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Prepared for Dept. Of Civil Engineering by Susan C.M. Harper September 2007
Table of Contents
Course Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Station/Drawer Supply List (if supplies are missing, please report) . . . . . 2 First Session: Laboratory Safety and Orientation . . . . . . . . . . . . . . . . . . . . 3
L ABORATORY R EPORT W RITE U P G UIDELINES . . . . . . . . . . . 5
Laboratory Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Laboratory 1: Solids Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Laboratory 2: COD, DO and BOD 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Laboratory 3: Bacteriological Examination of Sewage . . . . . . . . . . . . . . . 17 Laboratory 4: Chloride Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Laboratory 5: Chlorine and Chlorine Demand . . . . . . . . . . . . . . . . . . . . . 34 Laboratory 6: Alkalinity, Acidity, pH, Hardness, Colour, Turbidity . . . . 39 Laboratory 7a: Coagulation and 7b: Coagulation & Softening . . . . . . . . . 47
Appendices see: CIVL407Manual_CourseNotes.pdf . . . . . . . . . . . . . . . . . 54
(Environmental Laboratory Analysis - 1-3-0, 0-0-0) Testing procedures used in water quality studies and in the operation of water and wastewater treatment plants. Prerequisite Chem 151. Sc h e d u le Lecture CEME1204: Laboratory CEME1301: Wednesday 1:00 pm 1) Tuesday (time to be arranged by consensus) 2) Wednesday 2:00 to 5:00 pm 3) Thursday (time to be arranged by consensus) 4) Friday (time to be arranged by consensus) (Note: Lab Session must have at least 4 students to run)
Subject No Lab No Lab Laboratory Safety & Orientation (~1 hr) #1 Solids determination #2 Dissolved oxygen, COD, BOD No lab - Thanksgiving week - catch up #3 Bacteriological Examination of waste water #4 Chloride #5 Chlorine demand #6 Acidity, alkalinity, hardness, colour, turbidity No lab - Remembrance day - catch up #7a Coagulation #7b Coagulation and Softening
Date 2007 Sep. 4 - 7 Sep. 11 - 14 Sep. 19 Sep. 25 - 28 Oct 2 - 5 Oct 9 - 12 Oct. 16 - 19 Oct. 23 - 26 Oct. 30 - Nov.2 Nov. 6 - 9 Nov. 13 - 16 Nov. 20- 23 Nov. 27 - 30
Lecturer: Lab instructor: T.A.s: Lab: Marker:
Victor Lo, email: Susan Harper, email: Margaret Parsotan, email: Kazi Parvez Fattah, email:
email@example.com firstname.lastname@example.org email@example.com firstname.lastname@example.org
(604) 822-4880 (604) 822-4397
September 2007 CIVL 407 Lab M anual
Sta tio n /D ra w e r Su p p ly Lis t (if s u p p lie s a re m is s in g , p le a s e re p o rt)
The following items will be maintained in each stations supply drawer or at station on bench when required: 2 pair safety glasses 2 permanent marker pens 2 stir bars and a bar retriever 1 stir plate 2 buret funnels 4 30 ml beakers 4 50 ml beakers 2 100 ml beakers 2 150 ml beakers 2 250 ml beakers 2 1 L plastic beakers 2 600 ml plastic beakers 2 400 ml plastic beakers 2 10 ml grad cylinders 2 25 ml grad cylinders 2 50 ml grad cylinders 2 100 ml grad cylinders 1 buret stand 1 double buret clamp 2 50 ml burets 2 25 ml burets 2 10 ml burets 0-14 pH strips Thermometer 2 Stir stick or spatula Filter funnel (Nalgene magnetic) Tweezers (Millipore or Gelman for membrane filters)) On benches each row: Latex gloves- small, medium and large Non-latex gloves (keep on reserve for latex sensitive persons only) Filter racks with funnels #4 Whatman filter paper or equivalent Vacuum flasks and hoses 8 1 L grad cylinders 8 3 L plastic beakers
September 2007 CIVL 407 Lab M anual
If you cut yourself. Safety Shower. Sanitation: antibiotic soap for hand washing. calculators.clean and dry. 2. These should be used whenever standard solutions are measured or when upmost accuracy is required. Remember to empty and rinse burets as well. Hygiene: At times you will be working with sewage. DO NOT PUT BROKEN GLASS INTO THE REGULAR GARBAGE CONTAINER. 25. Putting pens in your mouth that may have been on the bench or handled with gloves could be hazardous. Be aware that handling your pens.0. They are calibrated by weighing the volume of distilled water that will flow from them by gravity. disinfectant for bench surfaces. gloves as required. 15. Please take your labcoat away in a plastic bag. Clean-up: You must leave your work station as you found it . Please ask for assistance if chemicals are spilled. if there is room. with the tip against the side of the receiving vessel. Broken Glass: Be careful not to break glass. Do not put lids and small items onto racks that may slip through to the filthy drip tray.0. no matter how minor. Pipettes must be placed with tips pointing up into the pipet basket supplied. 20. Some pipettes may be “to 3 September 2007 CIVL 407 Lab M anual . Spills: Wipe up all spills immediately. Personal Protective Equipment: Lab coat (long). Most pipettes are calibrated “to deliver” or TD and should never be blown out. No sandals or open-toe or heel shoes.. but if you do please ask for assistance. Safety Equipment: Eye Wash Station. packs. with contaminated gloves will contaminate those items. MSDS: is a supplier information sheet that must be available for each product and describe the hazards and necessary handling requisites. eye protection.2 First Session: Laboratory Safety and Orientation 1) 2) 3) 4) NO Food or drink. 1.. Pipettes/ graduated cylinders/ beakers/ flasks: 5) 6) 7) 8) 9) 10) Volumetric pipettes are the most accurate way to measure volume. 50 and 100 ml. please get attendant help. You must be informed of the hazards of a substance before using it.5. etc.up to 10. WHMIS: Workplace Hazardous Materials Information System is a government regulation. wash down and dry the bench. Wearing lab coats out of the lab is forbidden. A small amount of liquid always remains in the tip and must not be blown out. They are available in 0. 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.0 ml.
especially at smaller volumes. please rinse out immediately and set aside to dry. Chemicals will be put out as required for each lab and should be used sparingly. some are not meant to drain completely . Some are blow-out. markings are not accurate at all . Pipettes are in drawers indicated. 250 mls. 14) Glassware/supplies: Each station has a drawer where some glassware and other useful tools will be kept. Use the size of cylinder CLOSEST TO THE VOLUME YOU WISH to measure for best accuracy. They are calibrated the same way as TD except that the remaining drop is blown into the receiving vessel. Used to hold/transfer approximate volumes of samples. 500 mls.pay attention to the markings. Flasks: Vo lu m e tric flasks are very accurate and should be used when diluting standards (together with volumetric pipettes). 50 mls. NEVER mouth pipette.merely approximations. Do not squish bulbs in the direction of another person. 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. Erle n m e y e r or conical flask markings are not accurate. 2 & 4 L Beakers: are cylindrical in shape with straight sides and flat bottoms. Additional supplies are available in the supply room (around the corner). Chemical residues can be splashed in you face. They can be unpredictable in force and are close to eye level. 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. 12) Cup sinks: Please do not use these sinks for the disposal or delivery of liquids. Cylinders are available: 5 mls. They are useful for measuring samples 20 mls .deliver with Blow-Out”. Graduated cylinders: Not as accurate as pipettes. 10 mls. Ie If you want to measure 20 mls. YOU WILL USUALLY NEED THE INFORMATION GIVEN TO COMPLETE YOUR LAB WRITE-UPS. 11) Gas/air/vacuum taps: Do not touch these unless instructed to do so. 13) Stir bars: Please remove from containers before pouring contents down the drain. not wastefully as quantities are limited. They are usually identified by a double etched or coloured band at the top of the pipette. 1. 100 mls. Even the force of air from the air taps can be dangerous because it can contain grit or water. LISTEN AND MAKE NOTE OF VERBAL AND CHALK BOARD INSTRUCTIONS. Make sure that you label beakers and flasks to avoid confusion and waste. A demonstration will be given. 4 September 2007 CIVL 407 Lab M anual .. do not use a 1 L graduated cylinder. Undetected gas leaks can be deadly. chemicals or for titrations. They are containers best suited for “swirling” liquid ie to mix reagents with samples in a colourimetric determination (Lab 4 or 6).1000 mls. 25 mls.
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. Required  Required  Required  Required  Required  Required  Required   5 September 2007 CIVL 407 Lab M anual . 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. Total Mark L ONG R EPORT L AB 7 Required Required Required Include a brief description of the procedure. The short report is intended to be a concise yet complete laboratory report whereas the long report is more elaborate. The mark allocation for some sections for both reporting format is indicated in parenthesis in the table below. 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. Sufficient detail should be provided so that what was done could be understood and the experiment could be repeated from the procedure reported. 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.
subtract an estimated volume from the measured volume of matter. 2. graduated cylinders. Alternatively a place will be provided in the lab to store your coat. and record the volume of settleable solids. distilled water bottles. balance. If large pockets of liquid form between the particles of settled matter. Do not include any surface floating material as settled solids. evaporating dishes. Use pipette bulbs. A. if necessary.. MATERIALS: -Imhoff cones. tin dishes. Settle for 45 minutes. settle for 15 minutes longer. 6 September 2007 CIVL 407 Lab M anual . SETTLEABLE SOLIDS 1. desiccators. final effluent. sludge. gently dislodge any solids that have clung to the sides using a stirring rod. A 1 L graduated cylinder may be used in place of the Imhoff cone for the sludge sample.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. Do not wear lab coats outside of lab and do not take away from lab unless stored in a plastic bag. samples of raw sewage. wipe up all spills and wash with disinfectant. to measure sewage treatment plant efficiency in removing residue. oven muffle furnace. keep hands and pencils away from your mouth and wash hands frequently. Mix the samples thoroughly and fill individually marked Imhoff cones to the 1 litre mark. 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.
sample source and sample volume. rapidly pour sample into a 50 ml graduated cylinder to approximately equal 35-40 mls (if using 50 ml dishes). the dish will be transferred to a desiccator. 6. Mix the sample in the carboy thoroughly. . Carefully remove filter from filtration apparatus and transfer it back to the aluminum dish. rinse the cylinder. 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. Conversely. Obtain the tare weight of a properly prepared porcelain evaporating dish (ie one that has been washed. Once dishes are at room temperature they can be re-weighed. 3. Pinch sides of dish in a bit to protect the filter from oven drafts. immediately after stirring the beaker.]. prior to session). tare weight. The samples will be fired at 550° C for one hour. 2. Wet filter with a small volume of distilled water to seat it. Suggested portions: 100-200 ml of final effluent.] Using very small amounts of distilled water. Subtract the tare weight to obtain the total solids weight and calculate the mg/L value. The furnace will then be allowed to cool significantly before dishes are removed to a desiccator.[Note: it may be harder to wash out solids if you allow them to settle before pouring into the dish . Obtain the tare weights of three aluminum dishes each containing a glass fibre filter (already pre-washed. position the filter and begin suction (vacuum tap). Assemble filtering apparatus. Do not write on crucibles as high temperatures during the next steps will burn writing off. dried. stir the beaker contents and then rapidly (so that it doesn't settle) measure a volume of sample into the appropriate graduated cylinder. pre-fired and desiccated . SVI= settled sludge volume (ml/L) x 1000/suspended solids (mg/L)] B. Record the total volume filtered. dried and fired). TOTAL SOLIDS and TOTAL VOLATILE AND FIXED SOLIDS AT 550o C 1. 2. Put the dish in the 103-105 °C oven for drying. 3. SUSPENDED SOLIDS . The loss in weight represents the volatile solids lost from the originally measured sample volume. The dish containing the dried total solids can now be placed in a muffle furnace or. 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.TOTAL AND VOLATILE 1. 5.done for you.so be quick. Calculate the mg/L volatile and fixed solids. [Hint: pour out small portions (say 25 mls at a time for Effl.] Rinse the graduated cylinder with small amounts of distilled water and add to filter. 4.not beakers. This is a different test to the one we are doing using Imhoff cones and should not be confused. if instructed. pour about 500 mls (for Part B & Part C) into a beaker and then. adding the rinsings to the dish. 10 mls for Raw & 5 mls for sludge) of well-mixed sample and filter entire portion before pouring another portion. [Notes: to allow for rinsing and to avoid spillage when transferring dish to oven don't pour more than 40 mls].[Note: Sludge Volume Index is the volume occupied by 1 gram of sludge after 30 minutes of settling. 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 . Using the sample poured out for Part B (for consistency). Quickly record the exact volume and pour the sample into the dish. the solids remaining represent the fixed solids. C. on a cart in preparation for staff to do so when all are ready. After drying overnight. when filtering slows significantly do not add more.
From the above determinations it will now be possible to obtain a value for the dissolved solids present in the sample. % reduction in settleable matter _________ % reduction in total solids _________ % reduction in volatile solids _________ % reduction in fixed solids _________ % reduction in suspended solids _________ % reduction volatile susp.5. volatile. After firing the dishes will be moved to a desiccator. What.50. Calculate as the mg/L total suspended solids. The aluminum dish and filter (from #4) must now be fired in a muffle furnace at 550°C for at least 30 minutes (or until a constant weight is achieved). Lab personnel will perform the timed-firing after the class. 6. 8. what is the percentage reduction in solids. where the volatile solids are reduced from 65% to 40%. Classify each of the following into its proper solids category(s) i. What two techniques can be used to overcome or correct for the loss of material from filter pads when firing at 550 oC? 4. DO NOT DISCARD! The gain in weight represents the total suspended solids of the sample. A raw sewage sludge goes through an anaerobic digestion process. For the time being. 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. Please return tomorrow to obtain final weight. compute the sludge volume index (SVI). if any. fixed (use more than one adjective to describe each substance. What major types of solids are removed in primary treatment? in secondary treatment? 2. 7. If sludge is used in Part A. cool and weigh. Your hands may have moisture and natural and/or applied oil/lotion on them. 6.e. Calculate the percent solids reduction through the treatment plant. dissolved. Calculate the mg/L total volatile solids and total fixed suspended solids. Transfer dish to a desiccator. place the dishes on a cart designated by the instructor. solids _________ % reduction fixed suspended solids _________ % reduction dissolved solids _________ Lab 1 Questions 1. suspended. Discuss the meaning and significance of this index. 3.) a) a bacterium b) sodium chloride c) sugar d) clay 8 September 2007 CIVL 407 Lab M anual . Enter all data in the tables on following page.3 and fixed solids 2. If all of the volatile solids reduced is given off as gas and if the specific gravity of the volatile solids is 1. They must be allowed to cool completely before re-weighing them to determine the loss on ignition or volatile suspended solids. overnight).
DATA AND RESULTS Raw Sewage Aerobic Sludge A. Suspended Solids Tin dish marking (do not use ink .fired (G) Loss on ignition (G) Volatile suspended solids (mg/L) Fixed suspended solids (mg/L) Dissolved solids (mg/L) 9 September 2007 CIVL 407 Lab M anual Final Effluent .oven dry (G) Tin dish with filter tare weight (G) Total suspended solids (G) Total suspended solids (mg/L) Gross weight .oven dry (G) Dish tare weight (G) Total solids (G) Total solids (mg/L) Gross weight . Total Solids Dish marking (do not use ink .fired (G) Loss on ignition (G) Volatile solids (mg/L) Fixed solids (mg/L) Percentage fixed residue C. Settleable Matter After 1 hour (mL/L) B.use permanent mark on dish) Sample volume (mL) Gross weight .use embossed mark) Sample volume (ml) Gross weight .
and to demonstrate the use of dissolved oxygen probes. 10 September 2007 CIVL 407 Lab M anual . etc out of your mouth. Colourimetric Method NOTE: Open reflux method is the most accurate means of determining COD because a large sample size is used (20 mls). Wash hands frequently with soap. potassium hydrogen phthalate standards: 800. 100 and 50 mg/L as COD. Wipe up all spills immediately. pipettes. Always use and store pipettes with the top higher than the tip. as well as handling samples probably containing pathogenic bacteria. 200. pencils. I. The closed reflux method is more economical but because a small sample volume (2 mls) is used. Hach block digester. spectrophotometer. Digests from either method can be titrated or read colourimetrically. vials with pre-measured reagents. OBJECT: -to familiarize you with the commonest tests run for assessing treatment plant efficiency and pollution loading to receiving waters. COD . to illustrate the shortcomings of these tests. Keep fingers. 400. wash/rinse bench tops with water/disinfectant. D O a n d B O D 5 WARNING You will be using concentrated acid and other corrosive and toxic chemicals. homogenization of samples containing suspended solids may be necessary in order to get a representative sample and to improve reproducibility. etc. otherwise reagents will run to the bulb end and make pipetting difficult and contamination likely. NEVER pour water into acid.Closed Reflux.La b o ra to ry 2: CO D . Materials: Raw sewage and final effluent samples.
Raw Sewage (Infl) and/or Effluent (Effl) in the upper 1/4 of the tube using permanent markers. The probe has been previously calibrated. Read your samples and the set of COD standards: 800. which is set at 600 nm to read absorbance of light by the sample. concentrated sulphuric acid. into the appropriate tubes and screw cap on snugly. If sample is known to be outside the standard range (above 800 mg/L). 50 and 0 COD as mg O 2 /L. 10-ml graduated pipettes. Note: You are going to measure DO on four different liquids using two different methods. Make sure sample is well mixed with the acid contents of the vial. The four liquids are cold tap water. BOD bottles. burette stand with burettes. aerated dilution water. 2.noting the location of your well-labelled. You will use a chemical titration (Winkler . 11 September 2007 CIVL 407 Lab M anual . 4. before the lab. Prepare calibration curve. Put identifying labels on each tube ie your initials. dilution water. except without mercuric sulfate). absorbance versus concentration and calculate the COD in samples. and are at the spectrophotometer. 400. 250-ml graduated cylinder. Allow samples to digest for 30 minutes (normally this would be 2 hours). raw sewage and final effluent. Remove the vials and allow them to cool to room temperature (use cold water to speed up if necessary).025 N sodium thiosulphate. II. [Note: it should look homogenous and be careful it will be very hot!] 3. Pipette 2 mls of sample (using volumetric pipettes unless particulate is present. 0. manganous sulfate. The instructor will demonstrate the use of the spectrophotometer. 6. At your station you will find four vials with pre-measured solutions containing potassium dichromate and sulfuric acid (prepared according to standard methods. in which case.Procedure: 1. Take vials to fumehood and place in block digester . well-mixed samples in the 25-position digester. 100. Standards have been digested for you. DO NOT ADJUST THE SETTINGS ON THE METER. DISSOLVED OXYGEN (DO) Materials: Raw sewage and final effluent samples. use wide mouth graduated pipettes) in duplicate. 5. a smaller aliquot (making volume up to 2 mls with distilled water) or 2 mls of a pre-diluted sample can be used.the azide modification of the iodometric method) and a dissolved oxygen probe and meter. thermometers. a volumetric correction must be made to the value obtained from the curve. 500-ml Erlenmeyer flasks. alkaline-iodide-azide reagent. The vials should be clean and dry on the outside. starch indicator. 200. If less than two mls of sample or diluted sample was used.
4. 9. When the floc has settled the second time to about 1/3 bottle volume. dropwise. add 1 ml of concentrated H 2 SO 4 and quickly restopper. (Hint: if you measured very low DO level with the meter. raw sewage or influent and final effluent). right to the top and place stopper on bottle. 6. tip the liquid out of the space around the stopper (caution -it may be corrosive) and invert several times. Take each stoppered bottle. Instructions on the use of the probe will be given in the lab. 2. Titrate this volume with 0. 2.) Transfer 201 ml of each bottle (do one at a time) to a 500 ml conical flask. Set back in sink. until the first complete disappearance of the blue colour. using the DO probe and meter. 7. again quickly replacing the lid.think about it!) Complete all four titrations.) You can save these bottles for B. Use the specially marked volumetric flask to dispense the 201 ml. (Note: biological solids originally present in the sample will not dissolve. For the cold tap water sample. WINKLER TITRATION (Azide modification) NOTE: Use only the droppers attached to the reagent bottles for dispensing into samples. 1. Allow the floc to settle to about 1/3 the bottle volume. as demonstrated. Note: if solution already seems pale yellow add starch right away. to thoroughly mix contents. aerated dilution water. The chemical floc should completely dissolve. (Fill carefully. Place the bottles in the sink and one at a time remove the stopper. Stopper the bottles without trapping any air bubbles. then the same sample will require little or no titre and may not turn blue when starch is added . Neglect any reappearance of the blue colour. . 12 September 2007 CIVL 407 Lab M anual 3. B. Fill four BOD bottles with the four samples (cold tap water. remove the stopper. Determine DO.025 N Na 2 S2 O 3 until only a faint yellow or "straw" colour remains.A. Fill a BOD bottle with each of the four samples (or use ones saved from A ). add 1 ml of alkaline-iodide-azide solution the same way. taking care not to entrain additional air. 5. 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. 1. 8. Tip out the liquid from the area around the stopper and invert several times to mix thoroughly. Record the volume of titre. (Note: DO meter temperature is usually not accurate. Do not mix droppers and do not allow tap water into the reagent bottles. Determine the percent saturation for each sample. DISSOLVED OXYGEN PROBE NOTE: DO NOT add any reagents to the samples used for the probe method. Add enough starch solution to give a strong blue colour (one dropper full) and continue to titrate. invert several times again and allow to settle again. for each of the four samples. Record the temperature of each sample using the DO meter or a thermometer. submerge the hose in the bottle and allow the water to overflow for 15-30 seconds). To the same bottles. The DO concentration in mg/L is equal to the number of ml of titre as long as 201 ml is titrated.
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. use the saturation chart above. Pair up the mg/l of dissolved oxygen you measured and the temperature of the water in degrees C. (mg/L) DO conc. Note that this nomogram assumes that the water is at sea level The saturation value can also vary slightly depending on barometric pressure with lower values expected when a storm front moves through as compared to bright and sunny "high pressure" days.Dissolved Oxygen Data Sample/Data Bottle # Sample Volume (mls) Initial Buret reading Final Buret reading Net Titre (mls) DO conc. Raw Sewage Final Effluent Tap Water Dil’n Water 13 September 2007 CIVL 407 Lab M anual .
groups). Place all bottle sets in the 20 o C incubator for five days (or 6 for Tues. Be sure to record the bottle numbers and sample volumes on the following table . All bottles should have liquid in the well around the stopper . graduated cylinders. it may indicate that probe may not have been calibrated properly or the dilution water had been contaminated. 14 September 2007 CIVL 407 Lab M anual . 5. fill one BOD bottle with DILUTION WATER alone. 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). 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. 6. Materials: Raw sewage and final effluent samples. Stopper the BOD bottles. The blank is used to check that the dilution water has negligible BOD. After five (or six)days. ie Rinse the filling tube if it has been in the sample. Be sure to note this in your lab write-up. Measure the Initial DO of the blank using the calibrated probe and meter. 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. showing sample calculations. 3. There should be negligible BOD in the Dilution water. to bring the levels above the ground glass neck of the bottle. thereby maintaining a water seal for each bottle. Dissolved oxygen meter and probe. 10-ml graduated pipettes. Place plastic caps over all bottles. Using the volumes and samples provided by the instructor.if not add a some dilution water (or distilled water) to the well. Carefully top up the BOD bottles with DILUTION WATER. remove bottles and measure the DO. If the dilution water BOD is greater than 1 ppm. 1. carefully measure the waste water samples into BOD bottles. being sure to exclude any air bubbles that might be trapped under the stopper by adding dilution water.III. In addition. Calculate the 5-day (or 6-day if unavoidable) BOD. dilution water. BOD bottles. BIOCHEMICAL OXYGEN DEMAND Note: Because of the logistics of organization some groups may have 6 day BOD 5 instead of the usual 5 day. be careful not to contaminate it with sample. but if there is a significant BOD then it will have to be subtracted before the dilution factor is applied in the calculation. Note: Each group should have 9 BOD bottles in the incubator. 4. These caps are designed to prevent the evaporation of the liquid around the stopper. Measure the Initial DO using a calibrated probe and meter. Because you want to be sure that the dilution water has no BOD.
and ratio of seed water in diluted sample to seed in seed control= (%seed in diluted sample) / (%seed in seed control). DO of diluted sample immediately after preparation. 15 September 2007 CIVL 407 Lab M anual . mg/L DO of seed control after incubation. mg/L.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. = = = = decimal volumetric fraction of sample used DO of seed control before incubation. mg/L = DO of diluted sample after 5 d incubation at 20 oC.
) Why is your COD result different from the BOD result? 5. the other 0. 6) 7) 8) 16 September 2007 CIVL 407 Lab M anual . Diln. titrimetric method. 1 Final Effluent. open reflux. Calculate theoretical oxygen demand for 300 mg/L of methyl alcohol. One waste has a K value of 0. 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. 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. Diln.17. if at all possible.Summary Raw Sewage. Both wastes have equal BOD 5 temperature and volume. 2) 3) 4) Show on a graph the approximate dissolved oxygen sag curves in a river receiving two wastes.25. what would you expect its I) BOD 5 and ii) BOD L to be. If an industrial waste has a COD of 450 mg/L. Diln. Diln. 2 Final Effluent. 1 Raw Sewage.
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 . 35oC incubator. OBJECT: -to demonstrate different methods for the determination of the coliform group of organisms and to acquaint you with bacteriological procedures. etc. filtering apparatus. 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 . Quebec colony counter. Students will do only sections marked ? MATERIALS: Multiple tube method: dilution tubes. lauryl tryptose broth tubes. 35 oC incubator.. Membrane filter method: dilution tubes. T h e re f o re . 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 . wipe up all spills and disinfect the area of the spill.leave it here or take away in a plastic bag to launder. agar tubes. Demonstration only SAMPLE: You will do either a raw sewage sample or a final effluent sample.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. sterile pipettes. DO NOT MOUTH PIPETTE. plates. Heterotrophic plate count: dilution tubes. lab coats. LES Endo agar plates. Get raw data from another group and include in your report. Wipe lab benches with disinfectant before and after each lab. membrane filters. pencils. Wash hands with soap before leaving. sterile dilution water. Keep hands. Demonstration only. sterile pipets. EC tubes and/or Hach A1 tubes. HACH P/A Broth with MUG. Brilliant Green Lactose Broth tubes. eye protection. not both. Do not wear lab coat out of lab . away from your mouth.
Note: these methods will be demonstrated but data must be recorded. All tubes have been examined for gas production. Colonies that are pink to dark red with a golden green metallic sheen are counted after incubation 20-22 hrs at 35oC. The broth will turn yellow indicating total coliform and fluoresce in the presence of E.coli.Pour Plate Method: Serial dilutions of samples mixed with agar and incubated for 48 hrs at 35oC. Bacterial numbers are calculated from the numbers of positive tubes. Gas formation indicates the possible presence of coliform organisms. Using aseptic technique. EC or A-1 tubes positives: 1. The number of colonies counted directly. Heterotrophic Plate Count .COLI in 4 to 24 hours. Demonstration. It is used as a faster alternative to the LTB/EC procedure for enumerating faecal coliforms in water. D.) Methods: ? Recording BGLB. 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. Express as Total coliforms.A. using a ready-to-use P/A Broth with MUG. 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. Parallel positive BGLB broth cultures with negative EC broth cultures indicate the presence of nonfaecal coliforms. streak one LES Endo agar plate from each tube of BGLB broth showing gas from the highest dilution and the second highest dilution. Another series of tubes are inoculated from these positive lauryl tryptose tubes. 1. 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). MUG produces a fluorogenic product when hydrolyzed by an enzyme specific to E. ? C. ? B. 3. Hach’s Most Probable Number Method 8368. Streak plates in a manner to insure presence of some discrete colonies. 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.7oC for 24 hours (confirmed test for faecal coliforms). E. Membrane Filter Technique: Serial dilutions of sample are collected on membrane filters and placed on M-Endo media. (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. Gas-positive Hach A-1 tubes indicate the presence of faecal coliform.5 oC) as a positive completed test response for both Total and faecal coliform. Presence/Absence Testing: HACH technique to simultaneously screen for total coliform and E. 18 September 2007 CIVL 407 Lab M anual . is USEPA-accepted for testing non-potable waters. 2. Calculate the MPN value from the number of positives using the Table 1. Results will be provided for the demonstration set. Use Table 2 on page 27.coli. Consider positive EC broths incubated at the elevated temperature (44. A-1 Medium. as demonstrated. Microscope Slides (Completed Phase of Multiple-Tube Fermentation): Single colonies arising from single cells are prepared and stained for microscope examination.
The sheen area may vary in size from a small pinhead to complete coverage of the colony surface. The sheen area may vary in size from a small pinhead to complete coverage of the colony surface. If you haven’t already. Do not push down on the filter except on the outside edge and use only sterile forceps . Incubate plates (inverted) at 35 oC for 24 ± 2 hr.1. Pour the full volume of dilution tube # 6 onto the filter. 4. Incubate inverted Petrie dishes for 20-22 hr at 35 oC. Be sure dishes are labelled with your name and dilutions. A high count of non-coliform colonies may interfere with the maximum development of coliforms. Verification is usually done by a test for lactose fermentation. Mount apparatus on suction flask with valve sideways to block suction. -Note: “typical” colonies count as coliforms in this procedure. Prepare Gram Stain slides for examination under a microscope. open the valve slowly and allow all the liquid to be pulled through. white. Close valve and rinse the funnel with three aliquots (portions) of sterile dilution water opening and closing valve after each aliquot. or colourless and are considered to be non-coliforms. Colonies that lack sheen may be pink. Use dishes with 20 to 80 coliform colonies and not more than 200 of all types. transfer 1 ml of sample from dilution tube # 6 (most dilute for Raw sewage) or #5 (for Effluent) to an empty (large) Petrie dish. 5. ?B. removing the cover just enough to insert pipette tip and dispense sample. Gram staining technique can be also be used. 19 September 2007 CIVL 407 Lab M anual . Heterotrophic Plate Count . 3.2. 2. from one edge. Verification of both types of colonies is advisable as some typical sheen colonies may be noncoliform and some atypical may be coliform. prepare one set of six dilution tubes following the instructions given under Multiple Tube Fermentation. ?C. Using sterile forceps. therefore the results table should specify #coliform colonies/100 ml. red. 2. Membrane Filter Technique -Total Coliform 1.Pour Plate Method 1. The typical coliform colony has a pink to dark-red colour with a golden green metallic surface sheen. Dilution tubes.. coliform bacteria are Gram-negative (pink) rods (sometimes coccus-like). 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. Atypical coliform colonies can be dark red or nucleated without sheen. Flame loop between second and third quadrants to improve colony isolation. Pipette about 10 ml of sterile dilution water over the surface of the filter with the suction valve still closed.applying gentle pressure. After 20-22 hr. then immediately replace the cover. The filtration apparatus has been sterilized by autoclaving. place a sterile membrane filter (grid side up) onto the filtration apparatus. Place the filter on a small LES Endo agar plate carefully. Using a sterile pipette. When filtering is complete and the valve is closed. In general. Prepare one set of six dilution tubes as described in Section A. Counting. Filtering. Inoculating. remove filter using flamesterilized forceps (allow forceps to cool or they will damage the filter). Dilution tubes. with a motion that excludes air as it comes in contact with the agar. choose appropriate dilutions for counting. Repeat for the rest of the dilution tubes to # 2.
place 1 loopful of sterile dilution water in the centre of a slide. count colonies in 13 squares (of the colony counter) of the most dilute plate having representative colony distribution.too hot for a bacterium. to mix the sample with the agar. Allow the agar to solidify in the dish before inverting. e. 20 September 2007 CIVL 407 Lab M anual . Compute bacterial density and report as “colony-forming units” (CFU) per ml. Take 5 tubes of melted agar from the incubator. Let them cool down from the 60C that they are kept at but not enough to solidify. Do not report as “too numerous to count” if all plates exceed 300 colonies.d e m o n s tra tio n -s ta rte d a h e a d o f la b 1. Add 100 mls of sample directly to pre-measured medium provided. Allow iodine to act for 30 seconds. Wash off stain with water and then with iodine solution (Lugol’s). E.3. Repeat with dilutions 5 to 2 (for Raw) and 4-1 (for Effluent). 2. Spread the colony in the drop of water so that you get a uniform dispersion over an area about the size of a dime. Counting. Air dry the slide high above the Bunsen burner and fix by passing slide briefly through a flame. If necessary. Wash slide in tap water. Examine for yellow colour (total coliforms) and fluorescence (E. ?3. ?4. on the bench. refer to Standard Methods for more instruction. Incubate at 35 oC for 24-48 hours. Multiply the number by 5 to compute estimated colonies per plate (66 cm 2 ). b. f. Slide staining. Microscope Slides: 1. Drain off excess iodine and wash freely with water and then alcohol-acetone decolourising solution. count four representative squares. A hand counter is also available. If more than 10/cm 2. Preferably select seven consecutive squares horizontally and six consecutive squares vertically. Test temperature on your wrist . Presence/Absence Testing . Repeat for the rest of the dilutions. pour and replace cover. marking (on edge of dish so as not to impede counting) and incubating for 48 hr at 35 oC. a. Label the slide using a grease pencil (T or A). Resterilise the loop and remove one colony from the surface of the Les Endo streaked plate (prepared previously). Record results of previously prepared test. Gently swirl the dish. average and multiply by 57 (for plastic Petrie dish). Flood slide with crystal violet solution and allow to act for 30 seconds. The aim of using several dilutions is to have at least one dilution giving colony counts between these limits. select the appropriate dilution to count. 4. After 48 hr incubation. 6. Slide the cover of a Petrie dish back just far enough to pour the agar. Slide preparation: With a flame sterilized inoculating loop.too hot for a baby . if fewer than 10/cm 2. Use the Quebec colony counter to aid in counting and a grease pencil to mark off colonies counted. d. Flood the slide with fresh alcohol-acetone solution to act for 30 seconds.coli). Instead. use the plate(s) that will give from 30 to 300 colonies/plate. 5. c. Work quickly or the agar will solidify in the tube before you get a chance to pour it. 2. D. Do this for a typical and an atypical colony. Ideally.
Microscopic examination. dull. .Come in and count Heterotrophic Plate colonies after 48 hours.5 . blot and then dry in air high above the Bunsen burner (so as not to burn away your efforts). cocci or rods. Examine the prepared slides . Note: in the staining process: before decolourisation all organisms appear Gram positive (blue). In general. Gram negative organisms are visualized (pink).Come in and count colonies on LES Endo Membrane Filter dishes. length to width ratio. After application of the counterstain. Plates ready on a weekend will be put in cold room until Monday. sometimes coccus-like and 0.counts will be provided on the board. .g.3 µm in size. usually motile. cell groupings (single.Prepare dilutions for Heterotrophic Plate Count and Membrane Filter technique. coliform are Gram-negative (pink). plump rods. pairs chains). h. .Examine prepared slides. transparency.Membrane filter prepared dilutions. and on the slide: typical or atypical. Day 3. Description of bacteria: Describe what you see on the plates: colonial morphology of each type of colony seen ie surface (shiny. metallic. nonsporing. rough. c. 21 September 2007 CIVL 407 Lab M anual .Record positives in all Multiple Tube Fermentation inoculations: LTB. in pairs.Prepare and incubate Heterotrophic (Pour) Plates using dilutions above. gram positive or negative. or in short chains. Day 2. concave). a. . . Wash in water. b. . BGLB and EC and AI tubes . Apply fuchsin or safranin (counter)stain for 30 seconds. odour. ?3. Cells occur singly. after decolourisation Gram negative organisms are no longer visible.Record Presence/absence testing results of Hach Prepared Media bottles. smooth. colour. .do not turn knobs except the positioning ones. place on LES Endo agar dishes and incubate. Schedule Summary Day 1. . short.
. 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 . Multiply the MPN index by the dilution factor from the series used when dilutions other than 1/1. 1 2 3 4 5 6 Heterotrophic Plate Count .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. 1/ 10 and 1/100 are used.
1 ml 5/5 4/5 1/5 0. MF results. With MUG reagent added to P/A Broth. you can simultaneously detect total coliforms and E. They are in concentrated form and merely require the addition of 100 ml of sample (or diluted sample) to the media and incubation.coli will fluoresce as early as 4-24 hours. use the results from only three of these in computing the MPN.Discussion: -sources of error? -compare MPN. Estimation of Bacterial Density . that in the denominator.coli. an enzyme specific to E. E. choose the highest dilution that gives positive results in all five portions tested (no lower dilution giving any negative results) and the two next succeeding higher dilutions. 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. Both media meet USEPA guidelines for testing of total coliforms in drinking water. MUG (4methylumbelliferyl-$-D-glucuronide) can be used to detect E. Use the results at these three volumes in computing the MPN index. the total tubes planted. The number in the numerator represents positive tubes. it is not necessary to enumerate.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 .coli because it produces a fluorogenic product when hydrolyzed by glucuronidase. To select the three dilutions to be used in determining the MPN index. Do they agree? Why/why not? -compare MPN/MF with Heterotrophic Plate Count. 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 0.01 ml 2/5 2/5 0/5 0.Most Probably Number (MPN) When more than three dilutions are used in a decimal series of dilutions. In the examples given below. If zero total coliform is the maximum contamination goal.coli. the significant dilution results are in boldface. A long-wave UV light is required to detect fluorescence.
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 .MPN Index and 95% Confidence Limits for Various Combinations of Positive Results when 5 Tubes are used per Dilution (10 mL. 1.1 mL) Comb.0 mL.Table 1 . Of Positiv es 0-0-0 0-0-1 0-1-0 0-2-0 1-0-0 1-0-1 1-1-0 1-1-1 1-2-0 2-0-0 2-0-1 2-1-1 2-2-0 2-3-0 3-0-0 3-0-1 3-1-0 3-1-1 3-2-0 3-2-1 4-0-0 4-0-1 4-1-0 4-1-1 4-1-2 MPN Index/ 100 ml 95% Confidence Limits Lower 1 1 1 1 1 1 2 2 1 2 3 3 5 3 4 4 6 6 7 5 7 7 9 12 Upper Comb. 0.
Hach .MPN Table 2 25 September 2007 CIVL 407 Lab M anual .
2) 3) 26 September 2007 CIVL 407 Lab M anual . What are the important pathogens transmitted by water? How is quality control achieved in microbiological analysis.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.com/ ¸Information Central/Learning Library/Microbiological Testing ASTM: (STP635) D.htm Lab 5 Questions: 1) Compare the advantages and disadvantages of the multiple tube fermentation and membrane filter techniques. Mara: -Bacterial Indicators/ Health Hazards Associated with Water -Bacteriology for Sanitary Engineers Microbiology 321: -Manual of Microbiological Techniques Website: http://www.D.org/microbelibrary/files/ccImages/Articleimages/keen/Gram stainkeen.http://www.hach. -The Use of Indicator Organisms to Assess Public Water Safety .microbelibrary.
chloride specific ion electrode. indicator-acidifier reagent. spectrophotometer. 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. 125 ml Erlenmeyer flasks. Hint: optimum ranges will coincide with the standards provided or will be indicated within the pertinent section. standard AgNO 3 solution. goggles or glasses and lab coats. graduated cylinders. standards. MATERIALS: -millivolt meter. 27 September 2007 CIVL 407 Lab M anual . pH meter with double-junction electrode and silver billet electrode. known addition and calibration techniques and colourimetric and potentiometric titrations. HNO 3. burettes. conc. OBJECT: -to acquaint you with the use of specific ion electrodes. That means you will probably have to dilute the sample by the most accurate means available. Note and consider carefully: Different methods of analysis have different optimum ranges. magnetic stirrer. immediately. indicator. sample. If chemicals are spilled alert instructors. volumetric pipettes. semi-log graph paper. standard chloride solution (0.La b o ra to ry 4: Ch lo rid e D e te rm in a tio n WARNING You will be using concentrated acid and other very dangerous chemicals. beakers. standard Hg(NO 3)2. NaHCO 3. buffer. to illustrate the differences in operation and accuracy between instrumental methods and wet chemical methods. reference electrode (double junction). beakers.5 mg/L). Use personal protective equipment: gloves. Mercuric thiocyanate is very toxic.
e. Determine the concentration of chloride in the sample. pour out 100 mls of each standard into separate. place the lowest standard on the stirrer and lower the electrodes into it. 2.E 1 volume of sample volume of stock solution added mg Cl. Calculate the chloride concentration. 4.in sample = mg/L Cl. 2 minutes) and use it for all standards and samples. CHLORIDE . mg Cl. stir and obtain a reading for it. Unless already done by a previous group. Standards of 1000.= ____________________ 28 September 2007 CIVL 407 Lab M anual . As probe tends to drift for some time it may be expedient to select a “standard time to read” (i. rinsing and blotting dry the electrodes between each. 6. Using semi-log paper or software equivalent.added to the sample "slope" = change in potential over 10 fold change in concentration of standard (use data from Step 2).I. Repeat for rest of standards. 5. plot the millivolt readings (linear axis) against the concentration of the standards (log axis) to produce a calibration curve. 100 and 10 ppm (mg/L) have been prepared for you and are at the meter. 3. 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. Add 2 mls of IONIC STRENGTH ADJUSTMENT (ISA) buffer to each using the plastic dropper (filled to the mark). Measure 100 mls of sample. 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 ). where: E1 = E2 = E = Vo = Vs = A = S = potential of sample (mV) potential of sample plus stock addition potential change = E 2 . labelled beakers. added 2 mls of ISA.SPECIFIC ION ELECTRODE Part One: Preparation and reading standards and samples 1.
The colour of the solution should be green-blue at this point. use a plastic dropper to transfer blank into a spectrophotometer cuvette (do not fill more than 3/4 ). 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). (Use extreme caution as this is a corrosive & toxic chemical.0 and 8. Add 1 ml (use pipette to measure accurately) of indicator-acidifier reagent to sample. Near the end-point.0 ppm).8. pure blue. a pH of greater than 3. it should be rerun from the start after diluting. MERCURIC NITRATE METHOD FOR CHLORIDE DETERMINATION The principle of this method is as follows: Cl. and Hg ++ (excess) + DPC º violet colour 3. the excess mercuric ions combine with diphenylcarbazone (DPC) to produce a violet colour (the end-point).II. CHLORIDE . Using a 25 ml graduated cylinder. Titrate with 0. III. After all Cl. 2. after a few more minutes.ions in the sample combine with added mercuric ions forming a soluble but virtually undissociate-able complex. 4. (Light green indicates a pH of less than 2.. insert in the spectrometer and record the absorbance displayed on the meter.) Every two or three minutes thereafter you will rinse the cuvette with the next standard or sample.+ Hg(SCN)2 º HgCl2 + 2SCN SCN .1. 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 .) Mix thoroughly by gently swirling the flask. 2. (Alternatively. Determine the chloride concentration in the sample from the calibration curve. 5.0. discard and refill at least twice to rinse cuvette. Pour 100 ml of sample (or a portion diluted such that the concentration is less than 100 mg/L) into a 250 ml beaker. [Note: this delay between additions is to allow you time to measure absorbance of each at the 10 minute time required . and the sample (diluted if necessary) into separate and labelled 125 ml Erlenmeyer flasks (5 in total). zero instrument on distilled water and obtain an absorbance value for the blank.ions have been bound.014N Hg(NO 3) 2 to a definite purple end-point. 6. Marking the time (T=0). which then must be subtracted from the standards and sample. Hg ++ + 2Cl. Wait two or three minutes and then add the reagent to the first (lowest) standard. 3. (No further adjustment is required after the blank or distilled water has been set to zero. 4. If sample is above the highest standard. fill. add 5 ml of the “combined reagent” (containing ferric alum and mercuric thiocyanate solutions) to the blank first. the three standards (2.] After 10 minutes has passed (since the combined reagent was added to the blank).0.) Prepare a calibration curve by plotting the absorbance readings versus the concentration of chloride in the standards. measure 25 mls of “zero” standard or “blank” (halide-free distilled water). the 1 ml of the indicator-acidifier reagent will adjust the pH to 2.BY COLOURIMETRIC METHOD The principle of this method is based on the following formula: 2Cl.5 ± 0. Insert cuvette into the spectrophotometer (wavelength must be set at 463 nm) and press “zero” or “ref set” (depending on model) to make the meter read zero. using the dispenser provided.+ Fe+++ º Fe(SCN) ++ (Reddish brown) 1. For most samples.they can’t all be at 10 minutes at once.º HgCl2 1.
0 ml of concentrated HNO 3 (use plastic dropper). 5. The end-point occurs at the greatest mV change per unit addition of AgNO 3. until past the endpoint when larger increments can be used again (ª mV/ml decreases). Calculate the Normality of the AgNO 3 using the following equation: 30 September 2007 CIVL 407 Lab M anual . Place 10. large increments (1-2 mls) may be added. and add 2. 6. At the start.4. Begin titrating.2 ml or drop-wise) should be added. with standard AgNO 3. 4. in increments. The titre from this step must be subtracted from the titre for the sample. Start the stirrer and set the millivolt reading to zero or record initial mv reading [Note: Some meters can not be set to zero]. Part one: Standard 1. 3. 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 . 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).0 ml (use volumetric pipette) of standard chloride solution in a 250 ml beaker. Take care that stirrer is not hitting the electrodes.. Nitric acid is in the sink and is very corrosive. place beaker on stir plate and lower electrodes into the solution. where: A = ml titrant for sample B = ml titrant for blank N = normality of titrant IV. then.1 to 0. 2. dilute to about 100 ml with distilled water. drops of the purple end-point. as the end-point of the reaction is approached (ª mV/ml increases). Calculate the chloride concentration: 5.. A reference set of data will be provided for comparison. Plot a differential titration curve to determine the exact end-point. This way each station will have data for each part. CHLORIDE BY POTENTIOMETRIC TITRATION Note: For this part form two groups comprised of 1 member from each station. recording the volume and millivolt reading for each increment. Immerse stir bar. Continue the titration about 5 ml past the end-point. Don’t forget to add the indicator. Rotate the work within the group so that different pairs are doing the titrating and recording of numbers. smaller increments (0.if one is necessary.
be careful to use the average value of ml of titrant on the abscissa.Part Two: Sample 1. b. in this case. Calculate the chloride concentration in the sample using the following equation: 3.) Plot three curves for this titration: a. which are merely first and second derivatives. 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 . (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. Millivolts per ml per ml versus ml of titrant. Millivolts per ml versus ml of titrant. if dilution is necessary into a 250 ml beaker (use a graduated cylinder to measure). Millivolt reading versus ml of titrant. It is not. 4. Add 2 ml of HNO 3 and proceed as described in Part One. c. 2. In plotting b and c. Measure 100 ml of sample or portion made up to 100 ml.
Chloride . 32 September 2007 CIVL 407 Lab M anual .Potentiometric Titration RAW DATA A Volume AgNO 3 Added (Vol) (ml) B Meter Reading (E) (mV) C FIRST DERIVATIVE D SECOND DERIVATIVE F G H ª (Vol) (ml) ª (E) (mV) E ave (AgNO 3 ) Volume (ml) ª (E)/ ª (Vol) (mV/ml) ª ( ª E/ ª Vol) (mV/ml) ª [Ave(Vol)] (ml) I Ave of Ave(Vol) (ml) J ª( ª E/ ª Vol) /ml (mV/ml/ml ) Extra pages are at the back of this lab manual in appendix.
specific ion electrodes are rarely usable in environmental samples. fluoride and bromide titrate as chloride. chromate. fluoride and bromide titrate as chloride. # Other interferences: ferricyanide. # 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. # Chromate. From your chloride results. hydrazine. MERCURIC NITRATE TITRATION METHOD # Iodide. strongly reducing solutions may form a surface layer of silver. It can only correct for substances that enhance or suppress the test response proportional to the amount of chloride ion present. 2. known addition cannot correct for any ions that test as chloride ion. Lab 3 Questions 1. in itself. # High levels of ions which form very insoluble salts of silver may deposit a layer of salt on the electrode membrane. In the above tests. and nitrite interfere. 3. cyanide. # Environmental samples usually require pretreatment.3 mg/L? Outline the advantages and disadvantages of all the methods used in this lab to measure chloride concentration. iodide. ferric ion. Also. dichromate.INTERFERENCES IN CHLORIDE DETERMINATIONS POTENTIOMETRIC METHOD # Iodide. # Pretreatment of environmental samples usually required. thiosulphate. KNOWN (STANDARD) ADDITION TECHNIQUE # The known addition techniques will only correct for certain types of interferences. Each sample type requires development of an appropriate method which. # In practice. can you see any advantage in suing the first or second derivative curve? Discuss. and sulfite ions interfere when concentration greater than 10 mg/L. SPECIFIC ION ELECTRODE # All halides interfere (the electrode is a halide electrode). This is a complex. ferric. # Mercury must be absent from samples. causing electrode malfunction. # Environmental samples usually require complex pre-treatment. # Colour may obscure or interfere with end-point determination. fluoride. PRETREATMENT TECHNIQUES # There are no "standard" pretreatment techniques. # Sample pH may need adjustment beyond the capacity of indicator-acidifier reagent. 33 September 2007 CIVL 407 Lab M anual . COLOURIMETRIC METHOD # Bromide. 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. requires knowledge of the compounds in the sample which are likely to interfere.
to illustrate the concepts of free and combined chlorine residuals. 1995. Materials: -600 ml beakers. and exactly 10 ml of 0. 2. When weighing chemicals at the balance. pipettes. potassium iodide (KI) crystals. 250 ml volumetric flasks. clean up all spills with a brush into a container and wash crystals down the drain. REFERENCES: Standard Methods 19th ed. gloves and lab coats. 3. 250 ml Erlenmeyer flasks. Take a 250 ml Erlenmeyer flask and add about 1 gram of potassium iodide. about 5 ml of glacial acetic acid (use a graduated cylinder). Personal protective equipment required at all times when working in the lab are: eye protection.N-diethyl-p-phenylenediamine (DPD) indicator solution. I. Titrate rapidly with the stock chlorine solution (in burette) until the appearance of a constant 34 September 2007 CIVL 407 Lab M anual .025 N sodium thiosulphate. 0. burettes and stands. OBJECT: -to acquaint you with disinfection of water supplies and to demonstrate techniques for the determination of chlorine residuals. about 50 ml of chlorine-free water (distilled water will do). PREPARATION OF STOCK CHLORINE SOLUTION (Iodometric method . chlorine demand and break-point chlorination. standard ferrous ammonium sulphate (FAS) solution. 100 ml graduated cylinder. Make up to the 250 ml and invert flask several times to mix. phosphate buffer solution.025 N sodium thiosulphate solution (use a volumetric pipette). (concentrated) glacial acetic acid. Mix well (on stir plate with stir bar). starch indicator. Bleach/chlorine solutions are strong oxidants. 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. chlorine free water.total chlorine residual) 1. Wipe up all liquid spills immediately. Rinse a burette and fill it with this stock chlorine solution. allow to mix again. N. add 1 ml of starch 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..
These 6 flasks can be prepared at once to make them ready for the next step. as there is lots to do .COORDINATE! IIa.). 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). refilling burette. 3. Total chlorine (Free plus Combined) = Reading C Free residual chlorine = Reading A Combined residual chlorine (dichloramine plus monochloramine) = Reading C . Continue titrating until red colour (if any) is discharged once again (Reading B).) Free and combined chlorine or total chlorine: To obtain total chlorine in one reading. mix to dissolve. For dichloramine concentrations greater than 1 mg/L. IIb. After 15 minutes of contact time. add full amount of KI at the start (ie 2 drops KI solution and 1 g of KI crystals) to a fresh sample. 35 September 2007 CIVL 407 Lab M anual . calculate the chlorine concentration in the stock solution (See Data and Results section for formula). 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. etc. Place 5 ml of phosphate buffer solution and 5 ml of DPD solution in a 250 ml conical (Erlenmeyer) flask and mix. 2. Add 100 ml of sample or diluted* sample using a 100 ml graduated cylinder and mix again. (*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. titrate to colourless again.Breakpoint Chlorination using Free and Combined Chlorine Residuals Values 1. d) Alternatively: (Not to be done in this lab. 2. 18th edition. let stand for two minutes and titrate until the red colour (if present) is discharged. Record the burette reading (Reading C). Work in groups of three or four for this part. Record the burette reading (mls) = Reading A. pp 4-43. if colour drifts back (indicating an incomplete reaction). From the amount of chlorine solution used. Determination of Free and Combined Chlorine by the DPD Method 1. 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). 4. 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). please refer to Standard Methods. Go to c. Method 4500-Cl F. let stand 2 min more if colour drifts back indicating incomplete reaction. Let stand 2 minutes more. Let stir for 2 min and then continue titrating until red colour (if any) is discharged (Reading C). c) Dichloramine: Add about 1 g KI (to the same sample) and mix to dissolve. 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.A Monochloramine= B-A Dichloramine= C-B For interferences. a) Free chlorine: titrate rapidly with standard FAS (Ferrous ammonium sulfate) titrant until the red colour is discharged.purplish-blue colour. b) Monochloramine: Add two drops of KI solution (to the same sample) and mix.
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. FAS titrant concentration is 1 ml = 100 µg Cl as Cl2 a. Make a plot of the three residual chlorine components against the applied chlorine dosage. The rate of kill of bacteria by chlorination follows first-order reaction kinetics. Given: HOCl W H + + OClK eqv = 2. Determine the concentration of total. Under what conditions is the practice of break point chlorination necessary? 4. 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. Discuss your results as a function of the residual forms of chlorine present at different chlorine dosages and at different contact times. what percentage of bacteria would be killed in 10 minutes at a chlorine residual of 0. Make a separate graph for the 15 minute and 1 hour contact time. Why is it important to determine chlorine residuals in domestic water supplies? 2. If this is true.5 mg/L if 50% are killed in 1½ minutes at this concentration? b. If a sewage sample contains 10 x 10 6 coliforms/100 ml. a. Guaranteed analysis is normally printed on the container and is usually 5-6%. c. II. how many coliforms would remain after a chlorine contact time of 10 minutes? 20 minutes? 36 September 2007 CIVL 407 Lab M anual .7 x 10-8 at 25 o C % HOCl = 27 What is the pH of the solution? 3. 250) then convert mg/L to g/100 mls = %.e.DATA AND RESULTS I. b. free and combined residual chlorine for each applied chlorine dose. d.
of Cl Soln.15 MINUTE CONTACT TIME BEAKER NUMBER Vol. (mls) (Stock=_____mg/L Cl as Cl2) Applied Cl dosage (mg/L) Volume of FAS to endpoint A Volume of FAS to endpoint B (includes A) Volume of FAS to endpoint C (includes A & B) Free chlorine (mg/L) (A) Monochloramine (mg/L) (B-A) Dichloramine (mg/L) (C-B) Combined residual chlorine (mg/L) (C-A) Total chlorine (mg/L) © Time of Cl addition Time of Residual measurement 1 2 3 4 5 6 37 September 2007 CIVL 407 Lab M anual .
of 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 . of FAS to endpoint A (mls) Vol. of FAS to endpoint B (mls) (includes A) Vol. (mls) (Stock=_____mg/L Cl as Cl2) Applied Cl Dosage (mg/L) Vol.60 MINUTE CONTACT TIME BEAKER NUMBER Vol. of Cl Soln.
place a beaker containing the sample and stir-bar on the stir-plate. metres and calibrated glassware are available in limited quantities and are very expensive to replace.) 2. bromcresol green. graduated cylinders. T u rb id ity WARNING Please use caution when handling all chemicals. turn on stirrer and obtain a stable reading. then use buffers 4 and 7 to calibrate. phenolphthalein. Use care when using lab equipment.(Do not allow the stir bar to hit the probe. I. Rinse probe when you are finished and leave immersed in pH 7 buffer for storage.) Record value in following table. burettes and stand. the two buffers that are chosen for calibration will bracket your sample ie if sample is less than 7. beakers. turbidimeters. Co lo u r. pH test paper. buffer solution. magnetic stirrer. MATERIALS: -pH meter. then use buffers 7 and 10. p H. Calibrate the pH meter by following the procedure outlined on the instruction sheet beside the meter unless told otherwise. Normally. 3. lower probe. OBJECT: -to familiarize you with the most common water treatment tests. standard acid. 39 September 2007 CIVL 407 Lab M anual . rinse & dry probe. water samples. hardness indicators. Ac id ity . Hard n e s s . to acquaint you with the use of pH metres. EDTA. Always wear personal protective equipment. 1 N NaOH. particularly those labelled with specific hazards. colour comparator. 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. if above 7. (Take care not to contaminate paper in case. pH test paper and other water quality assessment equipment. Probes. standard base. 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. 4. After calibration is complete. metacresol purple. bromphenol blue.Lab o rato ry 6: Alkalin ity . flasks. DETERMINATION OF pH 1.
Record burette reading (P). III. Record the volume of titrant required in the following table. it may be necessary to repeat the titration with the sample diluted 1 to 1 (again) with distilled water. sample-rinsed graduated cylinder and transfer to an Erlenmeyer flask. Add the last few drops at 3-5 second intervals so that you do not "overshoot" the end-point. (If solution is clear after addition of P. Rinse and fill another burette with standard base solution and label it. until the reddish tinge disappears from the solution and a pure blue remains.II. Titrate slowly with EDTA. 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. titrate until just colourless. Complete the titration within 5 minutes to minimize the tendency toward CaCO 3 precipitation. 40 September 2007 CIVL 407 Lab M anual . Add a full dropper of phenolphthalein (P) indicator and if the solution stays pink or red. In order to remove any free residual chlorine (which interferes with the indicator colour response). TOTAL HARDNESS DETERMINATION . while stirring. (If the solution is already blue after indicator. DETERMINATION OF ALKALINITY 1. The buffer elevates the pH to 10. Measure 100 ml of water sample into a clean. IV. Measure 100 ml of sample (with minimum agitation or exposure to air) using a graduated cylinder and transfer carefully to an Erlenmeyer flask.. if the approximate hardness is known (by unsuccessful attempts). 2. If sample is highly alkaline (titration goes over one burette full) dilute the sample and titrate again.?) 4. 4. 3. Add stir-bar. Add 1 drop of sodium thiosulphate solution. Rinse and fill a labelled burette with standard acid solution. add 1 drop of 0. 5. Measure out a new sample and add 1 drop of sodium thiosulphate and a full dropper of phenolphthalein. Apply dilution factor to obtain final result. Titrate to the first uniform pink colour (colour stays)..EDTA TITRIMETRIC METHOD 1. 2. add 90% of the titrant to the sample before adjusting the pH with buffer. Rinse and fill a burette with standard EDTA solution and record the initial burette reading. Add a stir-bar. Alternatively. Pour into a 250 ml Erlenmeyer flask.5).1 N sodium thiosulphate to the sample. If a ppt does form within the 5 minutes. 3. 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. DETERMINATION OF ACIDITY 1.think what that means!) 4. (BG or MO) 6.. White paper placed under the flask will facilitate this determination.. 3. 2. Measure out 25 mls of sample using a graduated cylinder and dilute to 50 ml with distilled water. A pH much above 10 promotes CaCO 3 precipitate. Add a full dropper of bromphenol blue indicator and titrate until the colour changes from yellow to blue ("MO" Acidity).. Record the burette reading in the following table.
. lint-free cloth to remove water spots and fingerprints. Use signal average mode if 41 September 2007 CIVL 407 Lab M anual . The instrument may suggest diluting if over range. Record the final burette reading in the following table. Select signal averaging mode by pressing the SIGNAL AVERAGE key. Press: I/O. Record the final burette reading in the following table. Values are reported as APHA Colour Units (eqv. except on an unfiltered sample. or a volume sufficient to produce a pH of 13-14 (designed to precipitate the magnesium as a hydroxide). 2. Add one aliquot of the Calcium Hardness Indicator and titrate with EDTA slowly. Refill the burette with EDTA titrant and note the initial reading. Record all colour data in the following table. Pour the supernatant into one of the cells (to the etched mark) and fill another with distilled water. The instrument will turn on but will turn off automatically after a short time so you should be ready. Close the lid.0 ml of 1 N NaOH solution (use 3 droppers full). TURBIDITY . The display will show AUTO RNG when the instrument is in automatic range. Place the cell containing water into the spectrophotometer and press Zero. Add a stir-bar and stir to mix.EDTA TITRIMETRIC METHOD 1. The display will show SIG AVG when the instrument is using signal averaging.BY HACH 2100P Turbidity Meter.5. 2.TRUE AND APPARENT PART A: TRUE COLOUR . 3. The reading might be higher so if you had to dilute in Part A dilute for Part B. taking care to handle the sample cell by the top. Select automatic range by pressing the RANGE key. Fill a sample cell to the line (about 15 mL). to mg/L platinum as chloroplatinate) PART B: APPARENT COLOUR 1. 3. 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. Measure out 50 ml of sample into an Erlenmeyer flask and add 2.Enter the stored program number for true colour=120 1. Cap the cell. 2. Filter about 20 mls of sample using a filter funnel and stand and the pre-fluted filter paper provided 2. 4. Place the cell containing sample in it and press Read.. . 3. 4. Use the technique outlined in Part A. COLOUR . 4. VII. CALCIUM HARDNESS DETERMINATION . VI. Wipe the cell with a soft. with continuous stirring to a pure blue end-point. 5. to determine the apparent colour (measures the influence of turbidity on the reading).HACH Spectrophotometer.. V. Demonstration of Jackson Candle and Hellige PART A: Hach Turbidimeter 1.
Carefully fill the 20 mm viewing depth tube to the mark with sample. 16th edition...adding liquid to a hot tube will cause it to shatter.demonstration only. PART B: Hellige Turbidimeter . If you take too long. 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. the turbidity may start to settle and condense on the bottom of the tube. 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. Close the door and switch on the light. Record the turbidity after the lamp symbol turns off. (This allows you to make a more gradual and accurate approach to the end-point. 3. record the number and refer to the appropriate graph to determine the turbidity as mg/L SiO 2. 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. Pinch all excess charred wick with fingers before lighting or soot may be deposited on the glass tubes. T = Total alkalinity. Read the scale on the dial.Demonstration only 1.NTU.U.. Press: READ The display will show .T.s.) Remove the glass tube from the holder and read the turbidity at the meniscus of the sample.the sample causes a noisy signal (display changes constantly). 42 September 2007 CIVL 407 Lab M anual .. Lower the plunger into the liquid making sure no bubbles are trapped under the plunger and the top is dry. 2. that the filter selector shows "none" and that bulb B is in use. Note that there are several different scales on the side of the tube. then the turbidity in NTU. PART C: Jackson Candle Turbidity . Be sure that the calibrated tubes are clean before adding samples to the tubes.. Record value in following table as J. Wipe the outside of the tube to dry it and place it in the turbidimeter (in the circular groove of the mirror unit). 6. 3. Take the reading where the dark central part first merges with the surrounding field. obscuring vision. DO NOT PLACE AN EMPTY TUBE IN THE TUBE HOLDER WITH THE CANDLE LIT . 1. Note that the rectangular door mirror is closed. Try to be quick and not burn the candles more than a few minutes at a time. 2. Pour sample into the tube in small increments until you can no longer distinguish the shape of the candle flame. See Standard Methods. page 272-273.
" 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.Sample volume Initial burette reading P. Alkalinity end-point reading (mls) __________ "M.O.DATA Sample 1 pH ." Alkalinity end-point (mls) Initial burette reading Net P.Sample volume "M.O.Initial pH by test paper Initial pH by pH meter ALKALINITY.O." Acidity end-point reading (mls)__________ TOTAL Hardness Sample volume (mls)__________ Calcium hardness as mg CaCO 3/L __________ 43 September 2007 CIVL 407 Lab M anual . Alkalinity titre (mls) Net "M.O." Alkalinity titre (mls) ACIDITY. Acidity end-point reading (mls) Initial burette reading Net "M.
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 .CALCULATIONS Calcium: Calcium Hardness: where: A= B= ml titrant for sample mg CaCO 3 equivalent to 1.0 Total Hardness (EDTA): where: A= B= ml titrant for sample mg CaCO 3 equivalent to 1.00 ml EDTA titrant at the calcium indicator endpoint = 1.00 ml EDTA titrant at the calcium indicator endpoint = 1.
RESULTS (sample) P.Alkalinity as CaCO 3 (mg/L) OH .Alkalinity as HCO 3 .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 .Units= N.Alkalinity as CaCO 3 (mg/L) CO 3 -2 Alkalinity as CaCO 3 (mg/L) HCO 3.Alkalinity as OH .ion (mg/L) CO 3 -2 Alkalinity as CO 3 -2 ion (mg/L) HCO 3.O.U.APHA Colour Units: True Apparent Turbidity . Alkalinity as CaCO 3 (mg/L) "M." Alkalinity as CaCO 3 (mg/L) OH .T.ion (mg/L) "M.Hach . ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 45 September 2007 CIVL 407 Lab M anual .
non-carbonate and total hardness of this water in mg/L as CaCO 3 ? in meq/L? Is the analysis complete? Why? 6. What errors can occur in the calcium hardness determination? Why? 3. 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. 46 September 2007 CIVL 407 Lab M anual . calculate the carbonate alkalinity in mg/L as CaCO 3 for the water sample analyzed during this lab.Lab 6 Questions 1. Does this agree with the results from Part II of this lab? Explain. given K 2 = 4. 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. From equations (17-12) and (17-13) in Sawyer and McCarty. Is this water suitable for a domestic water supply? Why? 2.7 x 10 -11 and K w = 10 -14. What sanitary significance does colour have? 4.
for the next session (b). MATERIALS: -Jar Test Apparatus. No. alum solution (Al2 (SO 4 )3 @18H 2 O). to be conducted over two lab periods. OBJECT: -to illustrate the principles of coagulation and water softening. 1000 ml beakers.6. You should have with you the part of Exercise 7 that gives instructions for the various tests. 7 (except Jackson Candle and Hellige Turbidimeter). soda ash solution (Na 2CO 3) and lime (Ca(OH)2) or sodium hydroxide (NaOH). 47 September 2007 CIVL 407 Lab M anual . No.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. 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. and all of the materials from Lab. In the second lab you will use the previously determined dosages to coagulate and soften the sample and then re-analyse the resultant water. Please try to coordinate your work with the rest of the class. The optimum alum dosage for coagulation of the sample will then be chosen. Several lime and soda ash dosages will also be selected for determining the efficiency of water softening. This is a two-step experiment.
decant the supernatant liquid carefully into a clean set of beaker (try not to stir up what has settled) and discard the floc. if available. if time permits. 2. Record observations on settling rate. alkalinity. large). 7. Add the assigned alum dosages as quickly as possible and then start the stirrer. Stop stirring. hazy. 3. Measure out six 1000 ml aliquots of sample (using graduated cylinder to measure. Carefully decant into a graduated cylinder 800 mls of the supernatant liquid and pour into fresh beakers. and clarity of supernatant liquid. acidity. use indicators as specified in Lab 6. In the lecture. cloudy). Complete the coagulation summary sheet during the lab period. 3. but because of equipment limitations. Stir at 100 rpm for 1 minute and at 20 rpm for 10 minutes. 4. Stop stirrer and allow floc to settle. decant the supernatant liquid carefully into another set of beakers (try not to stir up what has settled). true colour and turbidity (Hach). Brief Methods Summary for Analyses Alkalinity: @ 100 ml of sample. position them under stirrers. COAGULATION USING ALUM . and fill in the table on the board. Record relative floc sizes (pin-point. 6. Observe and make notes describing floc formation and note the time of appearance. Determine which dosages were most appropriate for this sample.Part A:. slow). Analyze these supernates plus a sample of raw water for pH. times of appearance and descriptions and record. After the flocs have settled (allow 15 min). 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). Stir at 100 rpm for 1 minute. 6. Place beakers on the jar tester apparatus. clear.1 N sodium thiosulphate 48 September 2007 CIVL 407 Lab M anual . 5. Stir at 100 rpm for 1 minute. hardness (total and calcium).5 to 1 cm off the bottom of the beaker. and settling characteristics of the floc (rapid.work in 2 or 3 teams to assess a total of 6 dosages for each team. acidity. clarity of supernatant liquid (very clear. Make relative estimates of floc sizes. Reduce the stirring rate to about 20 rpm for 10 minutes. 5. Discard the floc and clean beakers in preparation for a second decanting in Step 4. Add the designated lime (or NaOH) and soda ash additions as quickly as possible to the decanted samples. turbidity and colour. 4. alkalinity. floc volume. 2. Stop stirrer. drop of 0. 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. Lift paddles out of beakers and secure. You may need to use blocks to position the paddle 0.. After the floc has settled (allow 15 min). moderate. you will discuss the data and select the appropriate alum dosage to be used by all groups in the water softening step next week. small. hardness (total and calcium). Analyze the supernates for pH. A pH meter could be used for the alkalinity and acidity determinations. medium. Make sure it is centred. not a beaker ) and transfer to 1000 ml beakers. Add the appropriate alum dosage and immediately start stirring. (NB dosage now based on 800 ml sample size). Pour six 1000 ml aliquots of sample into 1000 ml beakers and place on the jar test stirrer apparatus. 1. 1. Part B: WATER SOFTENING PROCEDURE (to be done the following week) again work as teams of 2 or 3.
Compare removal of hardness components under the different doses . 2.3 and with bromcresol green (continuing with the same sample) to yellow .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 . Based on your chemical analysis of the raw water.02 N base with bromphenol blue to blue .02 N acid with phenolphthalein to colourless . @ calculation: Ca hardness (mg/L CaCO 3 )= mls titre x 1000/mls sample Lab 7 Questions A.7 ("methyl orange" acidity) and with phenolphthalein (using a fresh sample) to pink . 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.alkalinity (mg/L CaCO 3)= mls titre x N acid x 50. What treatment would you recommend to prepare this water for human consumption? Why? 49 September 2007 CIVL 407 Lab M anual .1 N sodium thiosulphate @ titrate with 0. @ 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 .5. 3. Coagulation 1.@ titrate with 0. drop of 0.pure blue).pH 4.pH 3.3 ("phenolphthalein" or total acidity) @ calculation . Compare your experimental softening results to the theoretical results based on solubility equations or mass balance equations.use graphical presentation. show by calculation the theoretical amount of lime and soda ash required for excess lime/soda ash softening. 2. Softening 1. Analyze the response of colour and turbidity removal as a function of alum dose.pH 8. @ calculation .pure blue).pH 8.000/mls sample Acidity: @ 100 ml of sample. 4.acidity (mg/L CaCO 3)= mls titre x N base x 50.
7 “MO” Net titre to pH 8.sample vol Net titre Notes: 50 September 2007 CIVL 407 Lab M anual .5 “TOT” Acidity .5 “P” Net titre to pH 4.5 “TOT” Total Hardness sample vol Net titre Ca Hardness .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.
) unfiltered Notes: 51 September 2007 CIVL 407 Lab M anual .P.U.A.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.H.) True Colour (A.T.) (filtered) Turbidity (N.P.A.H.
sample vol ml Net titre to pH 3.5 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 8.5 Total Hardness sample vol Net titre Ca Hardness .sample vol Net titre Notes: 52 September 2007 CIVL 407 Lab M anual .7 Net (Total) titre pH 8.5 Acidity .
P.U. -mg/L CaCO 3 Apparent Colour (A. Calculation 53 September 2007 CIVL 407 Lab M anual ** By .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.A.P.) (Filtered) Turbidity (N.H.H.T.) Note:* Total Alkalinity must include Phenolphthalein Alkalinity just as Total Acidity must include “MO” Acidity.Coagulation and Softening Summary Sheet Data Lime/NaOH mg/L Soda Ash mg/L Alum mg/L pH P.A.) True Colour (A. -mg/L CaCO 3 **Non-Carb Hard -mg/L CaCO 3 **Excess Alk.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Solids Removal in Wastewater Treatment . . pH . Known Addition Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 20. . . . . . . . . . . . Colour. . 57 3. . . . . . . . 74 13. . . . . COD . . . . . . . . . . 107 26. . . . . . . . . . . . . . . . . . . . . 71 10. . . . . . . . . . . . . . . . . . Alkalinity. . . . . . . Faecal Coliforms . . . . . . . . . . . . . . . . . . . . Hach Absorption Method . . . . . Instructions for Laboratory Report Writing . . . . . . . . . . . . . . Concepts and Definitions . . . . . . . . . . . . . . . . . . . . . . . 112 28. . . . 55 2. . . . . . . 118 54 September 2007 CIVL 407 Lab M anual . . . Bar Diagram Method for Water Softening Problems . . . . . . . . . . . . . . . . . . . . 70 9. . . . . . . . . . . . . . . . . 117 29. . . . . . . . . . 92 19. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical Requirements for Water Softening . . . . . . Introduction . . . . . . . . . . . . . Alkalinity Species as a Function of pH . . . . . . . . . . . . . . 91 18. . . . . . . . . . . . . . . . . . . . . . . . . . . Balancing an Oxidation Reduction Equation . . . Hardness Complexes . . . . . . . . . . . . . . . . . Bacteriology and Water-related diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Additional Notes . . . . . . . 69 8. . . . . 82 15. . . . . . . . . . . Normal Solutions and Equivalent Weights . . .Levels and Water Use . . . . . . . 96 21. . . . . . . . . . . . . . . . . . . . Titrations . . . . . . . . . . 104 24. . . . 100 22. . . . . . . . . . . . . 102 23. . . . . Turbidity. . . . . . 72 11. . . . . . . 84 17. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acidity. . 105 25. . . . . . . . . . . . . . . . Extra tables for Chlorine Lab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 27. . . . . . . . . . . . . . . . . . . . Residues . . . . . . . . . . . . Chlorine . Chloride Analytical Methods . . . . . . . . . . . . . . . . . . 67 6. . . . . . . . . . . . . . . . . . .4 Appendices see: CIVL407Manual_CourseNotes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Milliequivalent and mg/l as CaCO 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bacteriological Examination . . . . . . . . . . . . 83 16. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Typical Problems for Acid-Base and General Chemistry . . . . . . . . . . . . . . 80 14. . . . . . . . . . . . . .COD . . 68 7. . . . . . . . . . . . . . . . . . Equilibrium Relationships of Chlorine . . . . . . . . . . . . . . . . . 73 12. . . DO. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reading and Reference Material . . . . . Standard Solutions. 60 4. . . . . . . . . . . . . 64 5. . . . . . . . . . . . . . . . . . . . . . . . . . . . BOD. . . . . . . . . . . . . . . . . . Water Softening . . . . . . . . . . . . . . . Hardness. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Chlorine Demand . . . .pdf 1. . Periodic table . . . . . . . Coagulation . . . . . . . . . . . . . .
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