where: P = Phenolphthalein alkalinity.. See Standard Methods. 42 September 2007 CIVL 407 Lab M anual .s. Remove about 10 mls of sample by pipette and slowly dispense this aliquot back into the tube until the shape of the flame is no longer distinguishable. Wipe the outside of the tube to dry it and place it in the turbidimeter (in the circular groove of the mirror unit). Close the door and switch on the light. 2. then the turbidity in NTU. 16th edition. Carefully fill the 20 mm viewing depth tube to the mark with sample.adding liquid to a hot tube will cause it to shatter. Note that there are several different scales on the side of the tube. Pinch all excess charred wick with fingers before lighting or soot may be deposited on the glass tubes.. 1.U. Press: READ The display will show . 3. 6. If you take too long.. (This allows you to make a more gradual and accurate approach to the end-point.) Remove the glass tube from the holder and read the turbidity at the meniscus of the sample. the turbidity may start to settle and condense on the bottom of the tube. Read the scale on the dial. Be sure that the calibrated tubes are clean before adding samples to the tubes. obscuring vision. Pour sample into the tube in small increments until you can no longer distinguish the shape of the candle flame. Record value in following table as J. PART C: Jackson Candle Turbidity .the sample causes a noisy signal (display changes constantly). Immediately balance the light intensity of the central spot with the surrounding observation field by turning the dial on the right-hand side of the instrument. Lower the plunger into the liquid making sure no bubbles are trapped under the plunger and the top is dry.Demonstration only 1. Take the reading where the dark central part first merges with the surrounding field. PART B: Hellige Turbidimeter .
O. Acidity end-point reading (mls) Initial burette reading Net "M.Sample volume Initial burette reading P.O. Alkalinity titre (mls) Net "M.Initial pH by test paper Initial pH by pH meter ALKALINITY." titre (mls) Net Total acidity titre (mls) Net CO 2 Acidity titre (mls) Final burette reading (mls) Initial burette reading (mls) Net titre (mls) Total Hardness as mg/L CaCO 3 CALCIUM Sample volume (mls) Final burette reading (mls) Initial burette reading (mls) Net titre (mls) Calcium as mg Ca ++ /L __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ ___________ ___________ ___________ ___________ ___________ ___________ ___________ ___________ Sample 2 ___________ ___________ P.O.DATA Sample 1 pH ." Alkalinity end-point (mls) Initial burette reading Net P.O." Acidity end-point reading (mls)__________ TOTAL Hardness Sample volume (mls)__________ Calcium hardness as mg CaCO 3/L __________ 43 September 2007 CIVL 407 Lab M anual .Sample volume "M." Alkalinity titre (mls) ACIDITY. Alkalinity end-point reading (mls) __________ "M.
00 ml EDTA titrant at the calcium indicator endpoint = 1.CALCULATIONS Calcium: Calcium Hardness: where: A= B= ml titrant for sample mg CaCO 3 equivalent to 1.0 Alkalinity: where: A= N= ml standard acid used normality of standard acid Acidity: where:A = N= ml standard base used normality of standard base 44 September 2007 CIVL 407 Lab M anual .0 Total Hardness (EDTA): where: A= B= ml titrant for sample mg CaCO 3 equivalent to 1.00 ml EDTA titrant at the calcium indicator endpoint = 1.
" Acidity as CaCO 3 (mg/L) Total Acidity as CaCO 3 (mg/L) Free carbon dioxide as CO 2 (mg/L) Total Acidity as CO 2 (mg/L) Ca Hardness as mg Ca +2 (mg/L) Ca Hardness as CaCO 3 (mg/L) Mg Hardness as CaCO 3 (mg/L) Mg Hardness as Mg+2 (mg/L) Carbonate Hardness as CaCO 3 (mg/L) Non-carbonate Hardness as CaCO 3 (mg/L) Excess alkalinity as CaCO 3 (mg/L) Colour .T.Alkalinity as CaCO 3 (mg/L) CO 3 -2 Alkalinity as CaCO 3 (mg/L) HCO 3.APHA Colour Units: True Apparent Turbidity .O.RESULTS (sample) P.O. ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 45 September 2007 CIVL 407 Lab M anual .U.Alkalinity as OH .ion (mg/L) CO 3 -2 Alkalinity as CO 3 -2 ion (mg/L) HCO 3.Alkalinity as CaCO 3 (mg/L) OH ." Alkalinity as CaCO 3 (mg/L) OH .ion (mg/L) "M.Alkalinity as HCO 3 . Alkalinity as CaCO 3 (mg/L) "M.Units= N.Hach .
A water has the following analysis: Na + K+ Ca +2 15 mg/L 33 mg/L 12 mg/L ClSO 4-2 HCO 3 70 mg/L Mg+2 18 mg/L 42 mg/L 8 mg/L What is the carbonate. Note: You should bring a copy of this lab to the next two sessions as you will need the same instructions to perform the same tests in the Coagulation and Softening Labs. non-carbonate and total hardness of this water in mg/L as CaCO 3 ? in meq/L? Is the analysis complete? Why? 6.7 x 10 -11 and K w = 10 -14. calculate the carbonate alkalinity in mg/L as CaCO 3 for the water sample analyzed during this lab. What errors can occur in the calcium hardness determination? Why? 3.Lab 6 Questions 1. From equations (17-12) and (17-13) in Sawyer and McCarty. given K 2 = 4. Does this agree with the results from Part II of this lab? Explain. Is this water suitable for a domestic water supply? Why? 2. What sanitary significance does colour have? 4. 46 September 2007 CIVL 407 Lab M anual .
7 (except Jackson Candle and Hellige Turbidimeter). You should have with you the part of Exercise 7 that gives instructions for the various tests. OBJECT: -to illustrate the principles of coagulation and water softening. 1000 ml beakers. Several lime and soda ash dosages will also be selected for determining the efficiency of water softening. In the second lab you will use the previously determined dosages to coagulate and soften the sample and then re-analyse the resultant water. MATERIALS: -Jar Test Apparatus. and all of the materials from Lab. The optimum alum dosage for coagulation of the sample will then be chosen. to be conducted over two lab periods. The first step will be to analyse the raw water sample and the supernate from a number of alum dosages (to be announced in the class) using the techniques learned in Lab.Lab o rato ry 7a: Co ag u latio n an d 7b : Co ag u latio n & So fte n in g ATTENTION This lab will require patience and cooperation from all of you as we have only two jar testers with 6 paddle sites each. No. This is a two-step experiment. soda ash solution (Na 2CO 3) and lime (Ca(OH)2) or sodium hydroxide (NaOH).for the next session (b). No. alum solution (Al2 (SO 4 )3 @18H 2 O). 47 September 2007 CIVL 407 Lab M anual . Please try to coordinate your work with the rest of the class.6.
Make sure it is centred. and clarity of supernatant liquid. Lift paddles out of beakers and secure. but because of equipment limitations. Reduce the stirring rate to about 20 rpm for 10 minutes. hardness (total and calcium). Stir at 100 rpm for 1 minute. Add the designated lime (or NaOH) and soda ash additions as quickly as possible to the decanted samples. floc volume. In the lecture. turbidity and colour. 3. drop of 0. medium. cloudy). 1. times of appearance and descriptions and record. clarity of supernatant liquid (very clear. alkalinity. Stop stirrer and allow floc to settle. Analyze these supernates plus a sample of raw water for pH. true colour and turbidity (Hach). Observe and make notes describing floc formation and note the time of appearance. 7. Add the assigned alum dosages as quickly as possible and then start the stirrer. (NB dosage now based on 800 ml sample size). Complete the coagulation summary sheet during the lab period. A pH meter could be used for the alkalinity and acidity determinations. Stir at 100 rpm for 1 minute. 5. 4. Analyze the supernates for pH. then reduce stirring rate to about 20 rpm for 10 minutes (just fast enough to keep the floc suspended but not so fast that the floc is sheared). and fill in the table on the board. small. hazy. Stop stirring. acidity. Make relative estimates of floc sizes.5 to 1 cm off the bottom of the beaker. acidity. moderate. slow). You will also determine the optimum lime and soda ash dosages to soften this water and select a range of dosages surrounding this optimum for investigation next week.Part A:. Part B: WATER SOFTENING PROCEDURE (to be done the following week) again work as teams of 2 or 3. Measure out six 1000 ml aliquots of sample (using graduated cylinder to measure. not a beaker ) and transfer to 1000 ml beakers. After the floc has settled (allow 15 min). 4. if time permits. You may need to use blocks to position the paddle 0. Place beakers on the jar tester apparatus. use indicators as specified in Lab 6.1 N sodium thiosulphate 48 September 2007 CIVL 407 Lab M anual . Record observations on settling rate. Record relative floc sizes (pin-point. decant the supernatant liquid carefully into a clean set of beaker (try not to stir up what has settled) and discard the floc.. Brief Methods Summary for Analyses Alkalinity: @ 100 ml of sample. decant the supernatant liquid carefully into another set of beakers (try not to stir up what has settled).work in 2 or 3 teams to assess a total of 6 dosages for each team. 3. 5. 2. position them under stirrers. 1. 6. and settling characteristics of the floc (rapid. large). Discard the floc and clean beakers in preparation for a second decanting in Step 4. Pour six 1000 ml aliquots of sample into 1000 ml beakers and place on the jar test stirrer apparatus. if available. Add the appropriate alum dosage and immediately start stirring. Carefully decant into a graduated cylinder 800 mls of the supernatant liquid and pour into fresh beakers. After the flocs have settled (allow 15 min). you will discuss the data and select the appropriate alum dosage to be used by all groups in the water softening step next week. hardness (total and calcium). 2. Stop stirrer. Stir at 100 rpm for 1 minute and at 20 rpm for 10 minutes. Determine which dosages were most appropriate for this sample. 6. alkalinity. COAGULATION USING ALUM . clear.
3 and with bromcresol green (continuing with the same sample) to yellow .pure blue).02 N acid with phenolphthalein to colourless . 3.pure blue).7 ("methyl orange" acidity) and with phenolphthalein (using a fresh sample) to pink . 2. What treatment would you recommend to prepare this water for human consumption? Why? 49 September 2007 CIVL 407 Lab M anual . show by calculation the theoretical amount of lime and soda ash required for excess lime/soda ash softening. @ calculation: hardness (mg/L CaCO 3 )= mls titre x 1000/mls sample Calcium Hardness: @ 50 ml sample @ add 1 ml 1 N NaOH (or to pH 13) and Ca Hardness Indicator (Hydroxy Naphthol Blue) @ titrate quickly with EDTA to blue end-point (no pink/purple tinge .3 ("phenolphthalein" or total acidity) @ calculation . Softening 1.alkalinity (mg/L CaCO 3)= mls titre x N acid x 50. Based on your chemical analysis of the raw water.02 N base with bromphenol blue to blue .@ titrate with 0.5.use graphical presentation.pH 4. @ calculation . 2. Compare your experimental softening results to the theoretical results based on solubility equations or mass balance equations.pH 3. Compare removal of hardness components under the different doses .pH 8. @ calculation: Ca hardness (mg/L CaCO 3 )= mls titre x 1000/mls sample Lab 7 Questions A.000/mls of sample Total Hardness: @ 25 ml sample diluted to 50 ml @ add 1 ml buffer and Total Hardness Indicator (Calmagite) @ titrate quickly with EDTA to blue end-point (no pink/purple tinge .acidity (mg/L CaCO 3)= mls titre x N base x 50. drop of 0.000/mls sample Acidity: @ 100 ml of sample. How does the consumption of alkalinity by alum compare with theoretical calculation based on balanced equations? How much extra soda ash should you add to compensate for the alkalinity consumption by 70 mg/L alum? B. Coagulation 1. 4.pH 8. Analyze the response of colour and turbidity removal as a function of alum dose.1 N sodium thiosulphate @ titrate with 0.
sample vol Net titre Notes: 50 September 2007 CIVL 407 Lab M anual .Coagulation Raw Data Sheet DATA Raw Sample Alum mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L pH Alkalinity-sample vol ml Net titre to pH 8.5 “P” Net titre to pH 4.sample vol ml Net titre to pH 3.5 “TOT” Total Hardness sample vol Net titre Ca Hardness .5 “TOT” Acidity .7 “MO” Net titre to pH 8.
) True Colour (A.A.) unfiltered Notes: 51 September 2007 CIVL 407 Lab M anual .P.Coagulation Summary Sheet DATA Raw Sample Alum mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L pH Floc size Clarity Floc settling rate Alkalinity (mg/L CaCO 3 ) “P” Alkalinity (mg/L CaCO 3 ) “TOT” Acidity (mg/L CaCO 3 ) “MO” Acidity (mg/L CaCO 3 ) “TOT” Total Hardness (mg/L CaCO 3 ) Ca Hardness (mg/L CaCO 3 ) Apparent Colour (A.P.U.H.) (filtered) Turbidity (N.H.T.A.
5 Total Hardness sample vol Net titre Ca Hardness .5 Acidity .Coagulation and Softening Raw Data D ATA Lime/NaOH mg/L Soda Ash mg/L Alum mg/L pH Alkalinity-sample vol ml Net titre to pH 8.sample vol ml Net titre to pH 3.7 Net (Total) titre pH 8.sample vol Net titre Notes: 52 September 2007 CIVL 407 Lab M anual .5 Net (Total) titre pH 4.
-mg/L CaCO 3 **Non-Carb Hard -mg/L CaCO 3 **Excess Alk. -mg/L CaCO 3 Apparent Colour (A.P.) True Colour (A.T.Alkalinity -mg/L CaCO 3 *Total Alkalinity-mg/L CaCO 3 “MO” Acidity -mg/L CaCO 3 *Total Acidity -mg/L CaCO 3 Total Hardness -mg/L CaCO 3 Ca Hardness -mg/L CaCO 3 Mg Hardness -mg/L CaCO 3 **Carbonate Hard.Coagulation and Softening Summary Sheet Data Lime/NaOH mg/L Soda Ash mg/L Alum mg/L pH P.U.A.H. Calculation 53 September 2007 CIVL 407 Lab M anual ** By .) (Filtered) Turbidity (N.A.P.H.) Note:* Total Alkalinity must include Phenolphthalein Alkalinity just as Total Acidity must include “MO” Acidity.
. . . . . . . . . . . 118 54 September 2007 CIVL 407 Lab M anual . . . . . Hach Absorption Method . . . . . . . . . . . . . . . . . Equilibrium Relationships of Chlorine . . . 94 20. . . . . . . . . . . . . . . . . . . . Bacteriological Examination . Hardness Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 14. . . . 64 5. . . . . . . . . . . . . . . . . . . . 110 27. Reading and Reference Material . . . . . . . . . . . . . . . . . . . Standard Solutions. . . . . . . . . . . . Turbidity. . . . . . . . . . . . . . Solids Removal in Wastewater Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Colour. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 18. . . . . . . . . . . 104 24. . . Alkalinity. . . . . . . 71 10. . . . . . . . . . . . . . . . . . . . . . . Milliequivalent and mg/l as CaCO 3 . . . Extra tables for Chlorine Lab . . . . . . . . . . 70 9. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bacteriology and Water-related diseases . . . . 69 8. . . . . . . . . . . . . . . . . . . Chemical Requirements for Water Softening . . . . . 102 23. . . . . . . . . . . . . . . . . . . . . . . . . . Water Softening . . . . . . pH . . . . . . . . . . . . . . . . . . . . . . Titrations . . . . . . . . . . . . . . 73 12. . . 68 7. . . 96 21. . . . . . . . . . Typical Problems for Acid-Base and General Chemistry . . . . . . . . 92 19. . . . . . . . . . . . . . . . . . . . . . . Hardness. . . . . . . . . Introduction . . . . . . . . 74 13. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Known Addition Technique . 84 17. . . . . . . 72 11. Faecal Coliforms . . . . . . . . . . . . 55 2. . . Concepts and Definitions . . . . . . . 67 6. . . Normal Solutions and Equivalent Weights . . . . . . . . . . . . . . . . . . . . 83 16. . . . . . . . . . . . . . . . . . . . . . . DO. . . . . . . . . . . . . . . . . . . . . . . . . 112 28. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Levels and Water Use . . . . . . . . . . . . . . . . . . . . . . . . Instructions for Laboratory Report Writing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chlorine . . . Chloride Analytical Methods . . . . . . . . . . . . . . Bar Diagram Method for Water Softening Problems . . . . . . Balancing an Oxidation Reduction Equation . . .4 Appendices see: CIVL407Manual_CourseNotes. . . . . . . . . . 57 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Coagulation . . . . . . . . . . 82 15. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 25. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 29. . . . . . . . . . . . . . . . . .Additional Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .COD . . . . . . . . . . . . . . . . Residues . . 100 22. . . . . . . . Periodic table . . . . . . . . . . . . . .pdf 1. . . . . . . . BOD. . . Alkalinity Species as a Function of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Chlorine Demand . . . . . . . . . . . . . . . . . 60 4. . . . COD . . . . Acidity. . . . . . . . . . . . . 107 26. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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