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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
5. with contaminated gloves will contaminate those items. Wearing lab coats out of the lab is forbidden.clean and dry. Spills: Wipe up all spills immediately. These should be used whenever standard solutions are measured or when upmost accuracy is required. but if you do please ask for assistance. 25. with the tip against the side of the receiving vessel. Sanitation: antibiotic soap for hand washing. etc.up to 10. please get attendant help. Please ask for assistance if chemicals are spilled. gloves as required. Clean-up: You must leave your work station as you found it . Some pipettes may be “to 3 September 2007 CIVL 407 Lab M anual . If you cut yourself. Pipettes/ graduated cylinders/ beakers/ flasks: 5) 6) 7) 8) 9) 10) Volumetric pipettes are the most accurate way to measure volume. eye protection. Personal Protective Equipment: Lab coat (long).0.. 2. Be aware that handling your pens. Broken Glass: Be careful not to break glass.0. Remember to empty and rinse burets as well. disinfectant for bench surfaces. Most pipettes are calibrated “to deliver” or TD and should never be blown out. Putting pens in your mouth that may have been on the bench or handled with gloves could be hazardous. You must be informed of the hazards of a substance before using it. wash down and dry the bench. no matter how minor. if there is room. Please take your labcoat away in a plastic bag. Pipettes must be placed with tips pointing up into the pipet basket supplied.0 ml. 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. Hygiene: At times you will be working with sewage. A small amount of liquid always remains in the tip and must not be blown out. They are calibrated by weighing the volume of distilled water that will flow from them by gravity. They are available in 0. calculators.. 20. packs. 15. 50 and 100 ml. No sandals or open-toe or heel shoes. Safety Equipment: Eye Wash Station. Safety Shower. WHMIS: Workplace Hazardous Materials Information System is a government regulation.2 First Session: Laboratory Safety and Orientation 1) 2) 3) 4) NO Food or drink. MSDS: is a supplier information sheet that must be available for each product and describe the hazards and necessary handling requisites. DO NOT PUT BROKEN GLASS INTO THE REGULAR GARBAGE CONTAINER. Do not put lids and small items onto racks that may slip through to the filthy drip tray. 1.
2 & 4 L Beakers: are cylindrical in shape with straight sides and flat bottoms. NEVER mouth pipette. 50 mls. Used to hold/transfer approximate volumes of samples. Cylinders are available: 5 mls. some are not meant to drain completely . Pipette bulbs or pumps must be used and great care must be taken not to contaminate bulbs. Flasks: Vo lu m e tric flasks are very accurate and should be used when diluting standards (together with volumetric pipettes). 1. They are useful for measuring samples 20 mls . not wastefully as quantities are limited. 500 mls. Chemicals will be put out as required for each lab and should be used sparingly. 10 mls. A demonstration will be given. 11) Gas/air/vacuum taps: Do not touch these unless instructed to do so. Even the force of air from the air taps can be dangerous because it can contain grit or water. Make sure that you label beakers and flasks to avoid confusion and waste. If liquid should enter a bulb or pump. markings are not accurate at all . do not use a 1 L graduated cylinder. 4 September 2007 CIVL 407 Lab M anual . They can be unpredictable in force and are close to eye level. 250 mls.1000 mls.pay attention to the markings. 14) Glassware/supplies: Each station has a drawer where some glassware and other useful tools will be kept. Undetected gas leaks can be deadly.merely approximations. Erle n m e y e r or conical flask markings are not accurate. chemicals or for titrations. Ie If you want to measure 20 mls. Chemical residues can be splashed in you face. LISTEN AND MAKE NOTE OF VERBAL AND CHALK BOARD INSTRUCTIONS. They are containers best suited for “swirling” liquid ie to mix reagents with samples in a colourimetric determination (Lab 4 or 6).. Graduated cylinders: Not as accurate as pipettes. Some are blow-out. please rinse out immediately and set aside to dry. 25 mls. 100 mls. They are usually identified by a double etched or coloured band at the top of the pipette. Additional supplies are available in the supply room (around the corner). 13) Stir bars: Please remove from containers before pouring contents down the drain. 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. YOU WILL USUALLY NEED THE INFORMATION GIVEN TO COMPLETE YOUR LAB WRITE-UPS. especially at smaller volumes.deliver with Blow-Out”. Use the size of cylinder CLOSEST TO THE VOLUME YOU WISH to measure for best accuracy. 12) Cup sinks: Please do not use these sinks for the disposal or delivery of liquids. They are calibrated the same way as TD except that the remaining drop is blown into the receiving vessel. Pipettes are in drawers indicated.
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. The mark allocation for some sections for both reporting format is indicated in parenthesis in the table below. Total Mark L ONG R EPORT L AB 7 Required Required Required Include a brief description of the procedure. Required  Required  Required  Required  Required  Required  Required   5 September 2007 CIVL 407 Lab M anual . State that the procedures were in accordance with CIVL 407 Lab Manual and note any deviations to the manual’s procedure. 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. 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.L ABORATORY R EPORT W RITE U P G UIDELINES Guidelines for laboratory report write up are also given in the CIVL407 Manual Course Notes and summarized here. The short report is intended to be a concise yet complete laboratory report whereas the long report is more elaborate. Sufficient detail should be provided so that what was done could be understood and the experiment could be repeated from the procedure reported.
Do not include any surface floating material as settled solids.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. MATERIALS: -Imhoff cones. oven muffle furnace. subtract an estimated volume from the measured volume of matter. settle for 15 minutes longer. A 1 L graduated cylinder may be used in place of the Imhoff cone for the sludge sample. and record the volume of settleable solids. if necessary.. If large pockets of liquid form between the particles of settled matter. graduated cylinders. Use pipette bulbs. OBJECT: -to become familiar with a number of the most common sewage treatment plant tests and to illustrate some of the difficulties in performing these tests. Settle for 45 minutes. gently dislodge any solids that have clung to the sides using a stirring rod. balance. to measure sewage treatment plant efficiency in removing residue. keep hands and pencils away from your mouth and wash hands frequently. final effluent. Mix the samples thoroughly and fill individually marked Imhoff cones to the 1 litre mark. wipe up all spills and wash with disinfectant. 6 September 2007 CIVL 407 Lab M anual . Alternatively a place will be provided in the lab to store your coat. samples of raw sewage. SETTLEABLE SOLIDS 1. 2. sludge. desiccators. evaporating dishes. Do not wear lab coats outside of lab and do not take away from lab unless stored in a plastic bag. tin dishes. distilled water bottles. A.
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. This is a different test to the one we are doing using Imhoff cones and should not be confused. 3. 3. Obtain the gross weight of the dish [Note: it should be room temperature before weighing.not beakers. Record the total volume filtered. The loss in weight represents the volatile solids lost from the originally measured sample volume. immediately after stirring the beaker. Suggested portions: 100-200 ml of final effluent. TOTAL SOLIDS and TOTAL VOLATILE AND FIXED SOLIDS AT 550o C 1. [Hint: pour out small portions (say 25 mls at a time for Effl. on a cart in preparation for staff to do so when all are ready. the dish will be transferred to a desiccator. Pinch sides of dish in a bit to protect the filter from oven drafts. the solids remaining represent the fixed solids. Mix the sample in the carboy thoroughly. After drying overnight. Calculate the mg/L volatile and fixed solids. Make sure that you have recorded all the pertinent details: Dish #. Obtain the tare weights of three aluminum dishes each containing a glass fibre filter (already pre-washed. sample source and sample volume. 5. 10 mls for Raw & 5 mls for sludge) of well-mixed sample and filter entire portion before pouring another portion. The dish containing the dried total solids can now be placed in a muffle furnace or. Put the dish in the 103-105 °C oven for drying. 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 . stir the beaker contents and then rapidly (so that it doesn't settle) measure a volume of sample into the appropriate graduated cylinder. Using the sample poured out for Part B (for consistency). dried and fired).[Note: Sludge Volume Index is the volume occupied by 1 gram of sludge after 30 minutes of settling. if instructed. when filtering slows significantly do not add more.] Using very small amounts of distilled water. Obtain the tare weight of a properly prepared porcelain evaporating dish (ie one that has been washed. pre-fired and desiccated . 6. SVI= settled sludge volume (ml/L) x 1000/suspended solids (mg/L)] B. Subtract the tare weight to obtain the total solids weight and calculate the mg/L value. pour about 500 mls (for Part B & Part C) into a beaker and then. Wet filter with a small volume of distilled water to seat it.done for you. prior to session). Once dishes are at room temperature they can be re-weighed. 2. Quickly record the exact volume and pour the sample into the dish. 4.] Rinse the graduated cylinder with small amounts of distilled water and add to filter. position the filter and begin suction (vacuum tap). Do not write on crucibles as high temperatures during the next steps will burn writing off.TOTAL AND VOLATILE 1. adding the rinsings to the dish. 2. C. SUSPENDED SOLIDS . The samples will be fired at 550° C for one hour. dried. Conversely. Carefully remove filter from filtration apparatus and transfer it back to the aluminum dish. The furnace will then be allowed to cool significantly before dishes are removed to a desiccator. [Notes: to allow for rinsing and to avoid spillage when transferring dish to oven don't pour more than 40 mls]. .[Note: it may be harder to wash out solids if you allow them to settle before pouring into the dish . tare weight.so be quick.]. rapidly pour sample into a 50 ml graduated cylinder to approximately equal 35-40 mls (if using 50 ml dishes). Assemble filtering apparatus.
Transfer dish to a desiccator. what is the percentage reduction in solids. If sludge is used in Part A. overnight). For the time being. if any. where the volatile solids are reduced from 65% to 40%. Lab personnel will perform the timed-firing after the class. 6.) a) a bacterium b) sodium chloride c) sugar d) clay 8 September 2007 CIVL 407 Lab M anual . fixed (use more than one adjective to describe each substance. What two techniques can be used to overcome or correct for the loss of material from filter pads when firing at 550 oC? 4. Classify each of the following into its proper solids category(s) i.e. What. After firing the dishes will be moved to a desiccator. cool and weigh. DO NOT DISCARD! The gain in weight represents the total suspended solids of the sample. What major types of solids are removed in primary treatment? in secondary treatment? 2. 7. 3. place the dishes on a cart designated by the instructor. suspended. Calculate the mg/L total volatile solids and total fixed suspended solids. 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? 5.3 and fixed solids 2. Your hands may have moisture and natural and/or applied oil/lotion on them.50.5. 6. Please return tomorrow to obtain final weight. A raw sewage sludge goes through an anaerobic digestion process. % reduction in settleable matter _________ % reduction in total solids _________ % reduction in volatile solids _________ % reduction in fixed solids _________ % reduction in suspended solids _________ % reduction volatile susp. 8. Discuss the meaning and significance of this index. Calculate the percent solids reduction through the treatment plant. Enter all data in the tables on following page. If all of the volatile solids reduced is given off as gas and if the specific gravity of the volatile solids is 1. 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). They must be allowed to cool completely before re-weighing them to determine the loss on ignition or volatile suspended solids. From the above determinations it will now be possible to obtain a value for the dissolved solids present in the sample. volatile. solids _________ % reduction fixed suspended solids _________ % reduction dissolved solids _________ Lab 1 Questions 1. dissolved. compute the sludge volume index (SVI). Calculate as the mg/L total suspended solids.
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 . Settleable Matter After 1 hour (mL/L) B.use embossed mark) Sample volume (ml) Gross weight . Total Solids Dish marking (do not use ink .use permanent mark on dish) Sample volume (mL) Gross weight .DATA AND RESULTS Raw Sewage Aerobic Sludge A.fired (G) Loss on ignition (G) Volatile solids (mg/L) Fixed solids (mg/L) Percentage fixed residue C.oven dry (G) Dish tare weight (G) Total solids (G) Total solids (mg/L) Gross weight . Suspended Solids Tin dish marking (do not use ink .oven dry (G) Tin dish with filter tare weight (G) Total suspended solids (G) Total suspended solids (mg/L) Gross weight .
Hach block digester. 200. Keep fingers. otherwise reagents will run to the bulb end and make pipetting difficult and contamination likely. spectrophotometer. pencils. Wipe up all spills immediately. NEVER pour water into acid.La b o ra to ry 2: CO D . 400. 100 and 50 mg/L as COD. OBJECT: -to familiarize you with the commonest tests run for assessing treatment plant efficiency and pollution loading to receiving waters. etc out of your mouth. etc. The closed reflux method is more economical but because a small sample volume (2 mls) is used.Closed Reflux. vials with pre-measured reagents. and to demonstrate the use of dissolved oxygen probes. as well as handling samples probably containing pathogenic bacteria. Colourimetric Method NOTE: Open reflux method is the most accurate means of determining COD because a large sample size is used (20 mls). I. Materials: Raw sewage and final effluent samples. homogenization of samples containing suspended solids may be necessary in order to get a representative sample and to improve reproducibility. potassium hydrogen phthalate standards: 800. D O a n d B O D 5 WARNING You will be using concentrated acid and other corrosive and toxic chemicals. to illustrate the shortcomings of these tests. pipettes. Always use and store pipettes with the top higher than the tip. Wash hands frequently with soap. 10 September 2007 CIVL 407 Lab M anual . wash/rinse bench tops with water/disinfectant. Digests from either method can be titrated or read colourimetrically. COD .
DISSOLVED OXYGEN (DO) Materials: Raw sewage and final effluent samples. [Note: it should look homogenous and be careful it will be very hot!] 3. dilution water. except without mercuric sulfate). starch indicator. 100.Procedure: 1. 500-ml Erlenmeyer flasks. 0. Prepare calibration curve. thermometers. 6. 2. The probe has been previously calibrated. in which case. 5. BOD bottles. 50 and 0 COD as mg O 2 /L. 10-ml graduated pipettes.025 N sodium thiosulphate. 250-ml graduated cylinder. raw sewage and final effluent. concentrated sulphuric acid. use wide mouth graduated pipettes) in duplicate. The vials should be clean and dry on the outside. The instructor will demonstrate the use of the spectrophotometer. before the lab. 200. If less than two mls of sample or diluted sample was used. aerated dilution water. 4. At your station you will find four vials with pre-measured solutions containing potassium dichromate and sulfuric acid (prepared according to standard methods. Put identifying labels on each tube ie your initials. 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 azide modification of the iodometric method) and a dissolved oxygen probe and meter. well-mixed samples in the 25-position digester.noting the location of your well-labelled. which is set at 600 nm to read absorbance of light by the sample. Remove the vials and allow them to cool to room temperature (use cold water to speed up if necessary). DO NOT ADJUST THE SETTINGS ON THE METER. Pipette 2 mls of sample (using volumetric pipettes unless particulate is present. alkaline-iodide-azide reagent. Raw Sewage (Infl) and/or Effluent (Effl) in the upper 1/4 of the tube using permanent markers. manganous sulfate. and are at the spectrophotometer. Take vials to fumehood and place in block digester . a smaller aliquot (making volume up to 2 mls with distilled water) or 2 mls of a pre-diluted sample can be used. 11 September 2007 CIVL 407 Lab M anual . burette stand with burettes. You will use a chemical titration (Winkler . a volumetric correction must be made to the value obtained from the curve. Read your samples and the set of COD standards: 800. Standards have been digested for you. into the appropriate tubes and screw cap on snugly. If sample is known to be outside the standard range (above 800 mg/L). 400. Allow samples to digest for 30 minutes (normally this would be 2 hours). II. absorbance versus concentration and calculate the COD in samples. The four liquids are cold tap water.
When the floc has settled the second time to about 1/3 bottle volume. Note: if solution already seems pale yellow add starch right away. (Fill carefully. (Hint: if you measured very low DO level with the meter. The DO concentration in mg/L is equal to the number of ml of titre as long as 201 ml is titrated. remove the stopper.think about it!) Complete all four titrations. as demonstrated. using the DO probe and meter. Fill four BOD bottles with the four samples (cold tap water. 12 September 2007 CIVL 407 Lab M anual 3. Tip out the liquid from the area around the stopper and invert several times to mix thoroughly. tip the liquid out of the space around the stopper (caution -it may be corrosive) and invert several times. Titrate this volume with 0. Stopper the bottles without trapping any air bubbles. raw sewage or influent and final effluent). Add enough starch solution to give a strong blue colour (one dropper full) and continue to titrate. to thoroughly mix contents. 6. 1. Place the bottles in the sink and one at a time remove the stopper. Determine the percent saturation for each sample. DISSOLVED OXYGEN PROBE NOTE: DO NOT add any reagents to the samples used for the probe method. 9. B. . 2.) Transfer 201 ml of each bottle (do one at a time) to a 500 ml conical flask. (Note: DO meter temperature is usually not accurate.025 N Na 2 S2 O 3 until only a faint yellow or "straw" colour remains. then the same sample will require little or no titre and may not turn blue when starch is added . add 1 ml of alkaline-iodide-azide solution the same way. 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. Do not mix droppers and do not allow tap water into the reagent bottles. 8. for each of the four samples. Determine DO. Neglect any reappearance of the blue colour. submerge the hose in the bottle and allow the water to overflow for 15-30 seconds). 2. aerated dilution water.) You can save these bottles for B. Instructions on the use of the probe will be given in the lab. add 1 ml of concentrated H 2 SO 4 and quickly restopper. right to the top and place stopper on bottle. Allow the floc to settle to about 1/3 the bottle volume.A. 7. To the same bottles. again quickly replacing the lid. Record the temperature of each sample using the DO meter or a thermometer. invert several times again and allow to settle again. Take each stoppered bottle. 5. dropwise. (Note: biological solids originally present in the sample will not dissolve. Set back in sink. taking care not to entrain additional air. For the cold tap water sample. 1. Fill a BOD bottle with each of the four samples (or use ones saved from A ). Use the specially marked volumetric flask to dispense the 201 ml. until the first complete disappearance of the blue colour. Record the volume of titre. The chemical floc should completely dissolve. 4. WINKLER TITRATION (Azide modification) NOTE: Use only the droppers attached to the reagent bottles for dispensing into samples.
use the saturation chart above. (mg/L) DO conc. The percent saturation is the value where the line intercepts the saturation scale. Draw a straight line between the water temperature and the mg/l of dissolved oxygen. 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. Pair up the mg/l of dissolved oxygen you measured and the temperature of the water in degrees C. (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.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 .
4. BIOCHEMICAL OXYGEN DEMAND Note: Because of the logistics of organization some groups may have 6 day BOD 5 instead of the usual 5 day. preferably using the same meter and probe used to get IDO reading. Using the volumes and samples provided by the instructor. to bring the levels above the ground glass neck of the bottle. If the dilution water BOD is greater than 1 ppm.if not add a some dilution water (or distilled water) to the well. Carefully top up the BOD bottles with DILUTION WATER. There should be negligible BOD in the Dilution water. BOD bottles. 14 September 2007 CIVL 407 Lab M anual . thereby maintaining a water seal for each bottle. Calculate the 5-day (or 6-day if unavoidable) BOD. graduated cylinders. 10-ml graduated pipettes. 1. Place plastic caps over all bottles. remove bottles and measure the DO. Measure the Initial DO of the blank using the calibrated probe and meter. groups). it may indicate that probe may not have been calibrated properly or the dilution water had been contaminated. 6. Because you want to be sure that the dilution water has no BOD.do not write on the bottles and do not use the numbers on the lids. These caps are designed to prevent the evaporation of the liquid around the stopper. 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. In addition. 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). Be sure to record the bottle numbers and sample volumes on the following table . ie Rinse the filling tube if it has been in the sample. The blank is used to check that the dilution water has negligible BOD. being sure to exclude any air bubbles that might be trapped under the stopper by adding dilution water. carefully measure the waste water samples into BOD bottles. Dissolved oxygen meter and probe. Note: Each group should have 9 BOD bottles in the incubator. be careful not to contaminate it with sample. fill one BOD bottle with DILUTION WATER alone. 3. Stopper the BOD bottles. Measure the Initial DO using a calibrated probe and meter. Materials: Raw sewage and final effluent samples. All bottles should have liquid in the well around the stopper .III. Place all bottle sets in the 20 o C incubator for five days (or 6 for Tues. After five (or six)days. but if there is a significant BOD then it will have to be subtracted before the dilution factor is applied in the calculation. Be sure to note this in your lab write-up. dilution water. showing sample calculations. 5.
mg/L = DO of diluted sample after 5 d incubation at 20 oC.BIOCHEMICAL OXYGEN DEMAND (BOD) DATA Sample source: ________________________________ Raw Sewage Bottle number (not lid #) Sample volume (ml) Initial DO (mg/L) Final DO (mg/L) Final Effluent Bottle number (not lid #) Sample volume (ml) Initial DO (mg/L) Final DO (mg/L) Dilution water Bottle number (not lid #) Sample volume (ml) Initial DO (mg/L) Final DO (mg/L) _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ BOD Calculation When dilution water is not seeded (our water is not seeded for this exercise): When seeded water is used: where: D 1 = D2 P B1 B2 f mg/L. mg/L DO of seed control after incubation. = = = = decimal volumetric fraction of sample used DO of seed control before incubation. DO of diluted sample immediately after preparation. mg/L. 15 September 2007 CIVL 407 Lab M anual . and ratio of seed water in diluted sample to seed in seed control= (%seed in diluted sample) / (%seed in seed control).
25.17. 1 Raw Sewage. 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. Both wastes have equal BOD 5 temperature and volume. Calculate theoretical oxygen demand for 300 mg/L of methyl alcohol. 1 Final Effluent. open reflux. One waste has a K value of 0. 2 Final Effluent. Diln. Diln. 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.) Why is your COD result different from the BOD result? 5. titrimetric method. If an industrial waste has a COD of 450 mg/L. the other 0. 6) 7) 8) 16 September 2007 CIVL 407 Lab M anual . Diln. 2) 3) 4) Show on a graph the approximate dissolved oxygen sag curves in a river receiving two wastes. what would you expect its I) BOD 5 and ii) BOD L to be. if at all possible. Diln.Summary Raw Sewage.
Do not wear lab coat out of lab . agar tubes. Wipe lab benches with disinfectant before and after each lab. Heterotrophic plate count: dilution tubes. etc. EC tubes and/or Hach A1 tubes. DO NOT MOUTH PIPETTE. sterile pipets. 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 . Keep hands.. sterile dilution water. 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 . filtering apparatus. OBJECT: -to demonstrate different methods for the determination of the coliform group of organisms and to acquaint you with bacteriological procedures. HACH P/A Broth with MUG. Get raw data from another group and include in your report. wipe up all spills and disinfect the area of the spill. Demonstration only SAMPLE: You will do either a raw sewage sample or a final effluent sample.leave it here or take away in a plastic bag to launder. Quebec colony counter. pencils. 35oC incubator. Demonstration only.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 . sterile pipettes. membrane filters. T h e re f o re . Membrane filter method: dilution tubes. eye protection. away from your mouth. 35 oC incubator. plates. not both. lauryl tryptose broth tubes.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. lab coats. Wash hands with soap before leaving. Brilliant Green Lactose Broth tubes. LES Endo agar plates. Students will do only sections marked ? MATERIALS: Multiple tube method: dilution tubes.
Note: these methods will be demonstrated but data must be recorded. Streak plates in a manner to insure presence of some discrete colonies. Gas-positive Hach A-1 tubes indicate the presence of faecal coliform. A-1 Medium. Demonstration. Express as Total coliforms. Parallel positive BGLB broth cultures with negative EC broth cultures indicate the presence of nonfaecal coliforms. (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. The number of colonies counted directly. 18 September 2007 CIVL 407 Lab M anual . 2. streak one LES Endo agar plate from each tube of BGLB broth showing gas from the highest dilution and the second highest dilution. Consider positive EC broths incubated at the elevated temperature (44. Another series of tubes are inoculated from these positive lauryl tryptose tubes. Hach’s Most Probable Number Method 8368. Microscope Slides (Completed Phase of Multiple-Tube Fermentation): Single colonies arising from single cells are prepared and stained for microscope examination. All tubes have been examined for gas production. Gas formation indicates the possible presence of coliform organisms. Use Table 2 on page 27. It is used as a faster alternative to the LTB/EC procedure for enumerating faecal coliforms in water. E. Results will be provided for the demonstration set. Formation of gas in any amount in the inverted vial of the BGLB broth fermentation tube at any time within 48 hr ±3 hr constitutes a positive confirmed phase. Calculate the MPN value from the number of positives using the Table 1. 1. as demonstrated. ? B.coli.coli. Heterotrophic Plate Count .A. 3. 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. using a ready-to-use P/A Broth with MUG. ? C.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. EC or A-1 tubes positives: 1. D.) Methods: ? Recording BGLB. 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). Presence/Absence Testing: HACH technique to simultaneously screen for total coliform and E. Colonies that are pink to dark red with a golden green metallic sheen are counted after incubation 20-22 hrs at 35oC. 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. The broth will turn yellow indicating total coliform and fluoresce in the presence of E. Membrane Filter Technique: Serial dilutions of sample are collected on membrane filters and placed on M-Endo media.7oC for 24 hours (confirmed test for faecal coliforms). Using aseptic technique.COLI in 4 to 24 hours.5 oC) as a positive completed test response for both Total and faecal coliform. is USEPA-accepted for testing non-potable waters. MUG produces a fluorogenic product when hydrolyzed by an enzyme specific to E.
Use dishes with 20 to 80 coliform colonies and not more than 200 of all types. from one edge. 19 September 2007 CIVL 407 Lab M anual . In general. Mount apparatus on suction flask with valve sideways to block suction. with a motion that excludes air as it comes in contact with the agar. Inoculating. The filtration apparatus has been sterilized by autoclaving.. ?B. 4. 5.1. -Note: “typical” colonies count as coliforms in this procedure. prepare one set of six dilution tubes following the instructions given under Multiple Tube Fermentation. 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. removing the cover just enough to insert pipette tip and dispense sample. Using a sterile pipette. The typical coliform colony has a pink to dark-red colour with a golden green metallic surface sheen. transfer 1 ml of sample from dilution tube # 6 (most dilute for Raw sewage) or #5 (for Effluent) to an empty (large) Petrie dish. Flame loop between second and third quadrants to improve colony isolation. Do not push down on the filter except on the outside edge and use only sterile forceps .applying gentle pressure.Pour Plate Method 1. If you haven’t already. 2. Repeat for the rest of the dilution tubes to # 2. Prepare one set of six dilution tubes as described in Section A. Place the filter on a small LES Endo agar plate carefully. Dilution tubes. Verification is usually done by a test for lactose fermentation. Pipette about 10 ml of sterile dilution water over the surface of the filter with the suction valve still closed. open the valve slowly and allow all the liquid to be pulled through. then immediately replace the cover. Colonies that lack sheen may be pink. Membrane Filter Technique -Total Coliform 1. Filtering. or colourless and are considered to be non-coliforms. A high count of non-coliform colonies may interfere with the maximum development of coliforms. place a sterile membrane filter (grid side up) onto the filtration apparatus. Atypical coliform colonies can be dark red or nucleated without sheen. The total count of colonies (coliform and noncoliform) on Endo-type medium has no consistent relationship to the total number of bacteria present in the original sample. Heterotrophic Plate Count . Counting. coliform bacteria are Gram-negative (pink) rods (sometimes coccus-like). After 20-22 hr. Incubate plates (inverted) at 35 oC for 24 ± 2 hr. 2. Gram staining technique can be also be used. white.2. When filtering is complete and the valve is closed. Be sure dishes are labelled with your name and dilutions. therefore the results table should specify #coliform colonies/100 ml. Prepare Gram Stain slides for examination under a microscope. Dilution tubes. red. choose appropriate dilutions for counting. Using sterile forceps. 3. ?C. remove filter using flamesterilized forceps (allow forceps to cool or they will damage the filter). Close valve and rinse the funnel with three aliquots (portions) of sterile dilution water opening and closing valve after each aliquot. Verification of both types of colonies is advisable as some typical sheen colonies may be noncoliform and some atypical may be coliform. Incubate inverted Petrie dishes for 20-22 hr at 35 oC. Pour the full volume of dilution tube # 6 onto the filter.
too hot for a baby . Flood slide with crystal violet solution and allow to act for 30 seconds.coli). 5. f. E. c. Wash slide in tap water. Slide the cover of a Petrie dish back just far enough to pour the agar. Resterilise the loop and remove one colony from the surface of the Les Endo streaked plate (prepared previously). Add 100 mls of sample directly to pre-measured medium provided. Counting. Drain off excess iodine and wash freely with water and then alcohol-acetone decolourising solution. 4. pour and replace cover. Presence/Absence Testing . Incubate at 35 oC for 24-48 hours. Test temperature on your wrist . Label the slide using a grease pencil (T or A). 20 September 2007 CIVL 407 Lab M anual . Record results of previously prepared test. Examine for yellow colour (total coliforms) and fluorescence (E. to mix the sample with the agar. Do not report as “too numerous to count” if all plates exceed 300 colonies. marking (on edge of dish so as not to impede counting) and incubating for 48 hr at 35 oC. ?3. Allow iodine to act for 30 seconds. Let them cool down from the 60C that they are kept at but not enough to solidify. Compute bacterial density and report as “colony-forming units” (CFU) per ml. A hand counter is also available.3. Instead. Take 5 tubes of melted agar from the incubator. Multiply the number by 5 to compute estimated colonies per plate (66 cm 2 ). count four representative squares. Use the Quebec colony counter to aid in counting and a grease pencil to mark off colonies counted. average and multiply by 57 (for plastic Petrie dish).d e m o n s tra tio n -s ta rte d a h e a d o f la b 1. 2. 2. Work quickly or the agar will solidify in the tube before you get a chance to pour it. place 1 loopful of sterile dilution water in the centre of a slide. Allow the agar to solidify in the dish before inverting. Preferably select seven consecutive squares horizontally and six consecutive squares vertically. select the appropriate dilution to count. Microscope Slides: 1. Air dry the slide high above the Bunsen burner and fix by passing slide briefly through a flame. 6. If more than 10/cm 2. Wash off stain with water and then with iodine solution (Lugol’s).too hot for a bacterium. if fewer than 10/cm 2. b. a. Slide preparation: With a flame sterilized inoculating loop. Ideally. Flood the slide with fresh alcohol-acetone solution to act for 30 seconds. ?4. use the plate(s) that will give from 30 to 300 colonies/plate. The aim of using several dilutions is to have at least one dilution giving colony counts between these limits. Spread the colony in the drop of water so that you get a uniform dispersion over an area about the size of a dime. Do this for a typical and an atypical colony. Gently swirl the dish. After 48 hr incubation. d. count colonies in 13 squares (of the colony counter) of the most dilute plate having representative colony distribution. Repeat for the rest of the dilutions. D. If necessary. e. on the bench. refer to Standard Methods for more instruction. Repeat with dilutions 5 to 2 (for Raw) and 4-1 (for Effluent). Slide staining.
3 µm in size. BGLB and EC and AI tubes . .Come in and count Heterotrophic Plate colonies after 48 hours. gram positive or negative.Membrane filter prepared dilutions.Come in and count colonies on LES Endo Membrane Filter dishes. Microscopic examination.Record positives in all Multiple Tube Fermentation inoculations: LTB. after decolourisation Gram negative organisms are no longer visible. . . b.Examine prepared slides. in pairs. Description of bacteria: Describe what you see on the plates: colonial morphology of each type of colony seen ie surface (shiny. colour. . Gram negative organisms are visualized (pink). length to width ratio. plump rods. 21 September 2007 CIVL 407 Lab M anual . h. cocci or rods. odour. short. and on the slide: typical or atypical.g. dull. rough. . c. Note: in the staining process: before decolourisation all organisms appear Gram positive (blue).Record Presence/absence testing results of Hach Prepared Media bottles. Cells occur singly. After application of the counterstain. Day 3.do not turn knobs except the positioning ones. metallic. place on LES Endo agar dishes and incubate. .counts will be provided on the board. concave).Prepare dilutions for Heterotrophic Plate Count and Membrane Filter technique. ?3. coliform are Gram-negative (pink). cell groupings (single. or in short chains.Prepare and incubate Heterotrophic (Pour) Plates using dilutions above. transparency. Schedule Summary Day 1. sometimes coccus-like and 0. Plates ready on a weekend will be put in cold room until Monday. Examine the prepared slides . nonsporing. . Wash in water. smooth. Apply fuchsin or safranin (counter)stain for 30 seconds. . a. pairs chains). usually motile. blot and then dry in air high above the Bunsen burner (so as not to burn away your efforts).5 . In general. Day 2.
pour plate method. 1/ 10 and 1/100 are used. Multiply the MPN index by the dilution factor from the series used when dilutions other than 1/1. 35 oC/48 hr Tube # 1 2 3 Mls of inoculum Infl Effl Plate count Infl Effl *CFU/ml Infl Effl *CFU= colony-forming units Membrane Filter Technique Tube # Dilution (mls) Infl Effl Coliform Colonies Infl Effl #Colonies/100 mls Infl Effl 1 2 3 4 5 6 4 5 6 22 September 2007 CIVL 407 Lab M anual . 1 2 3 4 5 6 Heterotrophic Plate Count .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..
use the results from only three of these in computing the MPN. If zero total coliform is the maximum contamination goal. 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. MUG (4methylumbelliferyl-$-D-glucuronide) can be used to detect E. In the examples given below. They are in concentrated form and merely require the addition of 100 ml of sample (or diluted sample) to the media and incubation. Estimation of Bacterial Density . 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.Most Probably Number (MPN) When more than three dilutions are used in a decimal series of dilutions. With MUG reagent added to P/A Broth.01 ml 2/5 2/5 0/5 0. that in the denominator. The number in the numerator represents positive tubes. an enzyme specific to E. the significant dilution results are in boldface. you can simultaneously detect total coliforms and E. the total tubes planted. A long-wave UV light is required to detect fluorescence.coli. E. MF results. To select the three dilutions to be used in determining the MPN index. Use the results at these three volumes in computing the MPN index.coli.coli will fluoresce as early as 4-24 hours. Do they agree? Why/why not? -compare MPN/MF with Heterotrophic Plate Count.1 ml 5/5 4/5 1/5 0.Discussion: -sources of error? -compare MPN. 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. Both media meet USEPA guidelines for testing of total coliforms in drinking water. 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.
1. Of Positives MPN Index/ 100 ml 22 26 27 33 34 23 30 40 30 50 60 50 70 90 80 110 140 170 130 170 220 280 350 240 300 500 900 1600 95% Confidence Limits Upper Lower <2 2 2 4 2 4 4 6 6 4 7 9 9 12 8 11 11 14 14 17 13 17 17 21 26 1 10 13 11 15 15 18 18 17 20 24 25 29 24 29 29 35 35 40 38 45 46 55 63 4-2-0 4-2-1 4-3-0 4-3-1 4-4-0 5-0-0 5-0-1 5-0-2 5-1-0 5-1-1 5-1-2 5-2-0 5-2-1 5-2-2 5-3-0 5-3-1 5-3-2 5-3-3 5-4-0 5-4-1 5-4-2 5-4-3 5-4-4 5-5-0 5-5-1 5-5-2 5-5-3 5-5-4 5-5-5 9 12 12 15 16 9 10 20 10 20 30 20 30 40 30 40 60 80 50 70 100 120 160 100 100 200 300 600 -— 56 65 67 77 80 86 110 140 120 150 180 170 210 250 250 300 360 410 390 480 580 690 820 940 1300 2000 2900 5300 ---- $1600 24 September 2007 CIVL 407 Lab M anual . 0. 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.1 mL) Comb.0 mL.MPN Index and 95% Confidence Limits for Various Combinations of Positive Results when 5 Tubes are used per Dilution (10 mL.Table 1 .
MPN Table 2 25 September 2007 CIVL 407 Lab M anual .Hach .
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. -The Use of Indicator Organisms to Assess Public Water Safety .microbelibrary. Mara: -Bacterial Indicators/ Health Hazards Associated with Water -Bacteriology for Sanitary Engineers Microbiology 321: -Manual of Microbiological Techniques Website: http://www. What are the important pathogens transmitted by water? How is quality control achieved in microbiological analysis. 2) 3) 26 September 2007 CIVL 407 Lab M anual .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.http://www.org/microbelibrary/files/ccImages/Articleimages/keen/Gram stainkeen.D.hach.
Mercuric thiocyanate is very toxic. If chemicals are spilled alert instructors. standards. conc. known addition and calibration techniques and colourimetric and potentiometric titrations. That means you will probably have to dilute the sample by the most accurate means available. NaHCO 3. pH meter with double-junction electrode and silver billet electrode. magnetic stirrer.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. indicator-acidifier reagent.5 mg/L). Use personal protective equipment: gloves. reference electrode (double junction). to illustrate the differences in operation and accuracy between instrumental methods and wet chemical methods. standard AgNO 3 solution. beakers. standard chloride solution (0. HNO 3. Hint: optimum ranges will coincide with the standards provided or will be indicated within the pertinent section. buffer. chloride specific ion electrode. 27 September 2007 CIVL 407 Lab M anual . semi-log graph paper. burettes. immediately. beakers. OBJECT: -to acquaint you with the use of specific ion electrodes. 125 ml Erlenmeyer flasks. sample. 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. indicator. goggles or glasses and lab coats. graduated cylinders. MATERIALS: -millivolt meter. Note and consider carefully: Different methods of analysis have different optimum ranges. spectrophotometer. standard Hg(NO 3)2. volumetric pipettes.
added to the sample "slope" = change in potential over 10 fold change in concentration of standard (use data from Step 2). 6. where: E1 = E2 = E = Vo = Vs = A = S = potential of sample (mV) potential of sample plus stock addition potential change = E 2 .in sample = mg/L Cl.SPECIFIC ION ELECTRODE Part One: Preparation and reading standards and samples 1. Calculate the chloride concentration. pour out 100 mls of each standard into separate. Add 2 mls of IONIC STRENGTH ADJUSTMENT (ISA) buffer to each using the plastic dropper (filled to the mark). Repeat for rest of standards. added 2 mls of ISA.= ____________________ 28 September 2007 CIVL 407 Lab M anual . 2. Determine the concentration of chloride in the sample. place the lowest standard on the stirrer and lower the electrodes into it. Standards of 1000. 100 and 10 ppm (mg/L) have been prepared for you and are at the meter. 2 minutes) and use it for all standards and samples. mg Cl. Measure 100 mls of sample. As probe tends to drift for some time it may be expedient to select a “standard time to read” (i. Unless already done by a previous group.e.I. Unless already done by a previous group (values written on the board). 4. stir and obtain a reading for it. 3. labelled beakers. Turn on stirrer and while stirring record the millivolt (mv) reading when the reading appears to be stable. Using semi-log paper or software equivalent. 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 ). plot the millivolt readings (linear axis) against the concentration of the standards (log axis) to produce a calibration curve.E 1 volume of sample volume of stock solution added mg Cl. 5. rinsing and blotting dry the electrodes between each. CHLORIDE .
Using a 25 ml graduated cylinder.] After 10 minutes has passed (since the combined reagent was added to the blank). which then must be subtracted from the standards and sample. 2. fill. 4. measure 25 mls of “zero” standard or “blank” (halide-free distilled water). Determine the chloride concentration in the sample from the calibration curve. add 5 ml of the “combined reagent” (containing ferric alum and mercuric thiocyanate solutions) to the blank first. 4. 6. insert in the spectrometer and record the absorbance displayed on the meter. Pour 100 ml of sample (or a portion diluted such that the concentration is less than 100 mg/L) into a 250 ml beaker. a pH of greater than 3.8. (Use extreme caution as this is a corrosive & toxic chemical. 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). For most samples. 3. [Note: this delay between additions is to allow you time to measure absorbance of each at the 10 minute time required .0 and 8.º HgCl2 1. pure blue. Marking the time (T=0).0 ppm).they can’t all be at 10 minutes at once. the excess mercuric ions combine with diphenylcarbazone (DPC) to produce a violet colour (the end-point). (Light green indicates a pH of less than 2.) Prepare a calibration curve by plotting the absorbance readings versus the concentration of chloride in the standards.ions have been bound.ions in the sample combine with added mercuric ions forming a soluble but virtually undissociate-able complex. zero instrument on distilled water and obtain an absorbance value for the blank. and Hg ++ (excess) + DPC º violet colour 3. 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. 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.II.0. CHLORIDE . The colour of the solution should be green-blue at this point. (Alternatively. 5. 2.+ Hg(SCN)2 º HgCl2 + 2SCN SCN .5 ± 0. using the dispenser provided.014N Hg(NO 3) 2 to a definite purple end-point. After all Cl. discard and refill at least twice to rinse cuvette. 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 . Hg ++ + 2Cl. Titrate with 0.1. Add 1 ml (use pipette to measure accurately) of indicator-acidifier reagent to sample. (No further adjustment is required after the blank or distilled water has been set to zero. after a few more minutes. Wait two or three minutes and then add the reagent to the first (lowest) standard.+ Fe+++ º Fe(SCN) ++ (Reddish brown) 1. and the sample (diluted if necessary) into separate and labelled 125 ml Erlenmeyer flasks (5 in total). Near the end-point.0.. If sample is above the highest standard. III. it should be rerun from the start after diluting. use a plastic dropper to transfer blank into a spectrophotometer cuvette (do not fill more than 3/4 ). the three standards (2.) Every two or three minutes thereafter you will rinse the cuvette with the next standard or sample.) Mix thoroughly by gently swirling the flask. MERCURIC NITRATE METHOD FOR CHLORIDE DETERMINATION The principle of this method is as follows: Cl.
where: A = ml titrant for sample B = ml titrant for blank N = normality of titrant IV. recording the volume and millivolt reading for each increment. 2. The titre from this step must be subtracted from the titre for the sample. Part one: Standard 1. smaller increments (0. 4. 5. The end-point occurs at the greatest mV change per unit addition of AgNO 3.. Begin titrating. Take care that stirrer is not hitting the electrodes. 3. dilute to about 100 ml with distilled water. Nitric acid is in the sink and is very corrosive. 6. A reference set of data will be provided for comparison. large increments (1-2 mls) may be added. Plot a differential titration curve to determine the exact end-point. Start the stirrer and set the millivolt reading to zero or record initial mv reading [Note: Some meters can not be set to zero]. 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 . At the start.2 ml or drop-wise) should be added. 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 of concentrated HNO 3 (use plastic dropper). Calculate the chloride concentration: 5.4. Don’t forget to add the indicator. This way each station will have data for each part. Rotate the work within the group so that different pairs are doing the titrating and recording of numbers. as the end-point of the reaction is approached (ª mV/ml increases). Immerse stir bar. place beaker on stir plate and lower electrodes into the solution. with standard AgNO 3. Place 10..1 to 0. until past the endpoint when larger increments can be used again (ª mV/ml decreases). drops of the purple end-point. Calculate the Normality of the AgNO 3 using the following equation: 30 September 2007 CIVL 407 Lab M anual . in increments. then. CHLORIDE BY POTENTIOMETRIC TITRATION Note: For this part form two groups comprised of 1 member from each station.0 ml (use volumetric pipette) of standard chloride solution in a 250 ml beaker. and add 2. Continue the titration about 5 ml past the end-point.if one is necessary.
(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. b. 2. 4. which are merely first and second derivatives. be careful to use the average value of ml of titrant on the abscissa.Part Two: Sample 1. Millivolts per ml per ml versus ml of titrant. Measure 100 ml of sample or portion made up to 100 ml. 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 dilution is necessary into a 250 ml beaker (use a graduated cylinder to measure). It is not. Calculate the chloride concentration in the sample using the following equation: 3. Millivolts per ml versus ml of titrant. c. In plotting b and c. Millivolt reading versus ml of titrant.) Plot three curves for this titration: a. Add 2 ml of HNO 3 and proceed as described in Part One. in this case.
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.Chloride .
In the above tests. thiosulphate. requires knowledge of the compounds in the sample which are likely to interfere. # Environmental samples usually require pretreatment. # Other interferences: ferricyanide. 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. # Mercury must be absent from samples. # Pretreatment of environmental samples usually required. causing electrode malfunction. fluoride. Also. COLOURIMETRIC METHOD # Bromide. can you see any advantage in suing the first or second derivative curve? Discuss. It can only correct for substances that enhance or suppress the test response proportional to the amount of chloride ion present. known addition cannot correct for any ions that test as chloride ion. 33 September 2007 CIVL 407 Lab M anual . timeconsuming process requiring a sound knowledge of chemistry and extensive experience working with environmental samples. in itself. iodide. 3. chromate. cyanide. PRETREATMENT TECHNIQUES # There are no "standard" pretreatment techniques. dichromate. # Environmental samples usually require complex pre-treatment. specific ion electrodes are rarely usable in environmental samples. This is a complex. # Chromate. ferric ion.INTERFERENCES IN CHLORIDE DETERMINATIONS POTENTIOMETRIC METHOD # Iodide. and nitrite interfere. # Colour in the sample may interfere in the absorbance measurement. SPECIFIC ION ELECTRODE # All halides interfere (the electrode is a halide electrode). Each sample type requires development of an appropriate method which. Lab 3 Questions 1. # Sample pH may need adjustment beyond the capacity of indicator-acidifier reagent. # Colour may obscure or interfere with end-point determination. # High levels of ions which form very insoluble salts of silver may deposit a layer of salt on the electrode membrane. fluoride and bromide titrate as chloride. # In practice. MERCURIC NITRATE TITRATION METHOD # Iodide. hydrazine. fluoride and bromide titrate as chloride. 2.3 mg/L? Outline the advantages and disadvantages of all the methods used in this lab to measure chloride concentration. and sulfite ions interfere when concentration greater than 10 mg/L. ferric. strongly reducing solutions may form a surface layer of silver. From your chloride results. KNOWN (STANDARD) ADDITION TECHNIQUE # The known addition techniques will only correct for certain types of interferences.
starch indicator. 3. allow to mix again. Bleach/chlorine solutions are strong oxidants. gloves and lab coats. burettes and stands. 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. 1995. Personal protective equipment required at all times when working in the lab are: eye protection. pages 4-56 to 4-57 or older/newer editions. 250 ml Erlenmeyer flasks. and exactly 10 ml of 0. chlorine demand and break-point chlorination. add 1 ml of starch solution. When weighing chemicals at the balance.N-diethyl-p-phenylenediamine (DPD) indicator solution. phosphate buffer solution.La b o ra to ry 5: Ch lo rin e a n d Ch lo rin e D e m a n d WARNING Glacial acetic acid is very corrosive. OBJECT: -to acquaint you with disinfection of water supplies and to demonstrate techniques for the determination of chlorine residuals. PREPARATION OF STOCK CHLORINE SOLUTION (Iodometric method .. about 5 ml of glacial acetic acid (use a graduated cylinder). Wipe up all liquid spills immediately.total chlorine residual) 1. potassium iodide (KI) crystals. I.025 N sodium thiosulphate solution (use a volumetric pipette). Take a 250 ml Erlenmeyer flask and add about 1 gram of potassium iodide. Make up to the 250 ml and invert flask several times to mix. clean up all spills with a brush into a container and wash crystals down the drain. about 50 ml of chlorine-free water (distilled water will do). bleach or hypochlorite solution. Rinse a burette and fill it with this stock chlorine solution. Materials: -600 ml beakers. standard ferrous ammonium sulphate (FAS) solution. Titrate rapidly with the stock chlorine solution (in burette) until the appearance of a constant 34 September 2007 CIVL 407 Lab M anual . 2.025 N sodium thiosulphate. 0. N. Mix well (on stir plate with stir bar). to illustrate the concepts of free and combined chlorine residuals. chlorine free water. 100 ml graduated cylinder. REFERENCES: Standard Methods 19th ed. (concentrated) glacial acetic acid. pipettes. 250 ml volumetric flasks.
For dichloramine concentrations greater than 1 mg/L. d) Alternatively: (Not to be done in this lab. These 6 flasks can be prepared at once to make them ready for the next step. mix to dissolve. as there is lots to do . b) Monochloramine: Add two drops of KI solution (to the same sample) and mix. IIb. etc. 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. 35 September 2007 CIVL 407 Lab M anual . Total chlorine (Free plus Combined) = Reading C Free residual chlorine = Reading A Combined residual chlorine (dichloramine plus monochloramine) = Reading C . (*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. 2. After 15 minutes of contact time. 4. 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). titrate to colourless again.) Free and combined chlorine or total chlorine: To obtain total chlorine in one reading. please refer to Standard Methods. c) Dichloramine: Add about 1 g KI (to the same sample) and mix to dissolve. refilling burette. Place 5 ml of phosphate buffer solution and 5 ml of DPD solution in a 250 ml conical (Erlenmeyer) flask and mix. Add 100 ml of sample or diluted* sample using a 100 ml graduated cylinder and mix again. Let stand 2 minutes more. Let stir for 2 min and then continue titrating until red colour (if any) is discharged (Reading C). a) Free chlorine: titrate rapidly with standard FAS (Ferrous ammonium sulfate) titrant until the red colour is discharged. if colour drifts back (indicating an incomplete reaction).). Continue titrating until red colour (if any) is discharged once again (Reading B). let stand for two minutes and titrate until the red colour (if present) is discharged. Method 4500-Cl F. Work in groups of three or four for this part. let stand 2 min more if colour drifts back indicating incomplete reaction. From the amount of chlorine solution used.Breakpoint Chlorination using Free and Combined Chlorine Residuals Values 1. Record the burette reading (mls) = Reading A. Determination of Free and Combined Chlorine by the DPD Method 1.COORDINATE! IIa. It is likely that the highest dose will require dilution. Record the burette reading (Reading C). 2.A Monochloramine= B-A Dichloramine= C-B For interferences. Go to c. 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).purplish-blue colour. 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). add full amount of KI at the start (ie 2 drops KI solution and 1 g of KI crystals) to a fresh sample. 3. calculate the chlorine concentration in the stock solution (See Data and Results section for formula). 18th edition. pp 4-43. 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.
If this is true. 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.e. If a sewage sample contains 10 x 10 6 coliforms/100 ml. II. 250) then convert mg/L to g/100 mls = %. d. 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. how many coliforms would remain after a chlorine contact time of 10 minutes? 20 minutes? 36 September 2007 CIVL 407 Lab M anual . what percentage of bacteria would be killed in 10 minutes at a chlorine residual of 0. free and combined residual chlorine for each applied chlorine dose.7 x 10-8 at 25 o C % HOCl = 27 What is the pH of the solution? 3.DATA AND RESULTS I. Determine the concentration of total. The rate of kill of bacteria by chlorination follows first-order reaction kinetics. Make a plot of the three residual chlorine components against the applied chlorine dosage. Why is it important to determine chlorine residuals in domestic water supplies? 2. Make a separate graph for the 15 minute and 1 hour contact time. a.5 mg/L if 50% are killed in 1½ minutes at this concentration? b. b. Under what conditions is the practice of break point chlorination necessary? 4. Given: HOCl W H + + OClK eqv = 2. Guaranteed analysis is normally printed on the container and is usually 5-6%. Discuss your results as a function of the residual forms of chlorine present at different chlorine dosages and at different contact times. c.
of Cl Soln. (mls) (Stock=_____mg/L Cl as Cl2) Applied Cl dosage (mg/L) Volume of FAS to endpoint A Volume of FAS to endpoint B (includes A) Volume of FAS to endpoint C (includes A & B) Free chlorine (mg/L) (A) Monochloramine (mg/L) (B-A) Dichloramine (mg/L) (C-B) Combined residual chlorine (mg/L) (C-A) Total chlorine (mg/L) © Time of Cl addition Time of Residual measurement 1 2 3 4 5 6 37 September 2007 CIVL 407 Lab M anual .15 MINUTE CONTACT TIME BEAKER NUMBER Vol.
of FAS to endpoint A (mls) Vol.60 MINUTE CONTACT TIME BEAKER NUMBER Vol. (mls) (Stock=_____mg/L Cl as Cl2) Applied Cl Dosage (mg/L) Vol. of Cl Soln. of FAS to endpoint B (mls) (includes A) Vol. 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 .
magnetic stirrer. the two buffers that are chosen for calibration will bracket your sample ie if sample is less than 7. 3.Lab o rato ry 6: Alkalin ity . MATERIALS: -pH meter. hardness indicators. standard base. Ac id ity . bromcresol green. burettes and stand. beakers. metacresol purple. Rinse probe when you are finished and leave immersed in pH 7 buffer for storage. Normally.) 2. then use buffers 4 and 7 to calibrate. (Take care not to contaminate paper in case. graduated cylinders. if above 7. Calibrate the pH meter by following the procedure outlined on the instruction sheet beside the meter unless told otherwise. to acquaint you with the use of pH metres. buffer solution. 1 N NaOH. metres and calibrated glassware are available in limited quantities and are very expensive to replace. place a beaker containing the sample and stir-bar on the stir-plate. bromphenol blue. 39 September 2007 CIVL 407 Lab M anual . pH test paper and other water quality assessment equipment. Use care when using lab equipment. phenolphthalein. colour comparator. water samples. I.) Record value in following table. standard acid. T u rb id ity WARNING Please use caution when handling all chemicals. p H. After calibration is complete. then use buffers 7 and 10. Co lo u r. lower probe. turbidimeters. 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. EDTA. Probes. Always wear personal protective equipment. DETERMINATION OF pH 1. rinse & dry probe. particularly those labelled with specific hazards. 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. pH test paper.(Do not allow the stir bar to hit the probe. turn on stirrer and obtain a stable reading. Hard n e s s . 4. OBJECT: -to familiarize you with the most common water treatment tests. flasks.
2. Measure out a new sample and add 1 drop of sodium thiosulphate and a full dropper of phenolphthalein. Measure out 25 mls of sample using a graduated cylinder and dilute to 50 ml with distilled water. Titrate slowly with EDTA. while stirring. Measure 100 ml of water sample into a clean.. White paper placed under the flask will facilitate this determination.. 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.5). Record burette reading (P). titrate until just colourless. Add the last few drops at 3-5 second intervals so that you do not "overshoot" the end-point. Add 1 drop of sodium thiosulphate solution. if the approximate hardness is known (by unsuccessful attempts). DETERMINATION OF ACIDITY 1. Record the burette reading in the following table. Add a full dropper of phenolphthalein (P) indicator and if the solution stays pink or red.think what that means!) 4. Complete the titration within 5 minutes to minimize the tendency toward CaCO 3 precipitation. 4. Add stir-bar. until the reddish tinge disappears from the solution and a pure blue remains. 3..EDTA TITRIMETRIC METHOD 1. Pour into a 250 ml Erlenmeyer flask. TOTAL HARDNESS DETERMINATION . 3. 5. Apply dilution factor to obtain final result. add 90% of the titrant to the sample before adjusting the pH with buffer. The buffer elevates the pH to 10. 3. 40 September 2007 CIVL 407 Lab M anual . (If the solution is already blue after indicator. it may be necessary to repeat the titration with the sample diluted 1 to 1 (again) with distilled water.II. If sample is highly alkaline (titration goes over one burette full) dilute the sample and titrate again. If a ppt does form within the 5 minutes. (If solution is clear after addition of P. Titrate to the first uniform pink colour (colour stays). Rinse and fill another burette with standard base solution and label it. Add a full dropper of bromphenol blue indicator and titrate until the colour changes from yellow to blue ("MO" Acidity). 2. III. A pH much above 10 promotes CaCO 3 precipitate.1 N sodium thiosulphate to the sample.. (BG or MO) 6. IV.. Measure 100 ml of sample (with minimum agitation or exposure to air) using a graduated cylinder and transfer carefully to an Erlenmeyer flask. 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. In order to remove any free residual chlorine (which interferes with the indicator colour response). 2. Add a stir-bar. add 1 drop of 0. Rinse and fill a burette with standard EDTA solution and record the initial burette reading. Record the volume of titrant required in the following table. Alternatively. Rinse and fill a labelled burette with standard acid solution. sample-rinsed graduated cylinder and transfer to an Erlenmeyer flask. DETERMINATION OF ALKALINITY 1.?) 4.
5.EDTA TITRIMETRIC METHOD 1. with continuous stirring to a pure blue end-point. Cap the cell. 2. to determine the apparent colour (measures the influence of turbidity on the reading). Press: I/O. V. Record all colour data in the following table. 4. Record the final burette reading in the following table. Record the final burette reading in the following table. VI. The display will show SIG AVG when the instrument is using signal averaging. The reading might be higher so if you had to dilute in Part A dilute for Part B. 4. Close the lid.HACH Spectrophotometer. 3.. 5. Select automatic range by pressing the RANGE key. 3. lint-free cloth to remove water spots and fingerprints.BY HACH 2100P Turbidity Meter. 3. TURBIDITY . Measure out 50 ml of sample into an Erlenmeyer flask and add 2. The display will show AUTO RNG when the instrument is in automatic range. Fill a sample cell to the line (about 15 mL). Values are reported as APHA Colour Units (eqv. VII. Place the cell containing water into the spectrophotometer and press Zero. . Wipe the cell with a soft. Select signal averaging mode by pressing the SIGNAL AVERAGE key. 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. CALCIUM HARDNESS DETERMINATION . The instrument may suggest diluting if over range. 4. 2. or a volume sufficient to produce a pH of 13-14 (designed to precipitate the magnesium as a hydroxide).Enter the stored program number for true colour=120 1. COLOUR . Use the technique outlined in Part A. Place the cell containing sample in it and press Read. Pour the supernatant into one of the cells (to the etched mark) and fill another with distilled water.. 2. Filter about 20 mls of sample using a filter funnel and stand and the pre-fluted filter paper provided 2. taking care to handle the sample cell by the top.0 ml of 1 N NaOH solution (use 3 droppers full). Add a stir-bar and stir to mix.. The instrument will turn on but will turn off automatically after a short time so you should be ready. to mg/L platinum as chloroplatinate) PART B: APPARENT COLOUR 1. Add one aliquot of the Calcium Hardness Indicator and titrate with EDTA slowly. except on an unfiltered sample. Demonstration of Jackson Candle and Hellige PART A: Hach Turbidimeter 1.TRUE AND APPARENT PART A: TRUE COLOUR . Use signal average mode if 41 September 2007 CIVL 407 Lab M anual . Refill the burette with EDTA titrant and note the initial reading.
16th edition. record the number and refer to the appropriate graph to determine the turbidity as mg/L SiO 2. the turbidity may start to settle and condense on the bottom of the tube.adding liquid to a hot tube will cause it to shatter. Record the turbidity after the lamp symbol turns off. Record value in following table as J..NTU.T. 2.s. Note that the rectangular door mirror is closed. that the filter selector shows "none" and that bulb B is in use. obscuring vision. 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. Take the reading where the dark central part first merges with the surrounding field.the sample causes a noisy signal (display changes constantly). Try to be quick and not burn the candles more than a few minutes at a time. Note that there are several different scales on the side of the tube. 3...Demonstration only 1. T = Total alkalinity. (This allows you to make a more gradual and accurate approach to the end-point. 1. Wipe the outside of the tube to dry it and place it in the turbidimeter (in the circular groove of the mirror unit). See Standard Methods. Pour sample into the tube in small increments until you can no longer distinguish the shape of the candle flame. PART C: Jackson Candle Turbidity . 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. Press: READ The display will show .. Carefully fill the 20 mm viewing depth tube to the mark with sample. 42 September 2007 CIVL 407 Lab M anual . page 272-273.U. Lower the plunger into the liquid making sure no bubbles are trapped under the plunger and the top is dry. PART B: Hellige Turbidimeter . then the turbidity in NTU.. Read the scale on the dial. 2. DO NOT PLACE AN EMPTY TUBE IN THE TUBE HOLDER WITH THE CANDLE LIT . Close the door and switch on the light. 3. 6.) Remove the glass tube from the holder and read the turbidity at the meniscus of the sample. Pinch all excess charred wick with fingers before lighting or soot may be deposited on the glass tubes. 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. Be sure that the calibrated tubes are clean before adding samples to the tubes.demonstration only. If you take too long.
DATA Sample 1 pH ." titre (mls) Net Total acidity titre (mls) Net CO 2 Acidity titre (mls) Final burette reading (mls) Initial burette reading (mls) Net titre (mls) Total Hardness as mg/L CaCO 3 CALCIUM Sample volume (mls) Final burette reading (mls) Initial burette reading (mls) Net titre (mls) Calcium as mg Ca ++ /L __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ __________ ___________ ___________ ___________ ___________ ___________ ___________ ___________ ___________ Sample 2 ___________ ___________ P.O. Acidity end-point reading (mls) Initial burette reading Net "M.O. Alkalinity end-point reading (mls) __________ "M.Sample volume Initial burette reading P.Sample volume "M." Alkalinity titre (mls) ACIDITY. Alkalinity titre (mls) Net "M." Alkalinity end-point (mls) Initial burette reading Net P." 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 .O.O.Initial pH by test paper Initial pH by pH meter ALKALINITY.
00 ml EDTA titrant at the calcium indicator endpoint = 1.00 ml EDTA titrant at the calcium indicator endpoint = 1.CALCULATIONS Calcium: Calcium Hardness: where: A= B= ml titrant for sample mg CaCO 3 equivalent to 1.0 Total Hardness (EDTA): where: A= B= ml titrant for sample mg CaCO 3 equivalent to 1.0 Alkalinity: where: A= N= ml standard acid used normality of standard acid Acidity: where:A = N= ml standard base used normality of standard base 44 September 2007 CIVL 407 Lab M anual .
Alkalinity as CaCO 3 (mg/L) OH .ion (mg/L) CO 3 -2 Alkalinity as CO 3 -2 ion (mg/L) HCO 3.Alkalinity as CaCO 3 (mg/L) CO 3 -2 Alkalinity as CaCO 3 (mg/L) HCO 3.Units= N.T.U.Alkalinity as HCO 3 . ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 45 September 2007 CIVL 407 Lab M anual ." Alkalinity as CaCO 3 (mg/L) OH .O. Alkalinity as CaCO 3 (mg/L) "M.Alkalinity as OH ." Acidity as CaCO 3 (mg/L) Total Acidity as CaCO 3 (mg/L) Free carbon dioxide as CO 2 (mg/L) Total Acidity as CO 2 (mg/L) Ca Hardness as mg Ca +2 (mg/L) Ca Hardness as CaCO 3 (mg/L) Mg Hardness as CaCO 3 (mg/L) Mg Hardness as Mg+2 (mg/L) Carbonate Hardness as CaCO 3 (mg/L) Non-carbonate Hardness as CaCO 3 (mg/L) Excess alkalinity as CaCO 3 (mg/L) Colour .Hach .O.APHA Colour Units: True Apparent Turbidity .ion (mg/L) "M.RESULTS (sample) P.
46 September 2007 CIVL 407 Lab M anual . From equations (17-12) and (17-13) in Sawyer and McCarty. 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.7 x 10 -11 and K w = 10 -14. Is this water suitable for a domestic water supply? Why? 2. 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. non-carbonate and total hardness of this water in mg/L as CaCO 3 ? in meq/L? Is the analysis complete? Why? 6.Lab 6 Questions 1. calculate the carbonate alkalinity in mg/L as CaCO 3 for the water sample analyzed during this lab. given K 2 = 4. Does this agree with the results from Part II of this lab? Explain. What errors can occur in the calcium hardness determination? Why? 3. What sanitary significance does colour have? 4.
7 (except Jackson Candle and Hellige Turbidimeter). and all of the materials from Lab. 47 September 2007 CIVL 407 Lab M anual . OBJECT: -to illustrate the principles of coagulation and water softening. You should have with you the part of Exercise 7 that gives instructions for the various tests. soda ash solution (Na 2CO 3) and lime (Ca(OH)2) or sodium hydroxide (NaOH). MATERIALS: -Jar Test Apparatus. Please try to coordinate your work with the rest of the class.for the next session (b). No.6.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. In the second lab you will use the previously determined dosages to coagulate and soften the sample and then re-analyse the resultant water. alum solution (Al2 (SO 4 )3 @18H 2 O). No. This is a two-step experiment. to be conducted over two lab periods. 1000 ml beakers. Several lime and soda ash dosages will also be selected for determining the efficiency of water softening. The optimum alum dosage for coagulation of the sample will then be chosen. 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.
alkalinity. Carefully decant into a graduated cylinder 800 mls of the supernatant liquid and pour into fresh beakers. Measure out six 1000 ml aliquots of sample (using graduated cylinder to measure. Record relative floc sizes (pin-point. medium. hazy. but because of equipment limitations. Lift paddles out of beakers and secure. COAGULATION USING ALUM . Make relative estimates of floc sizes. 5. Part B: WATER SOFTENING PROCEDURE (to be done the following week) again work as teams of 2 or 3. Place beakers on the jar tester apparatus. Stop stirrer. A pH meter could be used for the alkalinity and acidity determinations. drop of 0. cloudy). 1. Analyze the supernates for pH. Add the assigned alum dosages as quickly as possible and then start the stirrer. 4. 5. Add the designated lime (or NaOH) and soda ash additions as quickly as possible to the decanted samples. use indicators as specified in Lab 6. 6. 3. position them under stirrers. Analyze these supernates plus a sample of raw water for pH. You may need to use blocks to position the paddle 0. After the flocs have settled (allow 15 min). (NB dosage now based on 800 ml sample size). 3. and clarity of supernatant liquid. and fill in the table on the board.Part A:. true colour and turbidity (Hach). 4. acidity. 2. Observe and make notes describing floc formation and note the time of appearance.work in 2 or 3 teams to assess a total of 6 dosages for each team. Stop stirrer and allow floc to settle. Complete the coagulation summary sheet during the lab period.5 to 1 cm off the bottom of the beaker. Discard the floc and clean beakers in preparation for a second decanting in Step 4. 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. Stop stirring. hardness (total and calcium). turbidity and colour. alkalinity. clarity of supernatant liquid (very clear.. 7. not a beaker ) and transfer to 1000 ml beakers. clear. 6. Pour six 1000 ml aliquots of sample into 1000 ml beakers and place on the jar test stirrer apparatus. Stir at 100 rpm for 1 minute. and settling characteristics of the floc (rapid. if available. In the lecture. acidity. Determine which dosages were most appropriate for this sample. moderate. 2. hardness (total and calcium). After the floc has settled (allow 15 min). Stir at 100 rpm for 1 minute and at 20 rpm for 10 minutes. Reduce the stirring rate to about 20 rpm for 10 minutes. Add the appropriate alum dosage and immediately start stirring. if time permits. 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. Record observations on settling rate. slow). 1.1 N sodium thiosulphate 48 September 2007 CIVL 407 Lab M anual . decant the supernatant liquid carefully into a clean set of beaker (try not to stir up what has settled) and discard the floc. times of appearance and descriptions and record. floc volume. 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). large). Stir at 100 rpm for 1 minute. Make sure it is centred. Brief Methods Summary for Analyses Alkalinity: @ 100 ml of sample. decant the supernatant liquid carefully into another set of beakers (try not to stir up what has settled).
pH 3.pH 8. 3.02 N acid with phenolphthalein to colourless . show by calculation the theoretical amount of lime and soda ash required for excess lime/soda ash softening.use graphical presentation.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 .5. Compare removal of hardness components under the different doses . 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: Ca hardness (mg/L CaCO 3 )= mls titre x 1000/mls sample Lab 7 Questions A. @ calculation .@ titrate with 0.pH 8.000/mls sample Acidity: @ 100 ml of sample.3 and with bromcresol green (continuing with the same sample) to yellow .acidity (mg/L CaCO 3)= mls titre x N base x 50. 2.alkalinity (mg/L CaCO 3)= mls titre x N acid x 50. Compare your experimental softening results to the theoretical results based on solubility equations or mass balance equations. 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.02 N base with bromphenol blue to blue . 4. Coagulation 1.pure blue).7 ("methyl orange" acidity) and with phenolphthalein (using a fresh sample) to pink . 2. Softening 1. Analyze the response of colour and turbidity removal as a function of alum dose.pH 4. Based on your chemical analysis of the raw water. @ 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 . drop of 0.pure blue).3 ("phenolphthalein" or total acidity) @ calculation .
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 8.7 “MO” Net titre to pH 8.sample vol ml Net titre to pH 3.5 “TOT” Total Hardness sample vol Net titre Ca Hardness .5 “P” Net titre to pH 4.sample vol Net titre Notes: 50 September 2007 CIVL 407 Lab M anual .5 “TOT” Acidity .
P.H.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.) (filtered) Turbidity (N.T.A.) unfiltered Notes: 51 September 2007 CIVL 407 Lab M anual .U.) True Colour (A.P.H.
5 Net (Total) titre pH 4.7 Net (Total) titre pH 8.5 Acidity .5 Total Hardness sample vol Net titre Ca Hardness .sample vol ml Net titre to pH 3.sample vol Net titre Notes: 52 September 2007 CIVL 407 Lab M anual .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.
P.H.H. -mg/L CaCO 3 **Non-Carb Hard -mg/L CaCO 3 **Excess Alk.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. Calculation 53 September 2007 CIVL 407 Lab M anual ** By . -mg/L CaCO 3 Apparent Colour (A.U.) True Colour (A.A.) (Filtered) Turbidity (N.) 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.P.T.
. . . . . . . . Solids Removal in Wastewater Treatment . . 91 18. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Titrations . . . . . . Chlorine . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Periodic table . . . . . . . . . . . . . . . . . . . 107 26. . . . . . . . . . . . . . . . . . . . . . . . . . . . Alkalinity Species as a Function of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . Concepts and Definitions . . . . . . . . . . . . . . . . Chloride Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4. Equilibrium Relationships of Chlorine . . . . . Known Addition Technique . . . . . . . . 100 22. . . . . . . . . . . . . . . . . . . . . Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Standard Solutions. . . . . . Instructions for Laboratory Report Writing . . . . . 105 25. . . . . . . . . . . . . . . . . . . . . . . . Bacteriological Examination . . Typical Problems for Acid-Base and General Chemistry . . . . . pH . .Levels and Water Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 9. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 12. . . . . . . . Faecal Coliforms . . . . . . Acidity. . . . . Hardness Complexes . . . . . . . . . . . . . . . . . . 92 19. . . . . . . . . . . . . . . . 96 21. . . . . . . . . . . . . Alkalinity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical Requirements for Water Softening . . . . 84 17. . . . . . . . . . . . . . . . . . . . . . . . . . . Water Softening . . . . . . . . . Normal Solutions and Equivalent Weights . 57 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Turbidity. . . BOD. . . . . . . . . . . . . . . . . . . . 112 28. . . . . . . . . . . . . . . . . . COD . . 102 23. . . . . . . . 118 54 September 2007 CIVL 407 Lab M anual . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 13. . . . . . . . . . . . . . 80 14. . . . . . . . 71 10. . 104 24. . . . . . . . . . . 68 7. . . . . . . . . . . 82 15. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Coagulation . . . . . . . . . . . . . . . . . . . . . . . . . 64 5. . . . . . Hardness. . . . . . . . . . . . . . . . . Reading and Reference Material . . . Milliequivalent and mg/l as CaCO 3 . . . . . . . . . 110 27. . . . . .Chlorine Demand . . . . . . . . . .COD . . . . . . . . . . DO. . . . . . . . . . Extra tables for Chlorine Lab . . . . . . . . . . . . . . . . . . . . Balancing an Oxidation Reduction Equation . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 Appendices see: CIVL407Manual_CourseNotes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Colour. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bar Diagram Method for Water Softening Problems . . . . . . . . . . . . . . . . . . . . . . . . . . 72 11. . . . . . . . . . . . . . . 94 20. . . . . . . . Bacteriology and Water-related diseases . . . . . 55 2. . . . . . 117 29. . . . . .Additional Notes . . . . . . Hach Absorption Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 16. . . . 67 6. . . . 69 8. . . . . . . . . . . . . . . . . . . . .pdf 1. . . . . . . . . . . . . . . . . . . . . . . . . . . .