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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:
firstname.lastname@example.org email@example.com firstname.lastname@example.org email@example.com
(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
50 and 100 ml. calculators. Please ask for assistance if chemicals are spilled. please get attendant help. Wearing lab coats out of the lab is forbidden. packs.0. no matter how minor. Broken Glass: Be careful not to break glass.clean and dry. Personal Protective Equipment: Lab coat (long). Please take your labcoat away in a plastic bag. wash down and dry the bench. eye protection.2 First Session: Laboratory Safety and Orientation 1) 2) 3) 4) NO Food or drink. Sanitation: antibiotic soap for hand washing. A small amount of liquid always remains in the tip and must not be blown out. Be aware that handling your pens. If you cut yourself. 25. Safety Equipment: Eye Wash Station.. 1. Hygiene: At times you will be working with sewage. with the tip against the side of the receiving vessel.0 ml. They are calibrated by weighing the volume of distilled water that will flow from them by gravity. Clean-up: You must leave your work station as you found it . with contaminated gloves will contaminate those items. They are available in 0. DO NOT PUT BROKEN GLASS INTO THE REGULAR GARBAGE CONTAINER. Spills: Wipe up all spills immediately.0.up to 10. Pipettes/ graduated cylinders/ beakers/ flasks: 5) 6) 7) 8) 9) 10) Volumetric pipettes are the most accurate way to measure volume. No sandals or open-toe or heel shoes. if there is room. but if you do please ask for assistance. 20. You must be informed of the hazards of a substance before using it. Do not put lids and small items onto racks that may slip through to the filthy drip tray.5. gloves as required. These should be used whenever standard solutions are measured or when upmost accuracy is required. MSDS: is a supplier information sheet that must be available for each product and describe the hazards and necessary handling requisites. 2. Safety Shower. 15. Remember to empty and rinse burets as well. WHMIS: Workplace Hazardous Materials Information System is a government regulation. Most pipettes are calibrated “to deliver” or TD and should never be blown out. Some pipettes may be “to 3 September 2007 CIVL 407 Lab M anual . etc. Putting pens in your mouth that may have been on the bench or handled with gloves could be hazardous.. disinfectant for bench surfaces. 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. Pipettes must be placed with tips pointing up into the pipet basket supplied.
Additional supplies are available in the supply room (around the corner). some are not meant to drain completely . Chemical residues can be splashed in you face. Use the size of cylinder CLOSEST TO THE VOLUME YOU WISH to measure for best accuracy. 100 mls. They can be unpredictable in force and are close to eye level. They are containers best suited for “swirling” liquid ie to mix reagents with samples in a colourimetric determination (Lab 4 or 6). 10 mls. They are calibrated the same way as TD except that the remaining drop is blown into the receiving vessel. 250 mls. 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. not wastefully as quantities are limited. especially at smaller volumes. chemicals or for titrations.pay attention to the markings. 1.deliver with Blow-Out”. They are usually identified by a double etched or coloured band at the top of the pipette. 12) Cup sinks: Please do not use these sinks for the disposal or delivery of liquids. Pipettes are in drawers indicated. Graduated cylinders: Not as accurate as pipettes. Make sure that you label beakers and flasks to avoid confusion and waste. Erle n m e y e r or conical flask markings are not accurate.. markings are not accurate at all . Flasks: Vo lu m e tric flasks are very accurate and should be used when diluting standards (together with volumetric pipettes). YOU WILL USUALLY NEED THE INFORMATION GIVEN TO COMPLETE YOUR LAB WRITE-UPS. Even the force of air from the air taps can be dangerous because it can contain grit or water. Some are blow-out. They are useful for measuring samples 20 mls . 2 & 4 L Beakers: are cylindrical in shape with straight sides and flat bottoms. 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. If liquid should enter a bulb or pump.merely approximations. Do not squish bulbs in the direction of another person. 50 mls. LISTEN AND MAKE NOTE OF VERBAL AND CHALK BOARD INSTRUCTIONS. Cylinders are available: 5 mls. Undetected gas leaks can be deadly. 13) Stir bars: Please remove from containers before pouring contents down the drain. Ie If you want to measure 20 mls. 500 mls. 25 mls. 14) Glassware/supplies: Each station has a drawer where some glassware and other useful tools will be kept. A demonstration will be given. do not use a 1 L graduated cylinder. 4 September 2007 CIVL 407 Lab M anual . please rinse out immediately and set aside to dry. Chemicals will be put out as required for each lab and should be used sparingly. NEVER mouth pipette. Used to hold/transfer approximate volumes of samples.1000 mls.
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.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. Sufficient detail should be provided so that what was done could be understood and the experiment could be repeated from the procedure reported. 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. 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. 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.
SETTLEABLE SOLIDS 1. Mix the samples thoroughly and fill individually marked Imhoff cones to the 1 litre mark.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. distilled water bottles. Do not include any surface floating material as settled solids. gently dislodge any solids that have clung to the sides using a stirring rod. evaporating dishes. oven muffle furnace. if necessary. wipe up all spills and wash with disinfectant. 6 September 2007 CIVL 407 Lab M anual . A. tin dishes.. settle for 15 minutes longer. Do not wear lab coats outside of lab and do not take away from lab unless stored in a plastic bag. Settle for 45 minutes. MATERIALS: -Imhoff cones. Use pipette bulbs. 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. final effluent. If large pockets of liquid form between the particles of settled matter. and record the volume of settleable solids. graduated cylinders. to measure sewage treatment plant efficiency in removing residue. 2. A 1 L graduated cylinder may be used in place of the Imhoff cone for the sludge sample. sludge. keep hands and pencils away from your mouth and wash hands frequently. subtract an estimated volume from the measured volume of matter. samples of raw sewage. Alternatively a place will be provided in the lab to store your coat. balance. desiccators.
tare weight. Wet filter with a small volume of distilled water to seat it. 10 mls for Raw & 5 mls for sludge) of well-mixed sample and filter entire portion before pouring another portion. Obtain the tare weight of a properly prepared porcelain evaporating dish (ie one that has been washed. The samples will be fired at 550° C for one hour. position the filter and begin suction (vacuum tap). After drying overnight. TOTAL SOLIDS and TOTAL VOLATILE AND FIXED SOLIDS AT 550o C 1. stir the beaker contents and then rapidly (so that it doesn't settle) measure a volume of sample into the appropriate graduated cylinder. 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 . Suggested portions: 100-200 ml of final effluent. The dish containing the dried total solids can now be placed in a muffle furnace or. 2.not beakers. rinse the cylinder. 6. [Hint: pour out small portions (say 25 mls at a time for Effl. 4. rapidly pour sample into a 50 ml graduated cylinder to approximately equal 35-40 mls (if using 50 ml dishes). if instructed. dried and fired). Carefully remove filter from filtration apparatus and transfer it back to the aluminum dish. immediately after stirring the beaker. 3. The loss in weight represents the volatile solids lost from the originally measured sample volume. C. on a cart in preparation for staff to do so when all are ready. 5. 3.] Rinse the graduated cylinder with small amounts of distilled water and add to filter.[Note: Sludge Volume Index is the volume occupied by 1 gram of sludge after 30 minutes of settling.] Using very small amounts of distilled water. prior to session). pre-fired and desiccated . Record the total volume filtered. This is a different test to the one we are doing using Imhoff cones and should not be confused. pour about 500 mls (for Part B & Part C) into a beaker and then. Do not write on crucibles as high temperatures during the next steps will burn writing off. Once dishes are at room temperature they can be re-weighed. Pinch sides of dish in a bit to protect the filter from oven drafts. adding the rinsings to the dish. Make sure that you have recorded all the pertinent details: Dish #. Obtain the gross weight of the dish [Note: it should be room temperature before weighing. 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. SVI= settled sludge volume (ml/L) x 1000/suspended solids (mg/L)] B. Subtract the tare weight to obtain the total solids weight and calculate the mg/L value. Quickly record the exact volume and pour the sample into the dish.]. Assemble filtering apparatus.TOTAL AND VOLATILE 1. Calculate the mg/L volatile and fixed solids. Mix the sample in the carboy thoroughly. Obtain the tare weights of three aluminum dishes each containing a glass fibre filter (already pre-washed. the solids remaining represent the fixed solids. when filtering slows significantly do not add more. 2.so be quick.done for you. sample source and sample volume. [Notes: to allow for rinsing and to avoid spillage when transferring dish to oven don't pour more than 40 mls]. dried. Put the dish in the 103-105 °C oven for drying. The furnace will then be allowed to cool significantly before dishes are removed to a desiccator. Conversely. the dish will be transferred to a desiccator. .[Note: it may be harder to wash out solids if you allow them to settle before pouring into the dish . Using the sample poured out for Part B (for consistency). SUSPENDED SOLIDS .
if any. compute the sludge volume index (SVI). volatile. For the time being. overnight). Please return tomorrow to obtain final weight. What major types of solids are removed in primary treatment? in secondary treatment? 2. % reduction in settleable matter _________ % reduction in total solids _________ % reduction in volatile solids _________ % reduction in fixed solids _________ % reduction in suspended solids _________ % reduction volatile susp. From the above determinations it will now be possible to obtain a value for the dissolved solids present in the sample.50. 8. Lab personnel will perform the timed-firing after the class. What.) a) a bacterium b) sodium chloride c) sugar d) clay 8 September 2007 CIVL 407 Lab M anual . Transfer dish to a desiccator. If sludge is used in Part A. If all of the volatile solids reduced is given off as gas and if the specific gravity of the volatile solids is 1. 6. where the volatile solids are reduced from 65% to 40%. They must be allowed to cool completely before re-weighing them to determine the loss on ignition or volatile suspended solids. DO NOT DISCARD! The gain in weight represents the total suspended solids of the sample. Classify each of the following into its proper solids category(s) i. 6. Discuss the meaning and significance of this index.e. place the dishes on a cart designated by the instructor. 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). Calculate as the mg/L total suspended solids. A raw sewage sludge goes through an anaerobic digestion process. Your hands may have moisture and natural and/or applied oil/lotion on them. fixed (use more than one adjective to describe each substance. cool and weigh. Calculate the percent solids reduction through the treatment plant. dissolved. what is the percentage reduction in solids. After firing the dishes will be moved to a desiccator.5. What two techniques can be used to overcome or correct for the loss of material from filter pads when firing at 550 oC? 4. solids _________ % reduction fixed suspended solids _________ % reduction dissolved solids _________ Lab 1 Questions 1.3 and fixed solids 2. 7. 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. Enter all data in the tables on following page. suspended. Calculate the mg/L total volatile solids and total fixed suspended solids. 3.
Settleable Matter After 1 hour (mL/L) B.oven dry (G) Tin dish with filter tare weight (G) Total suspended solids (G) Total suspended solids (mg/L) Gross weight .fired (G) Loss on ignition (G) Volatile solids (mg/L) Fixed solids (mg/L) Percentage fixed residue C.DATA AND RESULTS Raw Sewage Aerobic Sludge A. Total Solids 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 embossed mark) Sample volume (ml) Gross weight . Suspended Solids Tin dish marking (do not use ink .oven dry (G) Dish tare weight (G) Total solids (G) Total solids (mg/L) Gross weight .use permanent mark on dish) Sample volume (mL) Gross weight .
Materials: Raw sewage and final effluent samples. homogenization of samples containing suspended solids may be necessary in order to get a representative sample and to improve reproducibility. D O a n d B O D 5 WARNING You will be using concentrated acid and other corrosive and toxic chemicals. Wipe up all spills immediately. otherwise reagents will run to the bulb end and make pipetting difficult and contamination likely. OBJECT: -to familiarize you with the commonest tests run for assessing treatment plant efficiency and pollution loading to receiving waters. 200.Closed Reflux. The closed reflux method is more economical but because a small sample volume (2 mls) is used. Always use and store pipettes with the top higher than the tip. as well as handling samples probably containing pathogenic bacteria. etc. 10 September 2007 CIVL 407 Lab M anual . 400. to illustrate the shortcomings of these tests. Wash hands frequently with soap. and to demonstrate the use of dissolved oxygen probes. Keep fingers.La b o ra to ry 2: CO D . Colourimetric Method NOTE: Open reflux method is the most accurate means of determining COD because a large sample size is used (20 mls). Digests from either method can be titrated or read colourimetrically. wash/rinse bench tops with water/disinfectant. NEVER pour water into acid. I. potassium hydrogen phthalate standards: 800. vials with pre-measured reagents. pipettes. 100 and 50 mg/L as COD. spectrophotometer. COD . etc out of your mouth. pencils. Hach block digester.
alkaline-iodide-azide reagent. thermometers. At your station you will find four vials with pre-measured solutions containing potassium dichromate and sulfuric acid (prepared according to standard methods. II. Read your samples and the set of COD standards: 800. 50 and 0 COD as mg O 2 /L. Standards have been digested for you. 2. Remove the vials and allow them to cool to room temperature (use cold water to speed up if necessary).the azide modification of the iodometric method) and a dissolved oxygen probe and meter. aerated dilution water.Procedure: 1. a volumetric correction must be made to the value obtained from the curve. a smaller aliquot (making volume up to 2 mls with distilled water) or 2 mls of a pre-diluted sample can be used. The four liquids are cold tap water. burette stand with burettes. into the appropriate tubes and screw cap on snugly. Allow samples to digest for 30 minutes (normally this would be 2 hours). 400. 500-ml Erlenmeyer flasks. 5. which is set at 600 nm to read absorbance of light by the sample. Take vials to fumehood and place in block digester . DO NOT ADJUST THE SETTINGS ON THE METER. and are at the spectrophotometer. Make sure sample is well mixed with the acid contents of the vial. The instructor will demonstrate the use of the spectrophotometer. The probe has been previously calibrated. dilution water. use wide mouth graduated pipettes) in duplicate.noting the location of your well-labelled. except without mercuric sulfate). manganous sulfate. [Note: it should look homogenous and be careful it will be very hot!] 3. concentrated sulphuric acid. Raw Sewage (Infl) and/or Effluent (Effl) in the upper 1/4 of the tube using permanent markers. 11 September 2007 CIVL 407 Lab M anual . in which case. 6. absorbance versus concentration and calculate the COD in samples. You will use a chemical titration (Winkler . starch indicator.025 N sodium thiosulphate. 0. raw sewage and final effluent. If less than two mls of sample or diluted sample was used. Note: You are going to measure DO on four different liquids using two different methods. well-mixed samples in the 25-position digester. before the lab. Prepare calibration curve. Pipette 2 mls of sample (using volumetric pipettes unless particulate is present. 100. The vials should be clean and dry on the outside. 250-ml graduated cylinder. 4. BOD bottles. Put identifying labels on each tube ie your initials. 10-ml graduated pipettes. DISSOLVED OXYGEN (DO) Materials: Raw sewage and final effluent samples. 200. If sample is known to be outside the standard range (above 800 mg/L).
Record the temperature of each sample using the DO meter or a thermometer. WINKLER TITRATION (Azide modification) NOTE: Use only the droppers attached to the reagent bottles for dispensing into samples. 7. Titrate this volume with 0. Use the specially marked volumetric flask to dispense the 201 ml. 8. using the DO probe and meter. as demonstrated. taking care not to entrain additional air. 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. (Fill carefully. The chemical floc should completely dissolve. Determine the percent saturation for each sample. (Note: biological solids originally present in the sample will not dissolve. Place the bottles in the sink and one at a time remove the stopper. Instructions on the use of the probe will be given in the lab. B. 9. 2. The DO concentration in mg/L is equal to the number of ml of titre as long as 201 ml is titrated.) You can save these bottles for B. 1. invert several times again and allow to settle again. (Hint: if you measured very low DO level with the meter. Stopper the bottles without trapping any air bubbles. Tip out the liquid from the area around the stopper and invert several times to mix thoroughly. until the first complete disappearance of the blue colour.think about it!) Complete all four titrations. 12 September 2007 CIVL 407 Lab M anual 3. then the same sample will require little or no titre and may not turn blue when starch is added . Fill a BOD bottle with each of the four samples (or use ones saved from A ). 2. 6. Take each stoppered bottle. 1.025 N Na 2 S2 O 3 until only a faint yellow or "straw" colour remains. Allow the floc to settle to about 1/3 the bottle volume. To the same bottles. (Note: DO meter temperature is usually not accurate. Do not mix droppers and do not allow tap water into the reagent bottles. For the cold tap water sample. When the floc has settled the second time to about 1/3 bottle volume. Set back in sink. Neglect any reappearance of the blue colour.A. Record the volume of titre. . to thoroughly mix contents. remove the stopper. submerge the hose in the bottle and allow the water to overflow for 15-30 seconds). raw sewage or influent and final effluent). 4. Add enough starch solution to give a strong blue colour (one dropper full) and continue to titrate. Determine DO. dropwise. DISSOLVED OXYGEN PROBE NOTE: DO NOT add any reagents to the samples used for the probe method. add 1 ml of alkaline-iodide-azide solution the same way. for each of the four samples. aerated dilution water.) Transfer 201 ml of each bottle (do one at a time) to a 500 ml conical flask. again quickly replacing the lid. 5. Note: if solution already seems pale yellow add starch right away. right to the top and place stopper on bottle. add 1 ml of concentrated H 2 SO 4 and quickly restopper. Fill four BOD bottles with the four samples (cold tap water. tip the liquid out of the space around the stopper (caution -it may be corrosive) and invert several times.
Pair up the mg/l of dissolved oxygen you measured and the temperature of the water in degrees C. 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. Raw Sewage Final Effluent Tap Water Dil’n Water 13 September 2007 CIVL 407 Lab M anual . use the saturation chart above. Draw a straight line between the water temperature and the mg/l of dissolved oxygen. (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. The percent saturation is the value where the line intercepts the saturation scale. (mg/L) DO conc.Dissolved Oxygen Data Sample/Data Bottle # Sample Volume (mls) Initial Buret reading Final Buret reading Net Titre (mls) DO conc.
groups). BIOCHEMICAL OXYGEN DEMAND Note: Because of the logistics of organization some groups may have 6 day BOD 5 instead of the usual 5 day. These caps are designed to prevent the evaporation of the liquid around the stopper. 4. remove bottles and measure the DO. Place all bottle sets in the 20 o C incubator for five days (or 6 for Tues. carefully measure the waste water samples into BOD bottles. ie Rinse the filling tube if it has been in the sample. Using the volumes and samples provided by the instructor. Calculate the 5-day (or 6-day if unavoidable) BOD. fill one BOD bottle with DILUTION WATER alone. Carefully top up the BOD bottles with DILUTION WATER. 3. All bottles should have liquid in the well around the stopper . 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). BOD bottles. Note: Each group should have 9 BOD bottles in the incubator. graduated cylinders. 5. thereby maintaining a water seal for each bottle. 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. to bring the levels above the ground glass neck of the bottle. preferably using the same meter and probe used to get IDO reading.do not write on the bottles and do not use the numbers on the lids. it may indicate that probe may not have been calibrated properly or the dilution water had been contaminated. Materials: Raw sewage and final effluent samples. There should be negligible BOD in the Dilution water. In addition. being sure to exclude any air bubbles that might be trapped under the stopper by adding dilution water. 1. Measure the Initial DO of the blank using the calibrated probe and meter. but if there is a significant BOD then it will have to be subtracted before the dilution factor is applied in the calculation.III. Because you want to be sure that the dilution water has no BOD. be careful not to contaminate it with sample. showing sample calculations.if not add a some dilution water (or distilled water) to the well. The blank is used to check that the dilution water has negligible BOD. Measure the Initial DO using a calibrated probe and meter. After five (or six)days. 14 September 2007 CIVL 407 Lab M anual . Be sure to note this in your lab write-up. If the dilution water BOD is greater than 1 ppm. 10-ml graduated pipettes. Dissolved oxygen meter and probe. dilution water. Stopper the BOD bottles. 6. Place plastic caps over all bottles. Be sure to record the bottle numbers and sample volumes on the following table .
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. mg/L = DO of diluted sample after 5 d incubation at 20 oC. mg/L. 15 September 2007 CIVL 407 Lab M anual .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. DO of diluted sample immediately after preparation.
If an industrial waste has a COD of 450 mg/L. Diln. the other 0.Summary Raw Sewage. 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. One waste has a K value of 0. Diln. Diln.25. what would you expect its I) BOD 5 and ii) BOD L to be. titrimetric method. 1 Raw Sewage. Both wastes have equal BOD 5 temperature and volume. 6) 7) 8) 16 September 2007 CIVL 407 Lab M anual .) Why is your COD result different from the BOD result? 5. 2 Final Effluent. Calculate theoretical oxygen demand for 300 mg/L of methyl alcohol. if at all possible.17. 2) 3) 4) Show on a graph the approximate dissolved oxygen sag curves in a river receiving two wastes. open reflux. Diln. 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.
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 . Students will do only sections marked ? MATERIALS: Multiple tube method: dilution tubes. OBJECT: -to demonstrate different methods for the determination of the coliform group of organisms and to acquaint you with bacteriological procedures. Brilliant Green Lactose Broth tubes.. Keep hands. sterile dilution water. T h e re f o re . DO NOT MOUTH PIPETTE. Wash hands with soap before leaving. membrane filters. etc. lab coats. wipe up all spills and disinfect the area of the spill. filtering apparatus. eye protection. agar tubes. Wipe lab benches with disinfectant before and after each lab. 35oC incubator. LES Endo agar plates. sterile pipettes. lauryl tryptose broth tubes. Heterotrophic plate count: dilution tubes. pencils. 35 oC incubator.leave it here or take away in a plastic bag to launder. not both.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 . Membrane filter method: dilution tubes. Get raw data from another group and include in your report. 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 . Quebec colony counter. Do not wear lab coat out of lab . plates. Demonstration only. EC tubes and/or Hach A1 tubes. Demonstration only SAMPLE: You will do either a raw sewage sample or a final effluent sample. HACH P/A Broth with MUG. away from your mouth. sterile pipets.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.
Demonstration. D. using a ready-to-use P/A Broth with MUG.COLI in 4 to 24 hours. Streak plates in a manner to insure presence of some discrete colonies. Note: these methods will be demonstrated but data must be recorded. Express as Total coliforms. streak one LES Endo agar plate from each tube of BGLB broth showing gas from the highest dilution and the second highest dilution. (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. Hach’s Most Probable Number Method 8368.) Methods: ? Recording BGLB.5 oC) as a positive completed test response for both Total and faecal coliform. Gas formation indicates the possible presence of coliform organisms. Microscope Slides (Completed Phase of Multiple-Tube Fermentation): Single colonies arising from single cells are prepared and stained for microscope examination.coli. The number of colonies counted directly. Heterotrophic Plate Count .A. 18 September 2007 CIVL 407 Lab M anual . Membrane Filter Technique: Serial dilutions of sample are collected on membrane filters and placed on M-Endo media. ? C. Colonies that are pink to dark red with a golden green metallic sheen are counted after incubation 20-22 hrs at 35oC. Another series of tubes are inoculated from these positive lauryl tryptose tubes.coli. A-1 Medium. 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. 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). 3. is USEPA-accepted for testing non-potable waters. 2. Bacterial numbers are calculated from the numbers of positive tubes. Consider positive EC broths incubated at the elevated temperature (44. It is used as a faster alternative to the LTB/EC procedure for enumerating faecal coliforms in water. E. Presence/Absence Testing: HACH technique to simultaneously screen for total coliform and E. Using aseptic technique. ? B. 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. 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. Results will be provided for the demonstration set. The broth will turn yellow indicating total coliform and fluoresce in the presence of E. MUG produces a fluorogenic product when hydrolyzed by an enzyme specific to E. Parallel positive BGLB broth cultures with negative EC broth cultures indicate the presence of nonfaecal coliforms.7oC for 24 hours (confirmed test for faecal coliforms). 1.Pour Plate Method: Serial dilutions of samples mixed with agar and incubated for 48 hrs at 35oC. Use Table 2 on page 27. Calculate the MPN value from the number of positives using the Table 1. as demonstrated. Gas-positive Hach A-1 tubes indicate the presence of faecal coliform. EC or A-1 tubes positives: 1. All tubes have been examined for gas production.
. Flame loop between second and third quadrants to improve colony isolation. -Note: “typical” colonies count as coliforms in this procedure. The typical coliform colony has a pink to dark-red colour with a golden green metallic surface sheen. The sheen area may vary in size from a small pinhead to complete coverage of the colony surface. Prepare one set of six dilution tubes as described in Section A. remove filter using flamesterilized forceps (allow forceps to cool or they will damage the filter). Use dishes with 20 to 80 coliform colonies and not more than 200 of all types. Heterotrophic Plate Count . removing the cover just enough to insert pipette tip and dispense sample. Verification of both types of colonies is advisable as some typical sheen colonies may be noncoliform and some atypical may be coliform.2. A high count of non-coliform colonies may interfere with the maximum development of coliforms. 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. Pour the full volume of dilution tube # 6 onto the filter. or colourless and are considered to be non-coliforms. After 20-22 hr. Colonies that lack sheen may be pink. 5. Atypical coliform colonies can be dark red or nucleated without sheen. Inoculating. Do not push down on the filter except on the outside edge and use only sterile forceps . place a sterile membrane filter (grid side up) onto the filtration apparatus. ?B. prepare one set of six dilution tubes following the instructions given under Multiple Tube Fermentation. coliform bacteria are Gram-negative (pink) rods (sometimes coccus-like). When filtering is complete and the valve is closed. The filtration apparatus has been sterilized by autoclaving. red. white.applying gentle pressure. Using sterile forceps. Dilution tubes. Dilution tubes. ?C. Gram staining technique can be also be used. Incubate inverted Petrie dishes for 20-22 hr at 35 oC. Prepare Gram Stain slides for examination under a microscope. Verification is usually done by a test for lactose fermentation. 19 September 2007 CIVL 407 Lab M anual . If you haven’t already. 4. transfer 1 ml of sample from dilution tube # 6 (most dilute for Raw sewage) or #5 (for Effluent) to an empty (large) Petrie dish. Place the filter on a small LES Endo agar plate carefully. 2. 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. choose appropriate dilutions for counting. Counting. In general. 2. with a motion that excludes air as it comes in contact with the agar. then immediately replace the cover. from one edge. Be sure dishes are labelled with your name and dilutions. Incubate plates (inverted) at 35 oC for 24 ± 2 hr. therefore the results table should specify #coliform colonies/100 ml. Mount apparatus on suction flask with valve sideways to block suction.1. Membrane Filter Technique -Total Coliform 1. Pipette about 10 ml of sterile dilution water over the surface of the filter with the suction valve still closed. open the valve slowly and allow all the liquid to be pulled through. Filtering. Repeat for the rest of the dilution tubes to # 2.Pour Plate Method 1. Using a sterile pipette. 3.
Do not report as “too numerous to count” if all plates exceed 300 colonies.too hot for a baby . D. Test temperature on your wrist . on the bench. Counting. Work quickly or the agar will solidify in the tube before you get a chance to pour it. After 48 hr incubation. Wash off stain with water and then with iodine solution (Lugol’s). Drain off excess iodine and wash freely with water and then alcohol-acetone decolourising solution. Repeat with dilutions 5 to 2 (for Raw) and 4-1 (for Effluent). Record results of previously prepared test. Label the slide using a grease pencil (T or A). Wash slide in tap water. E. Slide staining. Gently swirl the dish. Incubate at 35 oC for 24-48 hours. d. Flood slide with crystal violet solution and allow to act for 30 seconds. 4. pour and replace cover. place 1 loopful of sterile dilution water in the centre of a slide. 2. 2.d e m o n s tra tio n -s ta rte d a h e a d o f la b 1. 5. Repeat for the rest of the dilutions. refer to Standard Methods for more instruction. Allow iodine to act for 30 seconds. Let them cool down from the 60C that they are kept at but not enough to solidify. Resterilise the loop and remove one colony from the surface of the Les Endo streaked plate (prepared previously). a. Compute bacterial density and report as “colony-forming units” (CFU) per ml.too hot for a bacterium. f. b.coli). use the plate(s) that will give from 30 to 300 colonies/plate. ?4. marking (on edge of dish so as not to impede counting) and incubating for 48 hr at 35 oC. Do this for a typical and an atypical colony. average and multiply by 57 (for plastic Petrie dish). 20 September 2007 CIVL 407 Lab M anual . Add 100 mls of sample directly to pre-measured medium provided. if fewer than 10/cm 2. The aim of using several dilutions is to have at least one dilution giving colony counts between these limits. Spread the colony in the drop of water so that you get a uniform dispersion over an area about the size of a dime. Flood the slide with fresh alcohol-acetone solution to act for 30 seconds. Slide preparation: With a flame sterilized inoculating loop. If more than 10/cm 2. to mix the sample with the agar. A hand counter is also available. Air dry the slide high above the Bunsen burner and fix by passing slide briefly through a flame. Microscope Slides: 1. Preferably select seven consecutive squares horizontally and six consecutive squares vertically. select the appropriate dilution to count. Multiply the number by 5 to compute estimated colonies per plate (66 cm 2 ). Presence/Absence Testing . If necessary. Use the Quebec colony counter to aid in counting and a grease pencil to mark off colonies counted. count four representative squares. 6. Take 5 tubes of melted agar from the incubator. Ideally. Examine for yellow colour (total coliforms) and fluorescence (E. Allow the agar to solidify in the dish before inverting.3. e. ?3. c. Instead. count colonies in 13 squares (of the colony counter) of the most dilute plate having representative colony distribution. Slide the cover of a Petrie dish back just far enough to pour the agar.
after decolourisation Gram negative organisms are no longer visible.Examine prepared slides. Description of bacteria: Describe what you see on the plates: colonial morphology of each type of colony seen ie surface (shiny.5 . Gram negative organisms are visualized (pink). smooth. ?3.Come in and count colonies on LES Endo Membrane Filter dishes. In general. blot and then dry in air high above the Bunsen burner (so as not to burn away your efforts). .g. .do not turn knobs except the positioning ones. cell groupings (single. length to width ratio. . transparency. Examine the prepared slides . Cells occur singly. . plump rods.Record Presence/absence testing results of Hach Prepared Media bottles.Prepare and incubate Heterotrophic (Pour) Plates using dilutions above. . place on LES Endo agar dishes and incubate. coliform are Gram-negative (pink). Schedule Summary Day 1. Note: in the staining process: before decolourisation all organisms appear Gram positive (blue). nonsporing. Day 2.Come in and count Heterotrophic Plate colonies after 48 hours. .counts will be provided on the board. Apply fuchsin or safranin (counter)stain for 30 seconds. dull. b.3 µm in size.Membrane filter prepared dilutions.Prepare dilutions for Heterotrophic Plate Count and Membrane Filter technique. Day 3. or in short chains. h. colour. c. and on the slide: typical or atypical. Wash in water. short.Record positives in all Multiple Tube Fermentation inoculations: LTB. BGLB and EC and AI tubes . pairs chains). . concave). After application of the counterstain. sometimes coccus-like and 0. Plates ready on a weekend will be put in cold room until Monday. 21 September 2007 CIVL 407 Lab M anual . Microscopic examination. metallic. odour. usually motile. a. . rough. cocci or rods. in pairs. gram positive or negative.
1 2 3 4 5 6 Heterotrophic Plate Count .. 1/ 10 and 1/100 are used. Multiply the MPN index by the dilution factor from the series used when dilutions other than 1/1. 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 .pour plate method.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.
Most Probably Number (MPN) When more than three dilutions are used in a decimal series of dilutions. A long-wave UV light is required to detect fluorescence.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 . 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. 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. Use the results at these three volumes in computing the MPN index. The number in the numerator represents positive tubes. it is not necessary to enumerate. MF results. Estimation of Bacterial Density .coli.Discussion: -sources of error? -compare MPN.coli.coli will fluoresce as early as 4-24 hours. you can simultaneously detect total coliforms and E. that in the denominator. use the results from only three of these in computing the MPN. They are in concentrated form and merely require the addition of 100 ml of sample (or diluted sample) to the media and incubation. the significant dilution results are in boldface. Do they agree? Why/why not? -compare MPN/MF with Heterotrophic Plate Count. With MUG reagent added to P/A Broth.coli because it produces a fluorogenic product when hydrolyzed by glucuronidase. MUG (4methylumbelliferyl-$-D-glucuronide) can be used to detect E. an enzyme specific to E. Both media meet USEPA guidelines for testing of total coliforms in drinking water. To select the three dilutions to be used in determining the MPN index.1 ml 5/5 4/5 1/5 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. If zero total coliform is the maximum contamination goal.01 ml 2/5 2/5 0/5 0. E. the total tubes planted. In the examples given below.
Table 1 .1 mL) Comb. 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.MPN Index and 95% Confidence Limits for Various Combinations of Positive Results when 5 Tubes are used per Dilution (10 mL. 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 . 0. 1.0 mL.
Hach .MPN Table 2 25 September 2007 CIVL 407 Lab M anual .
com/ ¸Information Central/Learning Library/Microbiological Testing ASTM: (STP635) D. 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.org/microbelibrary/files/ccImages/Articleimages/keen/Gram stainkeen.http://www.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. 2) 3) 26 September 2007 CIVL 407 Lab M anual . -The Use of Indicator Organisms to Assess Public Water Safety . Mara: -Bacterial Indicators/ Health Hazards Associated with Water -Bacteriology for Sanitary Engineers Microbiology 321: -Manual of Microbiological Techniques Website: http://www.D.hach.microbelibrary.
sample. to illustrate the differences in operation and accuracy between instrumental methods and wet chemical methods. chloride specific ion electrode. If chemicals are spilled alert instructors. spectrophotometer. beakers. NaHCO 3. volumetric pipettes. OBJECT: -to acquaint you with the use of specific ion electrodes. goggles or glasses and lab coats. buffer. immediately. burettes. pH meter with double-junction electrode and silver billet electrode. 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. 27 September 2007 CIVL 407 Lab M anual . HNO 3. Note and consider carefully: Different methods of analysis have different optimum ranges. reference electrode (double junction). standard AgNO 3 solution. Mercuric thiocyanate is very toxic.5 mg/L). MATERIALS: -millivolt meter. Use personal protective equipment: gloves. That means you will probably have to dilute the sample by the most accurate means available. magnetic stirrer. indicator-acidifier reagent. beakers. conc. graduated cylinders. Hint: optimum ranges will coincide with the standards provided or will be indicated within the pertinent section.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. semi-log graph paper. known addition and calibration techniques and colourimetric and potentiometric titrations. standard chloride solution (0. indicator. standard Hg(NO 3)2. standards. 125 ml Erlenmeyer flasks.
5. pour out 100 mls of each standard into separate. labelled beakers.in sample = mg/L Cl. where: E1 = E2 = E = Vo = Vs = A = S = potential of sample (mV) potential of sample plus stock addition potential change = E 2 . Add 2 mls of IONIC STRENGTH ADJUSTMENT (ISA) buffer to each using the plastic dropper (filled to the mark). CHLORIDE . 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). 100 and 10 ppm (mg/L) have been prepared for you and are at the meter. Calculate the chloride concentration. As probe tends to drift for some time it may be expedient to select a “standard time to read” (i. 2. 4.= ____________________ 28 September 2007 CIVL 407 Lab M anual . 2 minutes) and use it for all standards and samples. plot the millivolt readings (linear axis) against the concentration of the standards (log axis) to produce a calibration curve.e.E 1 volume of sample volume of stock solution added mg Cl. added 2 mls of ISA. 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. mg Cl. 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 ).SPECIFIC ION ELECTRODE Part One: Preparation and reading standards and samples 1. 6.I. Using semi-log paper or software equivalent. place the lowest standard on the stirrer and lower the electrodes into it. 3. Measure 100 mls of sample. Standards of 1000. stir and obtain a reading for it. rinsing and blotting dry the electrodes between each. Repeat for rest of standards. Unless already done by a previous group.
) Prepare a calibration curve by plotting the absorbance readings versus the concentration of chloride in the standards. Near the end-point. (Light green indicates a pH of less than 2. 6.5 ± 0. after a few more minutes. insert in the spectrometer and record the absorbance displayed on the meter. If sample is above the highest standard. which then must be subtracted from the standards and sample. and the sample (diluted if necessary) into separate and labelled 125 ml Erlenmeyer flasks (5 in total). 2.) Mix thoroughly by gently swirling the flask. using the dispenser provided. [Note: this delay between additions is to allow you time to measure absorbance of each at the 10 minute time required .0 and 8.ions in the sample combine with added mercuric ions forming a soluble but virtually undissociate-able complex. (Alternatively. it should be rerun from the start after diluting. 5. Wait two or three minutes and then add the reagent to the first (lowest) standard.+ Fe+++ º Fe(SCN) ++ (Reddish brown) 1. 2. Using a 25 ml graduated cylinder.º HgCl2 1. 4.0. a pH of greater than 3. The colour of the solution should be green-blue at this point. 4. 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 . Determine the chloride concentration in the sample from the calibration curve.0 ppm). Marking the time (T=0).) Every two or three minutes thereafter you will rinse the cuvette with the next standard or sample.014N Hg(NO 3) 2 to a definite purple end-point. (Use extreme caution as this is a corrosive & toxic chemical. the excess mercuric ions combine with diphenylcarbazone (DPC) to produce a violet colour (the end-point). pure blue. fill. Hg ++ + 2Cl.they can’t all be at 10 minutes at once. zero instrument on distilled water and obtain an absorbance value for the blank. For most samples..8. discard and refill at least twice to rinse cuvette.II.ions have been bound. 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). Titrate with 0. 3.1. the 1 ml of the indicator-acidifier reagent will adjust the pH to 2. add 5 ml of the “combined reagent” (containing ferric alum and mercuric thiocyanate solutions) to the blank first. 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. III. CHLORIDE .0. After all Cl. (No further adjustment is required after the blank or distilled water has been set to zero. MERCURIC NITRATE METHOD FOR CHLORIDE DETERMINATION The principle of this method is as follows: Cl. and Hg ++ (excess) + DPC º violet colour 3. the three standards (2. use a plastic dropper to transfer blank into a spectrophotometer cuvette (do not fill more than 3/4 ). Add 1 ml (use pipette to measure accurately) of indicator-acidifier reagent to sample. Pour 100 ml of sample (or a portion diluted such that the concentration is less than 100 mg/L) into a 250 ml beaker.] After 10 minutes has passed (since the combined reagent was added to the blank).+ Hg(SCN)2 º HgCl2 + 2SCN SCN .BY COLOURIMETRIC METHOD The principle of this method is based on the following formula: 2Cl. measure 25 mls of “zero” standard or “blank” (halide-free distilled water).
This way each station will have data for each part. 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. 5. place beaker on stir plate and lower electrodes into the solution. 2. 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). Don’t forget to add the indicator.0 ml of concentrated HNO 3 (use plastic dropper). recording the volume and millivolt reading for each increment. and add 2.1 to 0. At the start. Place 10. Plot a differential titration curve to determine the exact end-point. Rotate the work within the group so that different pairs are doing the titrating and recording of numbers. in increments. A reference set of data will be provided for comparison. Continue the titration about 5 ml past the end-point. dilute to about 100 ml with distilled water. smaller increments (0.2 ml or drop-wise) should be added. large increments (1-2 mls) may be added. 6. Calculate the Normality of the AgNO 3 using the following equation: 30 September 2007 CIVL 407 Lab M anual . CHLORIDE BY POTENTIOMETRIC TITRATION Note: For this part form two groups comprised of 1 member from each station. The end-point occurs at the greatest mV change per unit addition of AgNO 3. Calculate the chloride concentration: 5. drops of the purple end-point. until past the endpoint when larger increments can be used again (ª mV/ml decreases). where: A = ml titrant for sample B = ml titrant for blank N = normality of titrant IV. Take care that stirrer is not hitting the electrodes.0 ml (use volumetric pipette) of standard chloride solution in a 250 ml beaker. 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 . 3. Part one: Standard 1.if one is necessary.4. then. as the end-point of the reaction is approached (ª mV/ml increases). The titre from this step must be subtracted from the titre for the sample. Immerse stir bar.. with standard AgNO 3.. Nitric acid is in the sink and is very corrosive.
Millivolts per ml versus ml of titrant. In plotting b and c.Part Two: Sample 1. if dilution is necessary into a 250 ml beaker (use a graduated cylinder to measure). Calculate the chloride concentration in the sample using the following equation: 3. (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. Measure 100 ml of sample or portion made up to 100 ml.) Plot three curves for this titration: a. Add 2 ml of HNO 3 and proceed as described in Part One. c. 2. Millivolts per ml per ml versus ml of titrant. be careful to use the average value of ml of titrant on the abscissa. b. in this case. 4. which are merely first and second derivatives. It is not. 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 . Millivolt reading versus ml of titrant.
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. 32 September 2007 CIVL 407 Lab M anual .Chloride .
strongly reducing solutions may form a surface layer of silver. COLOURIMETRIC METHOD # Bromide. 33 September 2007 CIVL 407 Lab M anual .3 mg/L? Outline the advantages and disadvantages of all the methods used in this lab to measure chloride concentration. # Colour in the sample may interfere in the absorbance measurement. timeconsuming process requiring a sound knowledge of chemistry and extensive experience working with environmental samples. # Colour may obscure or interfere with end-point determination. # Environmental samples usually require complex pre-treatment. # Chromate. In the above tests. # Environmental samples usually require pretreatment. From your chloride results. # Other interferences: ferricyanide.INTERFERENCES IN CHLORIDE DETERMINATIONS POTENTIOMETRIC METHOD # Iodide. dichromate. MERCURIC NITRATE TITRATION METHOD # Iodide. known addition cannot correct for any ions that test as chloride ion. iodide. # High levels of ions which form very insoluble salts of silver may deposit a layer of salt on the electrode membrane. KNOWN (STANDARD) ADDITION TECHNIQUE # The known addition techniques will only correct for certain types of interferences. This is a complex. ferric. requires knowledge of the compounds in the sample which are likely to interfere. Each sample type requires development of an appropriate method which. can you see any advantage in suing the first or second derivative curve? Discuss. Lab 3 Questions 1. fluoride and bromide titrate as chloride. fluoride and bromide titrate as chloride. hydrazine. ferric ion. specific ion electrodes are rarely usable in environmental samples. cyanide. # In practice. thiosulphate. # Pretreatment of environmental samples usually required. # Mercury must be absent from samples. 2. SPECIFIC ION ELECTRODE # All halides interfere (the electrode is a halide electrode). causing electrode malfunction. chromate. 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. fluoride. It can only correct for substances that enhance or suppress the test response proportional to the amount of chloride ion present. in itself. PRETREATMENT TECHNIQUES # There are no "standard" pretreatment techniques. and nitrite interfere. 3. Also. # Sample pH may need adjustment beyond the capacity of indicator-acidifier reagent. and sulfite ions interfere when concentration greater than 10 mg/L.
starch indicator. burettes and stands. Make up to the 250 ml and invert flask several times to mix. add 1 ml of starch solution..025 N sodium thiosulphate. Mix well (on stir plate with stir bar). 1995. pipettes. about 50 ml of chlorine-free water (distilled water will do). chlorine free water. phosphate buffer solution.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. Bleach/chlorine solutions are strong oxidants.N-diethyl-p-phenylenediamine (DPD) indicator solution. PREPARATION OF STOCK CHLORINE SOLUTION (Iodometric method . REFERENCES: Standard Methods 19th ed. allow to mix again.025 N sodium thiosulphate solution (use a volumetric pipette). about 5 ml of glacial acetic acid (use a graduated cylinder). 2. When weighing chemicals at the balance. 100 ml graduated cylinder. and exactly 10 ml of 0. standard ferrous ammonium sulphate (FAS) solution. clean up all spills with a brush into a container and wash crystals down the drain. chlorine demand and break-point chlorination. Personal protective equipment required at all times when working in the lab are: eye protection. Materials: -600 ml beakers. 250 ml Erlenmeyer flasks. Titrate rapidly with the stock chlorine solution (in burette) until the appearance of a constant 34 September 2007 CIVL 407 Lab M anual . bleach or hypochlorite solution. 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. pages 4-56 to 4-57 or older/newer editions. 3. 250 ml volumetric flasks. 0. Take a 250 ml Erlenmeyer flask and add about 1 gram of potassium iodide.total chlorine residual) 1. OBJECT: -to acquaint you with disinfection of water supplies and to demonstrate techniques for the determination of chlorine residuals. N. potassium iodide (KI) crystals. gloves and lab coats. Rinse a burette and fill it with this stock chlorine solution. (concentrated) glacial acetic acid. to illustrate the concepts of free and combined chlorine residuals. Wipe up all liquid spills immediately. I.
let stand 2 min more if colour drifts back indicating incomplete reaction. as there is lots to do . etc.) Free and combined chlorine or total chlorine: To obtain total chlorine in one reading. 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. refilling burette. calculate the chlorine concentration in the stock solution (See Data and Results section for formula). Determination of Free and Combined Chlorine by the DPD Method 1. a) Free chlorine: titrate rapidly with standard FAS (Ferrous ammonium sulfate) titrant until the red colour is discharged. 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. Total chlorine (Free plus Combined) = Reading C Free residual chlorine = Reading A Combined residual chlorine (dichloramine plus monochloramine) = Reading C . Method 4500-Cl F. Let stir for 2 min and then continue titrating until red colour (if any) is discharged (Reading C). d) Alternatively: (Not to be done in this lab. let stand for two minutes and titrate until the red colour (if present) is discharged.COORDINATE! IIa.purplish-blue colour. please refer to Standard Methods. 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). 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). b) Monochloramine: Add two drops of KI solution (to the same sample) and mix. IIb. It is likely that the highest dose will require dilution. 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). (*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. Work in groups of three or four for this part. 35 September 2007 CIVL 407 Lab M anual . After 15 minutes of contact time. Let stand 2 minutes more. pp 4-43. if colour drifts back (indicating an incomplete reaction).A Monochloramine= B-A Dichloramine= C-B For interferences. Add 100 ml of sample or diluted* sample using a 100 ml graduated cylinder and mix again. 18th edition. Record the burette reading (mls) = Reading A. Continue titrating until red colour (if any) is discharged once again (Reading B). c) Dichloramine: Add about 1 g KI (to the same sample) and mix to dissolve.). For dichloramine concentrations greater than 1 mg/L.Breakpoint Chlorination using Free and Combined Chlorine Residuals Values 1. Record the burette reading (Reading C). 4. 2. 2. titrate to colourless again. Go to c. These 6 flasks can be prepared at once to make them ready for the next step. From the amount of chlorine solution used. 3. mix to dissolve. add full amount of KI at the start (ie 2 drops KI solution and 1 g of KI crystals) to a fresh sample. Place 5 ml of phosphate buffer solution and 5 ml of DPD solution in a 250 ml conical (Erlenmeyer) flask and mix.
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.e. Given: HOCl W H + + OClK eqv = 2. how many coliforms would remain after a chlorine contact time of 10 minutes? 20 minutes? 36 September 2007 CIVL 407 Lab M anual . Make a plot of the three residual chlorine components against the applied chlorine dosage. Guaranteed analysis is normally printed on the container and is usually 5-6%. what percentage of bacteria would be killed in 10 minutes at a chlorine residual of 0. FAS titrant concentration is 1 ml = 100 µg Cl as Cl2 a. 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. Determine the concentration of total. Why is it important to determine chlorine residuals in domestic water supplies? 2. II. free and combined residual chlorine for each applied chlorine dose. Under what conditions is the practice of break point chlorination necessary? 4. 250) then convert mg/L to g/100 mls = %.7 x 10-8 at 25 o C % HOCl = 27 What is the pH of the solution? 3.DATA AND RESULTS I. If this is true. If a sewage sample contains 10 x 10 6 coliforms/100 ml. Make a separate graph for the 15 minute and 1 hour contact time. b. 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. c. d. Discuss your results as a function of the residual forms of chlorine present at different chlorine dosages and at different contact times. a.
(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 .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. of FAS to endpoint A (mls) Vol. of Cl Soln. of FAS to endpoint B (mls) (includes A) Vol. (mls) (Stock=_____mg/L Cl as Cl2) Applied Cl Dosage (mg/L) Vol.
bromphenol blue. 4. turn on stirrer and obtain a stable reading. p H. Normally. 39 September 2007 CIVL 407 Lab M anual . hardness indicators. MATERIALS: -pH meter. 1 N NaOH. DETERMINATION OF pH 1. rinse & dry probe. beakers. standard acid. buffer solution. particularly those labelled with specific hazards. pH test paper and other water quality assessment equipment. metres and calibrated glassware are available in limited quantities and are very expensive to replace.Lab o rato ry 6: Alkalin ity . Use care when using lab equipment. 3. T u rb id ity WARNING Please use caution when handling all chemicals. turbidimeters. colour comparator. I. 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. water samples. Ac id ity . EDTA. lower probe. metacresol purple. to acquaint you with the use of pH metres. 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. Hard n e s s . bromcresol green. then use buffers 7 and 10. Co lo u r. place a beaker containing the sample and stir-bar on the stir-plate.) 2. magnetic stirrer.(Do not allow the stir bar to hit the probe. if above 7. the two buffers that are chosen for calibration will bracket your sample ie if sample is less than 7. graduated cylinders. flasks. phenolphthalein. (Take care not to contaminate paper in case. Calibrate the pH meter by following the procedure outlined on the instruction sheet beside the meter unless told otherwise.) Record value in following table. then use buffers 4 and 7 to calibrate. Rinse probe when you are finished and leave immersed in pH 7 buffer for storage. standard base. pH test paper. burettes and stand. After calibration is complete. Probes. Always wear personal protective equipment. OBJECT: -to familiarize you with the most common water treatment tests.
. add 1 drop of 0. Add a stir-bar. Add stir-bar. Alternatively. Add the last few drops at 3-5 second intervals so that you do not "overshoot" the end-point. TOTAL HARDNESS DETERMINATION . III... 40 September 2007 CIVL 407 Lab M anual . In order to remove any free residual chlorine (which interferes with the indicator colour response). if the approximate hardness is known (by unsuccessful attempts). White paper placed under the flask will facilitate this determination. (If the solution is already blue after indicator. Record the volume of titrant required in the following table. (If solution is clear after addition of P. 3.5). If sample is highly alkaline (titration goes over one burette full) dilute the sample and titrate again. Add a full dropper of phenolphthalein (P) indicator and if the solution stays pink or red. Record the burette reading in the following table. Record burette reading (P). If a ppt does form within the 5 minutes. Titrate slowly with EDTA.?) 4. until the reddish tinge disappears from the solution and a pure blue remains. 3. Measure out a new sample and add 1 drop of sodium thiosulphate and a full dropper of phenolphthalein..EDTA TITRIMETRIC METHOD 1. A pH much above 10 promotes CaCO 3 precipitate. 4. Rinse and fill a labelled burette with standard acid solution. DETERMINATION OF ACIDITY 1. add 90% of the titrant to the sample before adjusting the pH with buffer. 2. sample-rinsed graduated cylinder and transfer to an Erlenmeyer flask. Rinse and fill a burette with standard EDTA solution and record the initial burette reading. while stirring. 2. IV. Measure 100 ml of water sample into a clean. Titrate to the first uniform pink colour (colour stays). Pour into a 250 ml Erlenmeyer flask. Measure out 25 mls of sample using a graduated cylinder and dilute to 50 ml with distilled water. Rinse and fill another burette with standard base solution and label it. Measure 100 ml of sample (with minimum agitation or exposure to air) using a graduated cylinder and transfer carefully to an Erlenmeyer flask. it may be necessary to repeat the titration with the sample diluted 1 to 1 (again) with distilled water. Add 1 drop of sodium thiosulphate solution.II.think what that means!) 4. Apply dilution factor to obtain final result. 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. Complete the titration within 5 minutes to minimize the tendency toward CaCO 3 precipitation. The buffer elevates the pH to 10. 3. 2. 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. (BG or MO) 6. titrate until just colourless. Add a full dropper of bromphenol blue indicator and titrate until the colour changes from yellow to blue ("MO" Acidity). DETERMINATION OF ALKALINITY 1. 5..1 N sodium thiosulphate to the sample.
except on an unfiltered sample. 4. COLOUR . Wipe the cell with a soft. Use signal average mode if 41 September 2007 CIVL 407 Lab M anual . Cap the cell.EDTA TITRIMETRIC METHOD 1. Record the final burette reading in the following table.5. V.. 4. Press: I/O. Measure out 50 ml of sample into an Erlenmeyer flask and add 2.BY HACH 2100P Turbidity Meter. Pour the supernatant into one of the cells (to the etched mark) and fill another with distilled water. Place the cell containing water into the spectrophotometer and press Zero. . Select automatic range by pressing the RANGE key. Refill the burette with EDTA titrant and note the initial reading. VI. 4. 3. Close the lid. The display will show SIG AVG when the instrument is using signal averaging. Record the final burette reading in the following table. 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. to mg/L platinum as chloroplatinate) PART B: APPARENT COLOUR 1. The instrument will turn on but will turn off automatically after a short time so you should be ready. Select signal averaging mode by pressing the SIGNAL AVERAGE key. 2. 3. Use the technique outlined in Part A. Place the cell containing sample in it and press Read. Add a stir-bar and stir to mix. The instrument may suggest diluting if over range. Filter about 20 mls of sample using a filter funnel and stand and the pre-fluted filter paper provided 2. TURBIDITY . VII. Add one aliquot of the Calcium Hardness Indicator and titrate with EDTA slowly. to determine the apparent colour (measures the influence of turbidity on the reading).TRUE AND APPARENT PART A: TRUE COLOUR . Fill a sample cell to the line (about 15 mL). 2. Record all colour data in the following table. lint-free cloth to remove water spots and fingerprints. 5.0 ml of 1 N NaOH solution (use 3 droppers full). Demonstration of Jackson Candle and Hellige PART A: Hach Turbidimeter 1.. The display will show AUTO RNG when the instrument is in automatic range.HACH Spectrophotometer. The reading might be higher so if you had to dilute in Part A dilute for Part B. Values are reported as APHA Colour Units (eqv. or a volume sufficient to produce a pH of 13-14 (designed to precipitate the magnesium as a hydroxide). taking care to handle the sample cell by the top. 2.Enter the stored program number for true colour=120 1.. 3. CALCIUM HARDNESS DETERMINATION . with continuous stirring to a pure blue end-point.
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.demonstration only. Pinch all excess charred wick with fingers before lighting or soot may be deposited on the glass tubes. 16th edition.. Note that there are several different scales on the side of the tube.) Remove the glass tube from the holder and read the turbidity at the meniscus of the sample. then the turbidity in NTU. Record the turbidity after the lamp symbol turns off. Take the reading where the dark central part first merges with the surrounding field.the sample causes a noisy signal (display changes constantly). Try to be quick and not burn the candles more than a few minutes at a time. Record value in following table as J.. (This allows you to make a more gradual and accurate approach to the end-point. Read the scale on the dial. Be sure that the calibrated tubes are clean before adding samples to the tubes. Close the door and switch on the light. 3.U. DO NOT PLACE AN EMPTY TUBE IN THE TUBE HOLDER WITH THE CANDLE LIT . 6.Demonstration only 1.. Carefully fill the 20 mm viewing depth tube to the mark with sample. If you take too long. 42 September 2007 CIVL 407 Lab M anual .T. Lower the plunger into the liquid making sure no bubbles are trapped under the plunger and the top is dry.. obscuring vision. PART C: Jackson Candle Turbidity .NTU. Press: READ The display will show .s. Wipe the outside of the tube to dry it and place it in the turbidimeter (in the circular groove of the mirror unit). record the number and refer to the appropriate graph to determine the turbidity as mg/L SiO 2. 1. page 272-273. the turbidity may start to settle and condense on the bottom of the tube. Note that the rectangular door mirror is closed. that the filter selector shows "none" and that bulb B is in use. 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.. T = Total alkalinity. 2. 3. 2. See Standard Methods. Pour sample into the tube in small increments until you can no longer distinguish the shape of the candle flame. PART B: Hellige Turbidimeter .adding liquid to a hot tube will cause it to shatter. 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.
O. Alkalinity titre (mls) Net "M.O." Alkalinity titre (mls) ACIDITY.DATA Sample 1 pH .Sample volume Initial burette reading P.O." Alkalinity end-point (mls) Initial burette reading Net P." 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.Initial pH by test paper Initial pH by pH meter ALKALINITY.Sample volume "M. Acidity end-point reading (mls) Initial burette reading Net "M. Alkalinity end-point reading (mls) __________ "M." 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 .
CALCULATIONS Calcium: Calcium Hardness: where: A= B= ml titrant for sample mg CaCO 3 equivalent to 1.00 ml EDTA titrant at the calcium indicator endpoint = 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 .00 ml EDTA titrant at the calcium indicator endpoint = 1.0 Total Hardness (EDTA): where: A= B= ml titrant for sample mg CaCO 3 equivalent to 1.
U.Alkalinity as CaCO 3 (mg/L) OH .ion (mg/L) CO 3 -2 Alkalinity as CO 3 -2 ion (mg/L) HCO 3.Units= N. Alkalinity as CaCO 3 (mg/L) "M.Alkalinity as OH .Alkalinity as CaCO 3 (mg/L) CO 3 -2 Alkalinity as CaCO 3 (mg/L) HCO 3.Alkalinity as HCO 3 .ion (mg/L) "M.T.O.RESULTS (sample) P. ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 45 September 2007 CIVL 407 Lab M anual ." Alkalinity as CaCO 3 (mg/L) OH .APHA Colour Units: True Apparent Turbidity .O." 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 .Hach .
What sanitary significance does colour have? 4. given K 2 = 4. non-carbonate and total hardness of this water in mg/L as CaCO 3 ? in meq/L? Is the analysis complete? Why? 6.Lab 6 Questions 1. calculate the carbonate alkalinity in mg/L as CaCO 3 for the water sample analyzed during this lab. From equations (17-12) and (17-13) in Sawyer and McCarty.7 x 10 -11 and K w = 10 -14. 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. What errors can occur in the calcium hardness determination? Why? 3. Is this water suitable for a domestic water supply? Why? 2. 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. Does this agree with the results from Part II of this lab? Explain. 46 September 2007 CIVL 407 Lab M anual .
The optimum alum dosage for coagulation of the sample will then be chosen. alum solution (Al2 (SO 4 )3 @18H 2 O). In the second lab you will use the previously determined dosages to coagulate and soften the sample and then re-analyse the resultant water. 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. Several lime and soda ash dosages will also be selected for determining the efficiency of water softening.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. to be conducted over two lab periods. OBJECT: -to illustrate the principles of coagulation and water softening. This is a two-step experiment. You should have with you the part of Exercise 7 that gives instructions for the various tests. No. soda ash solution (Na 2CO 3) and lime (Ca(OH)2) or sodium hydroxide (NaOH). 7 (except Jackson Candle and Hellige Turbidimeter). 1000 ml beakers. Please try to coordinate your work with the rest of the class.6. and all of the materials from Lab.for the next session (b). 47 September 2007 CIVL 407 Lab M anual . MATERIALS: -Jar Test Apparatus. No.
Observe and make notes describing floc formation and note the time of appearance. use indicators as specified in Lab 6. Brief Methods Summary for Analyses Alkalinity: @ 100 ml of sample. and settling characteristics of the floc (rapid.work in 2 or 3 teams to assess a total of 6 dosages for each team. if available. A pH meter could be used for the alkalinity and acidity determinations. Complete the coagulation summary sheet during the lab period. Add the designated lime (or NaOH) and soda ash additions as quickly as possible to the decanted samples. Carefully decant into a graduated cylinder 800 mls of the supernatant liquid and pour into fresh beakers. hardness (total and calcium). 5. if time permits. 3. Stir at 100 rpm for 1 minute. After the floc has settled (allow 15 min). moderate. alkalinity. Part B: WATER SOFTENING PROCEDURE (to be done the following week) again work as teams of 2 or 3. COAGULATION USING ALUM . decant the supernatant liquid carefully into a clean set of beaker (try not to stir up what has settled) and discard the floc. hardness (total and calcium). 5. Pour six 1000 ml aliquots of sample into 1000 ml beakers and place on the jar test stirrer apparatus.Part A:. medium. 4. Record relative floc sizes (pin-point. 7. clear. 4. In the lecture. Measure out six 1000 ml aliquots of sample (using graduated cylinder to measure. acidity. small. 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). Stop stirrer and allow floc to settle. Record observations on settling rate. 2. Make relative estimates of floc sizes. Discard the floc and clean beakers in preparation for a second decanting in Step 4.1 N sodium thiosulphate 48 September 2007 CIVL 407 Lab M anual . Stir at 100 rpm for 1 minute and at 20 rpm for 10 minutes. 1. not a beaker ) and transfer to 1000 ml beakers. clarity of supernatant liquid (very clear. 6. After the flocs have settled (allow 15 min). decant the supernatant liquid carefully into another set of beakers (try not to stir up what has settled). Make sure it is centred. alkalinity. Stop stirrer. Reduce the stirring rate to about 20 rpm for 10 minutes. position them under stirrers. you will discuss the data and select the appropriate alum dosage to be used by all groups in the water softening step next week. slow). turbidity and colour. Stop stirring. Add the assigned alum dosages as quickly as possible and then start the stirrer. and clarity of supernatant liquid. Place beakers on the jar tester apparatus. Lift paddles out of beakers and secure. floc volume. but because of equipment limitations. and fill in the table on the board. 3.. Analyze the supernates for pH. 1. 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. acidity. 2. Determine which dosages were most appropriate for this sample. drop of 0. cloudy). Stir at 100 rpm for 1 minute. times of appearance and descriptions and record. 6. (NB dosage now based on 800 ml sample size). Analyze these supernates plus a sample of raw water for pH. You may need to use blocks to position the paddle 0.5 to 1 cm off the bottom of the beaker. Add the appropriate alum dosage and immediately start stirring. hazy. large). true colour and turbidity (Hach).
alkalinity (mg/L CaCO 3)= mls titre x N acid x 50.pH 8.use graphical presentation.02 N base with bromphenol blue to blue . Analyze the response of colour and turbidity removal as a function of alum dose. 2. @ calculation: Ca hardness (mg/L CaCO 3 )= mls titre x 1000/mls sample Lab 7 Questions A. @ 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 .pure blue).000/mls sample Acidity: @ 100 ml of sample. Compare removal of hardness components under the different doses .7 ("methyl orange" acidity) and with phenolphthalein (using a fresh sample) to pink .acidity (mg/L CaCO 3)= mls titre x N base x 50.000/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 . Softening 1. 4. Based on your chemical analysis of the raw water. show by calculation the theoretical amount of lime and soda ash required for excess lime/soda ash softening.pH 3.1 N sodium thiosulphate @ titrate with 0. 3. @ calculation . What treatment would you recommend to prepare this water for human consumption? Why? 49 September 2007 CIVL 407 Lab M anual .02 N acid with phenolphthalein to colourless .pure blue).pH 4.@ titrate with 0. Coagulation 1.3 ("phenolphthalein" or total acidity) @ calculation . 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.pH 8. 2. Compare your experimental softening results to the theoretical results based on solubility equations or mass balance equations.5. drop of 0.3 and with bromcresol green (continuing with the same sample) to yellow .
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.sample vol ml Net titre to pH 3.sample vol Net titre Notes: 50 September 2007 CIVL 407 Lab M anual .5 “TOT” Acidity .7 “MO” Net titre to pH 8.5 “TOT” Total Hardness sample vol Net titre Ca Hardness .5 “P” Net titre to pH 4.
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.U.H.) True Colour (A.) unfiltered Notes: 51 September 2007 CIVL 407 Lab M anual .A.) (filtered) Turbidity (N.A.T.P.H.
5 Acidity .5 Total Hardness sample vol Net titre Ca Hardness .sample vol ml Net titre to pH 3.sample vol Net titre Notes: 52 September 2007 CIVL 407 Lab M anual .5 Net (Total) titre pH 4.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 8.7 Net (Total) titre pH 8.
U.Coagulation and Softening Summary Sheet Data Lime/NaOH mg/L Soda Ash mg/L Alum mg/L pH P. -mg/L CaCO 3 **Non-Carb Hard -mg/L CaCO 3 **Excess Alk.P.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.) Note:* Total Alkalinity must include Phenolphthalein Alkalinity just as Total Acidity must include “MO” Acidity.A. -mg/L CaCO 3 Apparent Colour (A.H.A. Calculation 53 September 2007 CIVL 407 Lab M anual ** By .) (Filtered) Turbidity (N.H.) True Colour (A.P.T.
. . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4. . . . . . . . . . . . . . . . . . . . . . . . .Chlorine Demand . . . . . . . . 100 22. . . . . . . . . . . . . . . . . . . Alkalinity Species as a Function of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chlorine . . . . . 82 15. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 14. . . . . . . . . . . . . Water Softening . . . . . . . . Milliequivalent and mg/l as CaCO 3 . . . . . . . . . . Periodic table . . . . . . . . . . . . . . . . . . 105 25. . . . . . . . . . . . . . . . . . . DO. . . . . . . . . . . . . . . . . . . .pdf 1. . . . . Normal Solutions and Equivalent Weights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 11. . . . Bacteriology and Water-related diseases . . . . . . . . . . . . . . . . . . . . 57 3. . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bacteriological Examination . . . . 74 13. . . . Typical Problems for Acid-Base and General Chemistry . . . . . . . . . . . . . . . . . . . . . . Alkalinity. 55 2. . . . Instructions for Laboratory Report Writing . . . . . . . . . . . . . . . . . . . . . .Additional Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bar Diagram Method for Water Softening Problems . BOD. . . . . . . Equilibrium Relationships of Chlorine . . . . . . . 112 28. . . . . . . . . . Coagulation . . . . . . . . . . . . . . . . . . . . 83 16. . . . . . . . . . . . 91 18. . . . . . . . . . . 117 29. . . . . . . . . . . . . . . . . . . 69 8. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 20. . . . . . . . . . . . . . pH . . Solids Removal in Wastewater Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chloride Analytical Methods . . . . . Extra tables for Chlorine Lab . . . . . . . . 71 10. . . . Colour. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Balancing an Oxidation Reduction Equation . 84 17. . . . . . . . . . Hach Absorption Method . . . . . . . . . . . . . . . . . . . . . 107 26.Levels and Water Use . . . . . Concepts and Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Turbidity. . . . . . . . 96 21. . . . . . . . . . 110 27. COD . . . . . . . . . . . . . . . . . . . 92 19. . . . . . . . 67 6. Acidity. . . . . . . . . . . . Faecal Coliforms . . . . . Standard Solutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 24. . . . 68 7.COD . . 102 23. . . . . . . . . . Chemical Requirements for Water Softening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hardness Complexes . . . . . . . . . . . . . . . . . .4 Appendices see: CIVL407Manual_CourseNotes. . . . . . . . . . . . . . . . . . 73 12. . . . . 64 5. Residues . . . . . . . . . . . . . . . . . . . . . . . . Reading and Reference Material . . Titrations . . . . . . 118 54 September 2007 CIVL 407 Lab M anual . . . . . . . . . . . . . . . . . . . . . . . . . . 70 9. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hardness. . . . . . . . . . . . . . . . . . . Known Addition Technique . . . . .
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