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Prepared for Dept. Of Civil Engineering by Susan C.M. Harper September 2007
Table of Contents
Course Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Station/Drawer Supply List (if supplies are missing, please report) . . . . . 2 First Session: Laboratory Safety and Orientation . . . . . . . . . . . . . . . . . . . . 3
L ABORATORY R EPORT W RITE U P G UIDELINES . . . . . . . . . . . 5
Laboratory Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Laboratory 1: Solids Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Laboratory 2: COD, DO and BOD 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Laboratory 3: Bacteriological Examination of Sewage . . . . . . . . . . . . . . . 17 Laboratory 4: Chloride Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Laboratory 5: Chlorine and Chlorine Demand . . . . . . . . . . . . . . . . . . . . . 34 Laboratory 6: Alkalinity, Acidity, pH, Hardness, Colour, Turbidity . . . . 39 Laboratory 7a: Coagulation and 7b: Coagulation & Softening . . . . . . . . . 47
Appendices see: CIVL407Manual_CourseNotes.pdf . . . . . . . . . . . . . . . . . 54
(Environmental Laboratory Analysis - 1-3-0, 0-0-0) Testing procedures used in water quality studies and in the operation of water and wastewater treatment plants. Prerequisite Chem 151. Sc h e d u le Lecture CEME1204: Laboratory CEME1301: Wednesday 1:00 pm 1) Tuesday (time to be arranged by consensus) 2) Wednesday 2:00 to 5:00 pm 3) Thursday (time to be arranged by consensus) 4) Friday (time to be arranged by consensus) (Note: Lab Session must have at least 4 students to run)
Subject No Lab No Lab Laboratory Safety & Orientation (~1 hr) #1 Solids determination #2 Dissolved oxygen, COD, BOD No lab - Thanksgiving week - catch up #3 Bacteriological Examination of waste water #4 Chloride #5 Chlorine demand #6 Acidity, alkalinity, hardness, colour, turbidity No lab - Remembrance day - catch up #7a Coagulation #7b Coagulation and Softening
Date 2007 Sep. 4 - 7 Sep. 11 - 14 Sep. 19 Sep. 25 - 28 Oct 2 - 5 Oct 9 - 12 Oct. 16 - 19 Oct. 23 - 26 Oct. 30 - Nov.2 Nov. 6 - 9 Nov. 13 - 16 Nov. 20- 23 Nov. 27 - 30
Lecturer: Lab instructor: T.A.s: Lab: Marker:
Victor Lo, email: Susan Harper, email: Margaret Parsotan, email: Kazi Parvez Fattah, email:
firstname.lastname@example.org email@example.com firstname.lastname@example.org email@example.com
(604) 822-4880 (604) 822-4397
September 2007 CIVL 407 Lab M anual
Sta tio n /D ra w e r Su p p ly Lis t (if s u p p lie s a re m is s in g , p le a s e re p o rt)
The following items will be maintained in each stations supply drawer or at station on bench when required: 2 pair safety glasses 2 permanent marker pens 2 stir bars and a bar retriever 1 stir plate 2 buret funnels 4 30 ml beakers 4 50 ml beakers 2 100 ml beakers 2 150 ml beakers 2 250 ml beakers 2 1 L plastic beakers 2 600 ml plastic beakers 2 400 ml plastic beakers 2 10 ml grad cylinders 2 25 ml grad cylinders 2 50 ml grad cylinders 2 100 ml grad cylinders 1 buret stand 1 double buret clamp 2 50 ml burets 2 25 ml burets 2 10 ml burets 0-14 pH strips Thermometer 2 Stir stick or spatula Filter funnel (Nalgene magnetic) Tweezers (Millipore or Gelman for membrane filters)) On benches each row: Latex gloves- small, medium and large Non-latex gloves (keep on reserve for latex sensitive persons only) Filter racks with funnels #4 Whatman filter paper or equivalent Vacuum flasks and hoses 8 1 L grad cylinders 8 3 L plastic beakers
September 2007 CIVL 407 Lab M anual
Do not put lids and small items onto racks that may slip through to the filthy drip tray.2 First Session: Laboratory Safety and Orientation 1) 2) 3) 4) NO Food or drink. These should be used whenever standard solutions are measured or when upmost accuracy is required. Safety Equipment: Eye Wash Station. wash down and dry the bench. Please take your labcoat away in a plastic bag.up to 10. Personal Protective Equipment: Lab coat (long). MSDS: is a supplier information sheet that must be available for each product and describe the hazards and necessary handling requisites. with contaminated gloves will contaminate those items. DO NOT PUT BROKEN GLASS INTO THE REGULAR GARBAGE CONTAINER. A small amount of liquid always remains in the tip and must not be blown out. Safety Shower. no matter how minor. Be aware that handling your pens. Pipettes must be placed with tips pointing up into the pipet basket supplied.clean and dry. Please ask for assistance if chemicals are spilled. Spills: Wipe up all spills immediately. 25. gloves as required.0 ml. 20. Putting pens in your mouth that may have been on the bench or handled with gloves could be hazardous. but if you do please ask for assistance. 1. Sanitation: antibiotic soap for hand washing. They are calibrated by weighing the volume of distilled water that will flow from them by gravity. No sandals or open-toe or heel shoes. Most pipettes are calibrated “to deliver” or TD and should never be blown out. if there is room.0. Some pipettes may be “to 3 September 2007 CIVL 407 Lab M anual . You must be informed of the hazards of a substance before using it. Remember to empty and rinse burets as well.. with the tip against the side of the receiving vessel. Broken Glass: Be careful not to break glass. packs. Hygiene: At times you will be working with sewage. 50 and 100 ml. Pipettes/ graduated cylinders/ beakers/ flasks: 5) 6) 7) 8) 9) 10) Volumetric pipettes are the most accurate way to measure volume. 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. calculators. disinfectant for bench surfaces. WHMIS: Workplace Hazardous Materials Information System is a government regulation. etc. 15.0. Wearing lab coats out of the lab is forbidden. 2. Clean-up: You must leave your work station as you found it .. If you cut yourself. please get attendant help.5. They are available in 0. eye protection.
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. Additional supplies are available in the supply room (around the corner). NEVER mouth pipette. Make sure that you label beakers and flasks to avoid confusion and waste. Do not squish bulbs in the direction of another person. Even the force of air from the air taps can be dangerous because it can contain grit or water.deliver with Blow-Out”. A demonstration will be given. YOU WILL USUALLY NEED THE INFORMATION GIVEN TO COMPLETE YOUR LAB WRITE-UPS. Chemical residues can be splashed in you face. please rinse out immediately and set aside to dry. If liquid should enter a bulb or pump. 14) Glassware/supplies: Each station has a drawer where some glassware and other useful tools will be kept. 10 mls. 50 mls. 4 September 2007 CIVL 407 Lab M anual . Pipette bulbs or pumps must be used and great care must be taken not to contaminate bulbs. Pipettes are in drawers indicated.merely approximations. 12) Cup sinks: Please do not use these sinks for the disposal or delivery of liquids. Erle n m e y e r or conical flask markings are not accurate.1000 mls. Chemicals will be put out as required for each lab and should be used sparingly. LISTEN AND MAKE NOTE OF VERBAL AND CHALK BOARD INSTRUCTIONS. 13) Stir bars: Please remove from containers before pouring contents down the drain. Undetected gas leaks can be deadly. They are useful for measuring samples 20 mls . Ie If you want to measure 20 mls. 1. They are containers best suited for “swirling” liquid ie to mix reagents with samples in a colourimetric determination (Lab 4 or 6). chemicals or for titrations. They are calibrated the same way as TD except that the remaining drop is blown into the receiving vessel. 250 mls. Cylinders are available: 5 mls. Graduated cylinders: Not as accurate as pipettes. Some are blow-out.. especially at smaller volumes. 11) Gas/air/vacuum taps: Do not touch these unless instructed to do so. They are usually identified by a double etched or coloured band at the top of the pipette.pay attention to the markings. 25 mls. They can be unpredictable in force and are close to eye level. do not use a 1 L graduated cylinder. Use the size of cylinder CLOSEST TO THE VOLUME YOU WISH to measure for best accuracy. 2 & 4 L Beakers: are cylindrical in shape with straight sides and flat bottoms. Used to hold/transfer approximate volumes of samples. Flasks: Vo lu m e tric flasks are very accurate and should be used when diluting standards (together with volumetric pipettes). 100 mls. 500 mls. not wastefully as quantities are limited. markings are not accurate at all . some are not meant to drain completely .
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. The short report is intended to be a concise yet complete laboratory report whereas the long report is more elaborate. Required  Required  Required  Required  Required  Required  Required   5 September 2007 CIVL 407 Lab M anual . R EPORT E LEMENTS Title page Objective Materials and Method S HORT R EPORT L ABS 1 TO 6 Required Required Details are not required. State that the procedures were in accordance with CIVL 407 Lab Manual and note any deviations to the manual’s procedure.L ABORATORY R EPORT W RITE U P G UIDELINES Guidelines for laboratory report write up are also given in the CIVL407 Manual Course Notes and summarized here. Please note that the marking outline is intended only as a guideline to assist in report write-up and that marks would also be allocated for overall presentation and clarity of the report. The mark allocation for some sections for both reporting format is indicated in parenthesis in the table below. Total Mark L ONG R EPORT L AB 7 Required Required Required Include a brief description of the procedure. Sufficient detail should be provided so that what was done could be understood and the experiment could be repeated from the procedure reported.
2.3 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. settle for 15 minutes longer. MATERIALS: -Imhoff cones. 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. 6 September 2007 CIVL 407 Lab M anual . samples of raw sewage. and record the volume of settleable solids. evaporating dishes. to measure sewage treatment plant efficiency in removing residue. A. keep hands and pencils away from your mouth and wash hands frequently. sludge. gently dislodge any solids that have clung to the sides using a stirring rod. A 1 L graduated cylinder may be used in place of the Imhoff cone for the sludge sample. desiccators. Do not include any surface floating material as settled solids. Alternatively a place will be provided in the lab to store your coat.. SETTLEABLE SOLIDS 1. Mix the samples thoroughly and fill individually marked Imhoff cones to the 1 litre mark. Use pipette bulbs. 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. if necessary. Settle for 45 minutes. oven muffle furnace. final effluent. balance. subtract an estimated volume from the measured volume of matter. If large pockets of liquid form between the particles of settled matter. graduated cylinders. wipe up all spills and wash with disinfectant.
so be quick. The loss in weight represents the volatile solids lost from the originally measured sample volume. [Notes: to allow for rinsing and to avoid spillage when transferring dish to oven don't pour more than 40 mls]. if instructed. Assemble filtering apparatus. immediately after stirring the beaker. This is a different test to the one we are doing using Imhoff cones and should not be confused.TOTAL AND VOLATILE 1. 2. rapidly pour sample into a 50 ml graduated cylinder to approximately equal 35-40 mls (if using 50 ml dishes). SUSPENDED SOLIDS .].[Note: it may be harder to wash out solids if you allow them to settle before pouring into the dish .] Rinse the graduated cylinder with small amounts of distilled water and add to filter. the dish will be transferred to a desiccator. dried and fired). tare weight. adding the rinsings to the dish. rinse the cylinder. SVI= settled sludge volume (ml/L) x 1000/suspended solids (mg/L)] B. Conversely. dried. Put the dish in the 103-105 °C oven for drying. Mix the sample in the carboy thoroughly. 2. Carefully remove filter from filtration apparatus and transfer it back to the aluminum dish. Using the sample poured out for Part B (for consistency). The dish containing the dried total solids can now be placed in a muffle furnace or.done for you.[Note: Sludge Volume Index is the volume occupied by 1 gram of sludge after 30 minutes of settling. sample source and sample volume. 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. 6. 4. Obtain the gross weight of the dish [Note: it should be room temperature before weighing. stir the beaker contents and then rapidly (so that it doesn't settle) measure a volume of sample into the appropriate graduated cylinder. C. Make sure that you have recorded all the pertinent details: Dish #. position the filter and begin suction (vacuum tap). The samples will be fired at 550° C for one hour. Once dishes are at room temperature they can be re-weighed. . TOTAL SOLIDS and TOTAL VOLATILE AND FIXED SOLIDS AT 550o C 1. The furnace will then be allowed to cool significantly before dishes are removed to a desiccator. Calculate the mg/L volatile and fixed solids. on a cart in preparation for staff to do so when all are ready. Record the total volume filtered. Obtain the tare weights of three aluminum dishes each containing a glass fibre filter (already pre-washed. pour about 500 mls (for Part B & Part C) into a beaker and then. Obtain the tare weight of a properly prepared porcelain evaporating dish (ie one that has been washed. Pinch sides of dish in a bit to protect the filter from oven drafts. 10 mls for Raw & 5 mls for sludge) of well-mixed sample and filter entire portion before pouring another portion. prior to session). Wet filter with a small volume of distilled water to seat it. 25-50 mls of raw sewage and 5-10 ml of sludge u s in g g ra d u a te d c y lin d e rs to measure .] Using very small amounts of distilled water. Quickly record the exact volume and pour the sample into the dish.not beakers. 3. Subtract the tare weight to obtain the total solids weight and calculate the mg/L value. 3. pre-fired and desiccated . when filtering slows significantly do not add more. Suggested portions: 100-200 ml of final effluent. Do not write on crucibles as high temperatures during the next steps will burn writing off. 5. [Hint: pour out small portions (say 25 mls at a time for Effl. the solids remaining represent the fixed solids. After drying overnight.
volatile.e. Discuss the meaning and significance of this index. % reduction in settleable matter _________ % reduction in total solids _________ % reduction in volatile solids _________ % reduction in fixed solids _________ % reduction in suspended solids _________ % reduction volatile susp. overnight). place the dishes on a cart designated by the instructor. A raw sewage sludge goes through an anaerobic digestion process. 3. 8. Your hands may have moisture and natural and/or applied oil/lotion on them. what is the percentage reduction in solids. What major types of solids are removed in primary treatment? in secondary treatment? 2. 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. where the volatile solids are reduced from 65% to 40%. 6. DO NOT DISCARD! The gain in weight represents the total suspended solids of the sample. For the time being. Calculate the percent solids reduction through the treatment plant. if any.3 and fixed solids 2. Calculate as the mg/L total suspended solids. solids _________ % reduction fixed suspended solids _________ % reduction dissolved solids _________ Lab 1 Questions 1. fixed (use more than one adjective to describe each substance. They must be allowed to cool completely before re-weighing them to determine the loss on ignition or volatile suspended solids. The aluminum dish and filter (from #4) must now be fired in a muffle furnace at 550°C for at least 30 minutes (or until a constant weight is achieved). What two techniques can be used to overcome or correct for the loss of material from filter pads when firing at 550 oC? 4. Transfer dish to a desiccator. suspended.50. From the above determinations it will now be possible to obtain a value for the dissolved solids present in the sample.) a) a bacterium b) sodium chloride c) sugar d) clay 8 September 2007 CIVL 407 Lab M anual . 6.5. Classify each of the following into its proper solids category(s) i. If all of the volatile solids reduced is given off as gas and if the specific gravity of the volatile solids is 1. Lab personnel will perform the timed-firing after the class. Please return tomorrow to obtain final weight. What. Enter all data in the tables on following page. cool and weigh. If sludge is used in Part A. 7. After firing the dishes will be moved to a desiccator. compute the sludge volume index (SVI). Calculate the mg/L total volatile solids and total fixed suspended solids. dissolved.
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 . Suspended Solids Tin dish marking (do not use ink .DATA AND RESULTS Raw Sewage Aerobic Sludge A.oven dry (G) Dish tare weight (G) Total solids (G) Total solids (mg/L) Gross weight . Total Solids Dish marking (do not use ink .use permanent mark on dish) Sample volume (mL) Gross weight .oven dry (G) Tin dish with filter tare weight (G) Total suspended solids (G) Total suspended solids (mg/L) Gross weight .fired (G) Loss on ignition (G) Volatile solids (mg/L) Fixed solids (mg/L) Percentage fixed residue C. Settleable Matter After 1 hour (mL/L) B.use embossed mark) Sample volume (ml) Gross weight .
wash/rinse bench tops with water/disinfectant. The closed reflux method is more economical but because a small sample volume (2 mls) is used. vials with pre-measured reagents. homogenization of samples containing suspended solids may be necessary in order to get a representative sample and to improve reproducibility. Hach block digester. etc out of your mouth. OBJECT: -to familiarize you with the commonest tests run for assessing treatment plant efficiency and pollution loading to receiving waters. Always use and store pipettes with the top higher than the tip. Wipe up all spills immediately. 200. to illustrate the shortcomings of these tests. and to demonstrate the use of dissolved oxygen probes.La b o ra to ry 2: CO D . Materials: Raw sewage and final effluent samples. Keep fingers. spectrophotometer. pencils. as well as handling samples probably containing pathogenic bacteria.Closed Reflux. etc. potassium hydrogen phthalate standards: 800. otherwise reagents will run to the bulb end and make pipetting difficult and contamination likely. 400. pipettes. 10 September 2007 CIVL 407 Lab M anual . Wash hands frequently with soap. 100 and 50 mg/L as COD. Colourimetric Method NOTE: Open reflux method is the most accurate means of determining COD because a large sample size is used (20 mls). D O a n d B O D 5 WARNING You will be using concentrated acid and other corrosive and toxic chemicals. COD . I. Digests from either method can be titrated or read colourimetrically. NEVER pour water into acid.
manganous sulfate. a volumetric correction must be made to the value obtained from the curve. well-mixed samples in the 25-position digester. The four liquids are cold tap water. Take vials to fumehood and place in block digester . starch indicator. Pipette 2 mls of sample (using volumetric pipettes unless particulate is present. 10-ml graduated pipettes. DISSOLVED OXYGEN (DO) Materials: Raw sewage and final effluent samples. The instructor will demonstrate the use of the spectrophotometer. Make sure sample is well mixed with the acid contents of the vial. Note: You are going to measure DO on four different liquids using two different methods. 400. Allow samples to digest for 30 minutes (normally this would be 2 hours). The probe has been previously calibrated. a smaller aliquot (making volume up to 2 mls with distilled water) or 2 mls of a pre-diluted sample can be used. II. Raw Sewage (Infl) and/or Effluent (Effl) in the upper 1/4 of the tube using permanent markers. and are at the spectrophotometer. alkaline-iodide-azide reagent. At your station you will find four vials with pre-measured solutions containing potassium dichromate and sulfuric acid (prepared according to standard methods. dilution water. 2. 100. [Note: it should look homogenous and be careful it will be very hot!] 3. aerated dilution water.noting the location of your well-labelled. If sample is known to be outside the standard range (above 800 mg/L). Remove the vials and allow them to cool to room temperature (use cold water to speed up if necessary). 11 September 2007 CIVL 407 Lab M anual . 6. Prepare calibration curve. 50 and 0 COD as mg O 2 /L.the azide modification of the iodometric method) and a dissolved oxygen probe and meter. DO NOT ADJUST THE SETTINGS ON THE METER. The vials should be clean and dry on the outside. Put identifying labels on each tube ie your initials. If less than two mls of sample or diluted sample was used. Standards have been digested for you. 0. 250-ml graduated cylinder. in which case. Read your samples and the set of COD standards: 800. which is set at 600 nm to read absorbance of light by the sample. before the lab. raw sewage and final effluent. use wide mouth graduated pipettes) in duplicate. into the appropriate tubes and screw cap on snugly. 500-ml Erlenmeyer flasks. concentrated sulphuric acid. 200. 5. 4.Procedure: 1. thermometers.025 N sodium thiosulphate. You will use a chemical titration (Winkler . BOD bottles. except without mercuric sulfate). burette stand with burettes. absorbance versus concentration and calculate the COD in samples.
Allow the floc to settle to about 1/3 the bottle volume. Record the temperature of each sample using the DO meter or a thermometer. 2. add 1 ml of concentrated H 2 SO 4 and quickly restopper. Fill a BOD bottle with each of the four samples (or use ones saved from A ). Determine DO. Record the volume of titre. add 1 ml of alkaline-iodide-azide solution the same way. When the floc has settled the second time to about 1/3 bottle volume. (Note: DO meter temperature is usually not accurate. 6. Determine the percent saturation for each sample. Neglect any reappearance of the blue colour. Stopper the bottles without trapping any air bubbles. aerated dilution water. raw sewage or influent and final effluent). For the cold tap water sample. Tip out the liquid from the area around the stopper and invert several times to mix thoroughly. Add enough starch solution to give a strong blue colour (one dropper full) and continue to titrate. 4. Titrate this volume with 0. Use the specially marked volumetric flask to dispense the 201 ml. dropwise. WINKLER TITRATION (Azide modification) NOTE: Use only the droppers attached to the reagent bottles for dispensing into samples. for each of the four samples. invert several times again and allow to settle again. To the same bottles. DISSOLVED OXYGEN PROBE NOTE: DO NOT add any reagents to the samples used for the probe method. again quickly replacing the lid. 8. 1. (Note: biological solids originally present in the sample will not dissolve. submerge the hose in the bottle and allow the water to overflow for 15-30 seconds). Do not mix droppers and do not allow tap water into the reagent bottles. (Hint: if you measured very low DO level with the meter. Place the bottles in the sink and one at a time remove the stopper. The DO concentration in mg/L is equal to the number of ml of titre as long as 201 ml is titrated. 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.) Transfer 201 ml of each bottle (do one at a time) to a 500 ml conical flask.) You can save these bottles for B. to thoroughly mix contents. (Fill carefully. right to the top and place stopper on bottle. Set back in sink. taking care not to entrain additional air. until the first complete disappearance of the blue colour. Instructions on the use of the probe will be given in the lab. remove the stopper. Fill four BOD bottles with the four samples (cold tap water. 9. using the DO probe and meter.A. 5. Take each stoppered bottle. B. Note: if solution already seems pale yellow add starch right away. 7. 1. then the same sample will require little or no titre and may not turn blue when starch is added . tip the liquid out of the space around the stopper (caution -it may be corrosive) and invert several times. 2.025 N Na 2 S2 O 3 until only a faint yellow or "straw" colour remains. . 12 September 2007 CIVL 407 Lab M anual 3.think about it!) Complete all four titrations. as demonstrated. The chemical floc should completely dissolve.
Pair up the mg/l of dissolved oxygen you measured and the temperature of the water in degrees C. (mg/L) DO conc. Note that this nomogram assumes that the water is at sea level The saturation value can also vary slightly depending on barometric pressure with lower values expected when a storm front moves through as compared to bright and sunny "high pressure" days.Dissolved Oxygen Data Sample/Data Bottle # Sample Volume (mls) Initial Buret reading Final Buret reading Net Titre (mls) DO conc. The percent saturation is the value where the line intercepts the saturation scale. Raw Sewage Final Effluent Tap Water Dil’n Water 13 September 2007 CIVL 407 Lab M anual . (Probe) Temp (oC) (thermometer) Saturation concentration Percent saturation DETERMINING PERCENT SATURATION THE "QUICK AND EASY" METHOD For a quick and easy determination of the percent saturation value for dissolved oxygen at a given temperature. use the saturation chart above. Draw a straight line between the water temperature and the mg/l of dissolved oxygen.
If the dilution water BOD is greater than 1 ppm. BOD bottles. being sure to exclude any air bubbles that might be trapped under the stopper by adding dilution water. 14 September 2007 CIVL 407 Lab M anual . Stopper the BOD bottles. Carefully top up the BOD bottles with DILUTION WATER. 10-ml graduated pipettes. 5. to bring the levels above the ground glass neck of the bottle. 4.if not add a some dilution water (or distilled water) to the well. Dissolved oxygen meter and probe. 1. carefully measure the waste water samples into BOD bottles. 3. it may indicate that probe may not have been calibrated properly or the dilution water had been contaminated. but if there is a significant BOD then it will have to be subtracted before the dilution factor is applied in the calculation. All bottles should have liquid in the well around the stopper . Be sure to record the bottle numbers and sample volumes on the following table . Measure the Initial DO of the blank using the calibrated probe and meter. Note: Each group should have 9 BOD bottles in the incubator. In addition. fill one BOD bottle with DILUTION WATER alone. remove bottles and measure the DO. Using the volumes and samples provided by the instructor. After five (or six)days.do not write on the bottles and do not use the numbers on the lids. 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. Measure the Initial DO using a calibrated probe and meter. Place all bottle sets in the 20 o C incubator for five days (or 6 for Tues. groups). There should be negligible BOD in the Dilution water. 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). Materials: Raw sewage and final effluent samples. thereby maintaining a water seal for each bottle. The blank is used to check that the dilution water has negligible BOD. Place plastic caps over all bottles. ie Rinse the filling tube if it has been in the sample. dilution water. 6. preferably using the same meter and probe used to get IDO reading. Calculate the 5-day (or 6-day if unavoidable) BOD. BIOCHEMICAL OXYGEN DEMAND Note: Because of the logistics of organization some groups may have 6 day BOD 5 instead of the usual 5 day. These caps are designed to prevent the evaporation of the liquid around the stopper. graduated cylinders. Be sure to note this in your lab write-up. showing sample calculations. be careful not to contaminate it with sample.III. Because you want to be sure that the dilution water has no BOD.
mg/L DO of seed control after incubation. 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). mg/L = DO of diluted sample after 5 d incubation at 20 oC. DO of diluted sample immediately after preparation.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. = = = = decimal volumetric fraction of sample used DO of seed control before incubation.
Diln. titrimetric method.) Why is your COD result different from the BOD result? 5. Diln. 1 Final Effluent. Diln. 2) 3) 4) Show on a graph the approximate dissolved oxygen sag curves in a river receiving two wastes.25. what would you expect its I) BOD 5 and ii) BOD L to be. 1 Raw Sewage.17. 2 Final Effluent. Calculate theoretical oxygen demand for 300 mg/L of methyl alcohol. One waste has a K value of 0. 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. 6) 7) 8) 16 September 2007 CIVL 407 Lab M anual . the other 0. What interference does the Azide Modification of the Winkler Dissolved Oxygen method overcome and why is it the most common method used for domestic sewage? Why is a blank titrated in the COD test? (Refer to standard methods. open reflux. Diln. if at all possible. If an industrial waste has a COD of 450 mg/L. Both wastes have equal BOD 5 temperature and volume.Summary Raw Sewage.
35oC incubator. OBJECT: -to demonstrate different methods for the determination of the coliform group of organisms and to acquaint you with bacteriological procedures. DO NOT MOUTH PIPETTE. lauryl tryptose broth tubes. etc. Membrane filter method: dilution tubes. not both. LES Endo agar plates. sterile pipets. plates. Heterotrophic plate count: dilution tubes. lab coats. Students will do only sections marked ? MATERIALS: Multiple tube method: dilution tubes.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 dilution water. Quebec colony counter. pencils. membrane filters.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. HACH P/A Broth with MUG. eye protection. 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 . Keep hands. wipe up all spills and disinfect the area of the spill. 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 . Wash hands with soap before leaving. EC tubes and/or Hach A1 tubes. Brilliant Green Lactose Broth tubes. Demonstration only SAMPLE: You will do either a raw sewage sample or a final effluent sample. sterile pipettes.. Demonstration only. away from your mouth. Get raw data from another group and include in your report. agar tubes. T h e re f o re . Do not wear lab coat out of lab . 35 oC incubator. filtering apparatus.leave it here or take away in a plastic bag to launder. Wipe lab benches with disinfectant before and after each lab.
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. Presence/Absence Testing: HACH technique to simultaneously screen for total coliform and E. Gas formation indicates the possible presence of coliform organisms. 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. Streak plates in a manner to insure presence of some discrete colonies. Express as Total coliforms. E.coli. A-1 Medium. Using aseptic technique. EC or A-1 tubes positives: 1. 3. MUG produces a fluorogenic product when hydrolyzed by an enzyme specific to E. It is used as a faster alternative to the LTB/EC procedure for enumerating faecal coliforms in water. ? B. (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. 18 September 2007 CIVL 407 Lab M anual .coli. D. Consider positive EC broths incubated at the elevated temperature (44. Multiple-tube Fermentation or Most Probable Number: Serial dilutions of sample are incubated in lauryl tryptose broth at 35oC for 48 hours (swirl after 24 hrs). as demonstrated. Gas-positive Hach A-1 tubes indicate the presence of faecal coliform.COLI in 4 to 24 hours. Another series of tubes are inoculated from these positive lauryl tryptose tubes. streak one LES Endo agar plate from each tube of BGLB broth showing gas from the highest dilution and the second highest dilution. Calculate the MPN value from the number of positives using the Table 1. 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. 2. is USEPA-accepted for testing non-potable waters.5 oC) as a positive completed test response for both Total and faecal coliform.) Methods: ? Recording BGLB. Heterotrophic Plate Count . Colonies that are pink to dark red with a golden green metallic sheen are counted after incubation 20-22 hrs at 35oC. Demonstration. using a ready-to-use P/A Broth with MUG. The number of colonies counted directly. 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. Hach’s Most Probable Number Method 8368. ? C. Membrane Filter Technique: Serial dilutions of sample are collected on membrane filters and placed on M-Endo media. Results will be provided for the demonstration set. Use Table 2 on page 27.Pour Plate Method: Serial dilutions of samples mixed with agar and incubated for 48 hrs at 35oC. The broth will turn yellow indicating total coliform and fluoresce in the presence of E. Note: these methods will be demonstrated but data must be recorded. Bacterial numbers are calculated from the numbers of positive tubes.7oC for 24 hours (confirmed test for faecal coliforms). Parallel positive BGLB broth cultures with negative EC broth cultures indicate the presence of nonfaecal coliforms. 1.A.
The typical coliform colony has a pink to dark-red colour with a golden green metallic surface sheen. Heterotrophic Plate Count . with a motion that excludes air as it comes in contact with the agar. therefore the results table should specify #coliform colonies/100 ml. Dilution tubes. 2. transfer 1 ml of sample from dilution tube # 6 (most dilute for Raw sewage) or #5 (for Effluent) to an empty (large) Petrie dish. 2. 5. Mount apparatus on suction flask with valve sideways to block suction. Gram staining technique can be also be used. Using sterile forceps. In general. choose appropriate dilutions for counting. place a sterile membrane filter (grid side up) onto the filtration apparatus. Pipette about 10 ml of sterile dilution water over the surface of the filter with the suction valve still closed. If you haven’t already. A high count of non-coliform colonies may interfere with the maximum development of coliforms. The filtration apparatus has been sterilized by autoclaving. 4. then immediately replace the cover. Place the filter on a small LES Endo agar plate carefully. ?C. Inoculating. Incubate plates (inverted) at 35 oC for 24 ± 2 hr.. 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. Prepare one set of six dilution tubes as described in Section A. or colourless and are considered to be non-coliforms. open the valve slowly and allow all the liquid to be pulled through. Do not push down on the filter except on the outside edge and use only sterile forceps . Be sure dishes are labelled with your name and dilutions. coliform bacteria are Gram-negative (pink) rods (sometimes coccus-like). Verification is usually done by a test for lactose fermentation. Membrane Filter Technique -Total Coliform 1. -Note: “typical” colonies count as coliforms in this procedure. 3. Using a sterile pipette. Incubate inverted Petrie dishes for 20-22 hr at 35 oC. red. remove filter using flamesterilized forceps (allow forceps to cool or they will damage the filter).Pour Plate Method 1.2. Pour the full volume of dilution tube # 6 onto the filter.applying gentle pressure. The sheen area may vary in size from a small pinhead to complete coverage of the colony surface. Prepare Gram Stain slides for examination under a microscope. Colonies that lack sheen may be pink. removing the cover just enough to insert pipette tip and dispense sample. After 20-22 hr. 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. Counting. Flame loop between second and third quadrants to improve colony isolation. from one edge. Close valve and rinse the funnel with three aliquots (portions) of sterile dilution water opening and closing valve after each aliquot. Dilution tubes.1. Use dishes with 20 to 80 coliform colonies and not more than 200 of all types. When filtering is complete and the valve is closed. ?B. Atypical coliform colonies can be dark red or nucleated without sheen. Filtering. Verification of both types of colonies is advisable as some typical sheen colonies may be noncoliform and some atypical may be coliform. Repeat for the rest of the dilution tubes to # 2. 19 September 2007 CIVL 407 Lab M anual . white.
Add 100 mls of sample directly to pre-measured medium provided. Presence/Absence Testing . average and multiply by 57 (for plastic Petrie dish). b. a. Do this for a typical and an atypical colony. Slide staining. Wash slide in tap water. Work quickly or the agar will solidify in the tube before you get a chance to pour it. Test temperature on your wrist . Resterilise the loop and remove one colony from the surface of the Les Endo streaked plate (prepared previously). on the bench. marking (on edge of dish so as not to impede counting) and incubating for 48 hr at 35 oC. 20 September 2007 CIVL 407 Lab M anual . Repeat for the rest of the dilutions.too hot for a baby . to mix the sample with the agar. select the appropriate dilution to count. E. Spread the colony in the drop of water so that you get a uniform dispersion over an area about the size of a dime. 2. 2. A hand counter is also available. ?3. c. pour and replace cover. e. Take 5 tubes of melted agar from the incubator.coli). 6. Gently swirl the dish. Drain off excess iodine and wash freely with water and then alcohol-acetone decolourising solution. Flood slide with crystal violet solution and allow to act for 30 seconds. use the plate(s) that will give from 30 to 300 colonies/plate. ?4. Wash off stain with water and then with iodine solution (Lugol’s). Examine for yellow colour (total coliforms) and fluorescence (E. Allow the agar to solidify in the dish before inverting. Use the Quebec colony counter to aid in counting and a grease pencil to mark off colonies counted. 5.d e m o n s tra tio n -s ta rte d a h e a d o f la b 1. Repeat with dilutions 5 to 2 (for Raw) and 4-1 (for Effluent). Flood the slide with fresh alcohol-acetone solution to act for 30 seconds. Air dry the slide high above the Bunsen burner and fix by passing slide briefly through a flame. Compute bacterial density and report as “colony-forming units” (CFU) per ml. refer to Standard Methods for more instruction. Multiply the number by 5 to compute estimated colonies per plate (66 cm 2 ). d. 4. Microscope Slides: 1. Incubate at 35 oC for 24-48 hours. count colonies in 13 squares (of the colony counter) of the most dilute plate having representative colony distribution. Instead. Allow iodine to act for 30 seconds. The aim of using several dilutions is to have at least one dilution giving colony counts between these limits. Slide the cover of a Petrie dish back just far enough to pour the agar. If more than 10/cm 2. if fewer than 10/cm 2.3. After 48 hr incubation. f. place 1 loopful of sterile dilution water in the centre of a slide. Counting. Record results of previously prepared test. Let them cool down from the 60C that they are kept at but not enough to solidify. Do not report as “too numerous to count” if all plates exceed 300 colonies. Ideally. Label the slide using a grease pencil (T or A). count four representative squares. If necessary. Preferably select seven consecutive squares horizontally and six consecutive squares vertically.too hot for a bacterium. D. Slide preparation: With a flame sterilized inoculating loop.
do not turn knobs except the positioning ones. coliform are Gram-negative (pink). BGLB and EC and AI tubes . 21 September 2007 CIVL 407 Lab M anual . .Examine prepared slides. b.g. sometimes coccus-like and 0. blot and then dry in air high above the Bunsen burner (so as not to burn away your efforts). Microscopic examination. metallic. odour. c. Day 2.Membrane filter prepared dilutions. rough.Prepare dilutions for Heterotrophic Plate Count and Membrane Filter technique. cocci or rods. Note: in the staining process: before decolourisation all organisms appear Gram positive (blue). Day 3. nonsporing. colour. Wash in water. ?3. usually motile. or in short chains.3 µm in size. and on the slide: typical or atypical. length to width ratio. h.Come in and count Heterotrophic Plate colonies after 48 hours. in pairs.Come in and count colonies on LES Endo Membrane Filter dishes.Record positives in all Multiple Tube Fermentation inoculations: LTB. After application of the counterstain. plump rods. Description of bacteria: Describe what you see on the plates: colonial morphology of each type of colony seen ie surface (shiny. Gram negative organisms are visualized (pink). smooth. pairs chains). . concave). . Examine the prepared slides . a.5 . . place on LES Endo agar dishes and incubate. . .Prepare and incubate Heterotrophic (Pour) Plates using dilutions above. after decolourisation Gram negative organisms are no longer visible. gram positive or negative. short. dull. Apply fuchsin or safranin (counter)stain for 30 seconds. In general.Record Presence/absence testing results of Hach Prepared Media bottles. Plates ready on a weekend will be put in cold room until Monday. Schedule Summary Day 1. . Cells occur singly. cell groupings (single.counts will be provided on the board. transparency. .
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 .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. Multiply the MPN index by the dilution factor from the series used when dilutions other than 1/1.pour plate method.. 1 2 3 4 5 6 Heterotrophic Plate Count . 1/ 10 and 1/100 are used.
the total tubes planted. you can simultaneously detect total coliforms and E.01 ml 2/5 2/5 0/5 0. To select the three dilutions to be used in determining the MPN index.coli because it produces a fluorogenic product when hydrolyzed by glucuronidase. MF results. Use the results at these three volumes in computing the MPN index. In the examples given below. Both media meet USEPA guidelines for testing of total coliforms in drinking water.coli will fluoresce as early as 4-24 hours. 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. 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. With MUG reagent added to P/A Broth. If zero total coliform is the maximum contamination goal. use the results from only three of these in computing the MPN.Most Probably Number (MPN) When more than three dilutions are used in a decimal series of dilutions. choose the highest dilution that gives positive results in all five portions tested (no lower dilution giving any negative results) and the two next succeeding higher dilutions. it is not necessary to enumerate.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. Do they agree? Why/why not? -compare MPN/MF with Heterotrophic Plate Count. that in the denominator.1 ml 5/5 4/5 1/5 0. E. They are in concentrated form and merely require the addition of 100 ml of sample (or diluted sample) to the media and incubation.Discussion: -sources of error? -compare MPN. MUG (4methylumbelliferyl-$-D-glucuronide) can be used to detect E. the significant dilution results are in boldface.coli. Estimation of Bacterial Density . The number in the numerator represents positive tubes. A long-wave UV light is required to detect fluorescence. an enzyme specific to E.
0 mL. 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.MPN Index and 95% Confidence Limits for Various Combinations of Positive Results when 5 Tubes are used per Dilution (10 mL. Of Positives MPN Index/ 100 ml 22 26 27 33 34 23 30 40 30 50 60 50 70 90 80 110 140 170 130 170 220 280 350 240 300 500 900 1600 95% Confidence Limits Upper Lower <2 2 2 4 2 4 4 6 6 4 7 9 9 12 8 11 11 14 14 17 13 17 17 21 26 1 10 13 11 15 15 18 18 17 20 24 25 29 24 29 29 35 35 40 38 45 46 55 63 4-2-0 4-2-1 4-3-0 4-3-1 4-4-0 5-0-0 5-0-1 5-0-2 5-1-0 5-1-1 5-1-2 5-2-0 5-2-1 5-2-2 5-3-0 5-3-1 5-3-2 5-3-3 5-4-0 5-4-1 5-4-2 5-4-3 5-4-4 5-5-0 5-5-1 5-5-2 5-5-3 5-5-4 5-5-5 9 12 12 15 16 9 10 20 10 20 30 20 30 40 30 40 60 80 50 70 100 120 160 100 100 200 300 600 -— 56 65 67 77 80 86 110 140 120 150 180 170 210 250 250 300 360 410 390 480 580 690 820 940 1300 2000 2900 5300 ---- $1600 24 September 2007 CIVL 407 Lab M anual . 1.Table 1 .
MPN Table 2 25 September 2007 CIVL 407 Lab M anual .Hach .
D. -The Use of Indicator Organisms to Assess Public Water Safety .com/ ¸Information Central/Learning Library/Microbiological Testing ASTM: (STP635) D.org/microbelibrary/files/ccImages/Articleimages/keen/Gram stainkeen.htm Lab 5 Questions: 1) Compare the advantages and disadvantages of the multiple tube fermentation and membrane filter techniques.microbelibrary.http://www. What are the important pathogens transmitted by water? How is quality control achieved in microbiological analysis.hach. Mara: -Bacterial Indicators/ Health Hazards Associated with Water -Bacteriology for Sanitary Engineers Microbiology 321: -Manual of Microbiological Techniques Website: http://www. 2) 3) 26 September 2007 CIVL 407 Lab M anual .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.
magnetic stirrer. NaHCO 3. indicator. sample.5 mg/L). chloride specific ion electrode. beakers. pH meter with double-junction electrode and silver billet electrode. burettes. HNO 3. goggles or glasses and lab coats. Use personal protective equipment: gloves. Mercuric thiocyanate is very toxic. 27 September 2007 CIVL 407 Lab M anual .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. conc. known addition and calibration techniques and colourimetric and potentiometric titrations. Note and consider carefully: Different methods of analysis have different optimum ranges. to illustrate the differences in operation and accuracy between instrumental methods and wet chemical methods. If chemicals are spilled alert instructors. MATERIALS: -millivolt meter. buffer. semi-log graph paper. 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. volumetric pipettes. OBJECT: -to acquaint you with the use of specific ion electrodes. Hint: optimum ranges will coincide with the standards provided or will be indicated within the pertinent section. graduated cylinders. standards. standard AgNO 3 solution. That means you will probably have to dilute the sample by the most accurate means available. 125 ml Erlenmeyer flasks. standard Hg(NO 3)2. spectrophotometer. reference electrode (double junction). indicator-acidifier reagent. beakers. standard chloride solution (0. immediately.
place the lowest standard on the stirrer and lower the electrodes into it.SPECIFIC ION ELECTRODE Part One: Preparation and reading standards and samples 1. Using semi-log paper or software equivalent. stir and obtain a reading for it. As probe tends to drift for some time it may be expedient to select a “standard time to read” (i. 2 minutes) and use it for all standards and samples. 4. labelled beakers. Measure 100 mls of sample. 2.E 1 volume of sample volume of stock solution added mg Cl. CHLORIDE . Turn on stirrer and while stirring record the millivolt (mv) reading when the reading appears to be stable. mg Cl.in sample = mg/L Cl. Calculate the chloride concentration. plot the millivolt readings (linear axis) against the concentration of the standards (log axis) to produce a calibration curve. 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 ).= ____________________ 28 September 2007 CIVL 407 Lab M anual . Unless already done by a previous group (values written on the board). where: E1 = E2 = E = Vo = Vs = A = S = potential of sample (mV) potential of sample plus stock addition potential change = E 2 . Standards of 1000. 3. Repeat for rest of standards. pour out 100 mls of each standard into separate.I. Add 2 mls of IONIC STRENGTH ADJUSTMENT (ISA) buffer to each using the plastic dropper (filled to the mark). rinsing and blotting dry the electrodes between each.e. Determine the concentration of chloride in the sample. 100 and 10 ppm (mg/L) have been prepared for you and are at the meter. Unless already done by a previous group.added to the sample "slope" = change in potential over 10 fold change in concentration of standard (use data from Step 2). 5. added 2 mls of ISA. 6.
2. Pour 100 ml of sample (or a portion diluted such that the concentration is less than 100 mg/L) into a 250 ml beaker. discard and refill at least twice to rinse cuvette. 4. Wait two or three minutes and then add the reagent to the first (lowest) standard. zero instrument on distilled water and obtain an absorbance value for the blank. it should be rerun from the start after diluting. (Light green indicates a pH of less than 2.0. (No further adjustment is required after the blank or distilled water has been set to zero. MERCURIC NITRATE METHOD FOR CHLORIDE DETERMINATION The principle of this method is as follows: Cl. using the dispenser provided.. insert in the spectrometer and record the absorbance displayed on the meter. 3. the 1 ml of the indicator-acidifier reagent will adjust the pH to 2. (Alternatively. 2.BY COLOURIMETRIC METHOD The principle of this method is based on the following formula: 2Cl. a pH of greater than 3. and Hg ++ (excess) + DPC º violet colour 3. CHLORIDE . fill. If sample is above the highest standard. which then must be subtracted from the standards and sample. 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. Near the end-point. Add 1 ml (use pipette to measure accurately) of indicator-acidifier reagent to sample. 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 . after a few more minutes. III.they can’t all be at 10 minutes at once. After all Cl.) Mix thoroughly by gently swirling the flask. the excess mercuric ions combine with diphenylcarbazone (DPC) to produce a violet colour (the end-point). Using a 25 ml graduated cylinder. (Use extreme caution as this is a corrosive & toxic chemical.8.) Every two or three minutes thereafter you will rinse the cuvette with the next standard or sample. Titrate with 0.II. pure blue. The colour of the solution should be green-blue at this point.º HgCl2 1. the three standards (2. Determine the chloride concentration in the sample from the calibration curve. Marking the time (T=0).5 ± 0. 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).0 and 8. 6. [Note: this delay between additions is to allow you time to measure absorbance of each at the 10 minute time required .) Prepare a calibration curve by plotting the absorbance readings versus the concentration of chloride in the standards.014N Hg(NO 3) 2 to a definite purple end-point.ions have been bound.0 ppm). For most samples. measure 25 mls of “zero” standard or “blank” (halide-free distilled water). add 5 ml of the “combined reagent” (containing ferric alum and mercuric thiocyanate solutions) to the blank first.ions in the sample combine with added mercuric ions forming a soluble but virtually undissociate-able complex.] After 10 minutes has passed (since the combined reagent was added to the blank). Hg ++ + 2Cl.0.+ Fe+++ º Fe(SCN) ++ (Reddish brown) 1. 5. use a plastic dropper to transfer blank into a spectrophotometer cuvette (do not fill more than 3/4 ).+ Hg(SCN)2 º HgCl2 + 2SCN SCN . 4. and the sample (diluted if necessary) into separate and labelled 125 ml Erlenmeyer flasks (5 in total).1.
0 ml (use volumetric pipette) of standard chloride solution in a 250 ml beaker. At the start.0 ml of concentrated HNO 3 (use plastic dropper). 5. 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).2 ml or drop-wise) should be added. Rotate the work within the group so that different pairs are doing the titrating and recording of numbers. Continue the titration about 5 ml past the end-point.if one is necessary. Start the stirrer and set the millivolt reading to zero or record initial mv reading [Note: Some meters can not be set to zero]. smaller increments (0. until past the endpoint when larger increments can be used again (ª mV/ml decreases). 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 . dilute to about 100 ml with distilled water. Immerse stir bar. The titre from this step must be subtracted from the titre for the sample.4. with standard AgNO 3. This way each station will have data for each part. Part one: Standard 1. drops of the purple end-point. Calculate the chloride concentration: 5. CHLORIDE BY POTENTIOMETRIC TITRATION Note: For this part form two groups comprised of 1 member from each station. Plot a differential titration curve to determine the exact end-point. where: A = ml titrant for sample B = ml titrant for blank N = normality of titrant IV. Place 10.. 3. Take care that stirrer is not hitting the electrodes. The end-point occurs at the greatest mV change per unit addition of AgNO 3. large increments (1-2 mls) may be added. Don’t forget to add the indicator. in increments. as the end-point of the reaction is approached (ª mV/ml increases). place beaker on stir plate and lower electrodes into the solution. Begin titrating. recording the volume and millivolt reading for each increment. then. and add 2..1 to 0. 2. Nitric acid is in the sink and is very corrosive. A reference set of data will be provided for comparison. Calculate the Normality of the AgNO 3 using the following equation: 30 September 2007 CIVL 407 Lab M anual . 4. 6.
Measure 100 ml of sample or portion made up to 100 ml.Part Two: Sample 1. b. 2. which are merely first and second derivatives. Millivolt reading versus ml of titrant. Millivolts per ml versus ml of titrant. Add 2 ml of HNO 3 and proceed as described in Part One. c. in this case. In plotting b and c. 4. It is not. Millivolts per ml per ml versus ml of titrant. be careful to use the average value of ml of titrant on the abscissa. Calculate the chloride concentration in the sample using the following equation: 3.) Plot three curves for this titration: a. if dilution is necessary into a 250 ml beaker (use a graduated cylinder to measure). (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. 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 .
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 . 32 September 2007 CIVL 407 Lab M anual .
causing electrode malfunction. # Mercury must be absent from samples. # Colour in the sample may interfere in the absorbance measurement. timeconsuming process requiring a sound knowledge of chemistry and extensive experience working with environmental samples. # High levels of ions which form very insoluble salts of silver may deposit a layer of salt on the electrode membrane.INTERFERENCES IN CHLORIDE DETERMINATIONS POTENTIOMETRIC METHOD # Iodide. PRETREATMENT TECHNIQUES # There are no "standard" pretreatment techniques. # Environmental samples usually require complex pre-treatment. hydrazine. 33 September 2007 CIVL 407 Lab M anual . # Colour may obscure or interfere with end-point determination. # In practice. known addition cannot correct for any ions that test as chloride ion. In the above tests. ferric ion. COLOURIMETRIC METHOD # Bromide. chromate. and sulfite ions interfere when concentration greater than 10 mg/L. in itself. 3. Each sample type requires development of an appropriate method which. specific ion electrodes are rarely usable in environmental samples. fluoride and bromide titrate as chloride. cyanide. dichromate. iodide. # Other interferences: ferricyanide. It can only correct for substances that enhance or suppress the test response proportional to the amount of chloride ion present. can you see any advantage in suing the first or second derivative curve? Discuss. and nitrite interfere. Also. 2. # Chromate. requires knowledge of the compounds in the sample which are likely to interfere. MERCURIC NITRATE TITRATION METHOD # Iodide. Lab 3 Questions 1. SPECIFIC ION ELECTRODE # All halides interfere (the electrode is a halide electrode). fluoride. # Sample pH may need adjustment beyond the capacity of indicator-acidifier reagent. # Environmental samples usually require pretreatment. From your chloride results. This is a complex. KNOWN (STANDARD) ADDITION TECHNIQUE # The known addition techniques will only correct for certain types of interferences. # Pretreatment of environmental samples usually required. 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. ferric. thiosulphate. fluoride and bromide titrate as chloride. strongly reducing solutions may form a surface layer of silver.3 mg/L? Outline the advantages and disadvantages of all the methods used in this lab to measure chloride concentration.
Rinse a burette and fill it with this stock chlorine solution. (concentrated) glacial acetic acid. standard ferrous ammonium sulphate (FAS) solution. phosphate buffer solution. 100 ml graduated cylinder.025 N sodium thiosulphate.N-diethyl-p-phenylenediamine (DPD) indicator solution. N. I. Wipe up all liquid spills immediately. 2. chlorine demand and break-point chlorination. clean up all spills with a brush into a container and wash crystals down the drain. allow to mix again.total chlorine residual) 1. Mix well (on stir plate with stir bar). Take a 250 ml Erlenmeyer flask and add about 1 gram of potassium iodide. Bleach/chlorine solutions are strong oxidants. 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. about 5 ml of glacial acetic acid (use a graduated cylinder). Titrate rapidly with the stock chlorine solution (in burette) until the appearance of a constant 34 September 2007 CIVL 407 Lab M anual . pages 4-56 to 4-57 or older/newer editions. 1995. burettes and stands. OBJECT: -to acquaint you with disinfection of water supplies and to demonstrate techniques for the determination of chlorine residuals. and exactly 10 ml of 0. Personal protective equipment required at all times when working in the lab are: eye protection. 3. REFERENCES: Standard Methods 19th ed. potassium iodide (KI) crystals. to illustrate the concepts of free and combined chlorine residuals. 0. 250 ml volumetric flasks. gloves and lab coats. about 50 ml of chlorine-free water (distilled water will do). pipettes. PREPARATION OF STOCK CHLORINE SOLUTION (Iodometric method .025 N sodium thiosulphate solution (use a volumetric pipette).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. 250 ml Erlenmeyer flasks.. add 1 ml of starch solution. chlorine free water. When weighing chemicals at the balance. bleach or hypochlorite solution. Make up to the 250 ml and invert flask several times to mix. Materials: -600 ml beakers. starch indicator.
Let stand 2 minutes more. mix to dissolve. if colour drifts back (indicating an incomplete reaction). d) Alternatively: (Not to be done in this lab. please refer to Standard Methods. For dichloramine concentrations greater than 1 mg/L. 3.COORDINATE! IIa. Determination of Free and Combined Chlorine by the DPD Method 1. let stand for two minutes and titrate until the red colour (if present) is discharged. Record the burette reading (Reading C). Continue titrating until red colour (if any) is discharged once again (Reading B). Work in groups of three or four for this part. calculate the chlorine concentration in the stock solution (See Data and Results section for formula). It is likely that the highest dose will require dilution. Place 5 ml of phosphate buffer solution and 5 ml of DPD solution in a 250 ml conical (Erlenmeyer) flask and mix. b) Monochloramine: Add two drops of KI solution (to the same sample) and mix. etc. Go to c. 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.). IIb. (*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. Add 100 ml of sample or diluted* sample using a 100 ml graduated cylinder and mix again. Record the burette reading (mls) = Reading A. Method 4500-Cl F. 4. 2.A Monochloramine= B-A Dichloramine= C-B For interferences. pp 4-43. These 6 flasks can be prepared at once to make them ready for the next step. as there is lots to do . From the amount of chlorine solution used.purplish-blue colour.) Free and combined chlorine or total chlorine: To obtain total chlorine in one reading. Let stir for 2 min and then continue titrating until red colour (if any) is discharged (Reading C). let stand 2 min more if colour drifts back indicating incomplete reaction. refilling burette. titrate to colourless again. Total chlorine (Free plus Combined) = Reading C Free residual chlorine = Reading A Combined residual chlorine (dichloramine plus monochloramine) = Reading C . After 15 minutes of contact time. a) Free chlorine: titrate rapidly with standard FAS (Ferrous ammonium sulfate) titrant until the red colour is discharged. 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). 35 September 2007 CIVL 407 Lab M anual . add full amount of KI at the start (ie 2 drops KI solution and 1 g of KI crystals) to a fresh sample. 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). 18th edition. 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).Breakpoint Chlorination using Free and Combined Chlorine Residuals Values 1. 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. c) Dichloramine: Add about 1 g KI (to the same sample) and mix to dissolve.
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. b. what percentage of bacteria would be killed in 10 minutes at a chlorine residual of 0. 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. Under what conditions is the practice of break point chlorination necessary? 4. c. Determine the concentration of total. The rate of kill of bacteria by chlorination follows first-order reaction kinetics. FAS titrant concentration is 1 ml = 100 µg Cl as Cl2 a. Make a plot of the three residual chlorine components against the applied chlorine dosage. Make a separate graph for the 15 minute and 1 hour contact time. d. If this is true. how many coliforms would remain after a chlorine contact time of 10 minutes? 20 minutes? 36 September 2007 CIVL 407 Lab M anual . II. 250) then convert mg/L to g/100 mls = %.DATA AND RESULTS I.7 x 10-8 at 25 o C % HOCl = 27 What is the pH of the solution? 3. Why is it important to determine chlorine residuals in domestic water supplies? 2. If a sewage sample contains 10 x 10 6 coliforms/100 ml.5 mg/L if 50% are killed in 1½ minutes at this concentration? b.e. Guaranteed analysis is normally printed on the container and is usually 5-6%. a. Discuss your results as a function of the residual forms of chlorine present at different chlorine dosages and at different contact times. Given: HOCl W H + + OClK eqv = 2. free and combined residual chlorine for each applied chlorine dose.
(mls) (Stock=_____mg/L Cl as Cl2) Applied Cl dosage (mg/L) Volume of FAS to endpoint A Volume of FAS to endpoint B (includes A) Volume of FAS to endpoint C (includes A & B) Free chlorine (mg/L) (A) Monochloramine (mg/L) (B-A) Dichloramine (mg/L) (C-B) Combined residual chlorine (mg/L) (C-A) Total chlorine (mg/L) © Time of Cl addition Time of Residual measurement 1 2 3 4 5 6 37 September 2007 CIVL 407 Lab M anual . of Cl Soln.15 MINUTE CONTACT TIME BEAKER NUMBER Vol.
of FAS to endpoint C (mls) Free chlorine (mg/L) (A) Monochloramine (mg/L) (B-A) Dichloramine (mg/L) (C-B) Combined residual chlorine (mg/L) (C-A) Total chlorine (mg/L) © Time of Cl addition Time of residual measurement 1 2 3 4 5 6 (includes A+B) 38 September 2007 CIVL 407 Lab M anual . of Cl Soln. of FAS to endpoint B (mls) (includes A) 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.
Rinse probe when you are finished and leave immersed in pH 7 buffer for storage. 3. Probes. then use buffers 7 and 10. EDTA. 1 N NaOH. to acquaint you with the use of pH metres. I. Ac id ity . p H. place a beaker containing the sample and stir-bar on the stir-plate. bromphenol blue. pH test paper.(Do not allow the stir bar to hit the probe. 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. flasks. Always wear personal protective equipment. metacresol purple. phenolphthalein. magnetic stirrer. rinse & dry probe. turn on stirrer and obtain a stable reading. beakers. standard acid. Use care when using lab equipment. metres and calibrated glassware are available in limited quantities and are very expensive to replace. the two buffers that are chosen for calibration will bracket your sample ie if sample is less than 7. Normally. pH test paper and other water quality assessment equipment. hardness indicators. 4. if above 7. bromcresol green. standard base. Be sure that buffers are being stirred when you perform the calibration and that when you change buffers you thoroughly rinse the probe into a waste beaker and blot dry so that you do not contaminate other buffers or your samples. Calibrate the pH meter by following the procedure outlined on the instruction sheet beside the meter unless told otherwise. Hard n e s s . DETERMINATION OF pH 1. (Take care not to contaminate paper in case. After calibration is complete. turbidimeters. lower probe. MATERIALS: -pH meter.) 2. T u rb id ity WARNING Please use caution when handling all chemicals. graduated cylinders. water samples. particularly those labelled with specific hazards. buffer solution.) Record value in following table. Co lo u r. OBJECT: -to familiarize you with the most common water treatment tests.Lab o rato ry 6: Alkalin ity . then use buffers 4 and 7 to calibrate. 39 September 2007 CIVL 407 Lab M anual . colour comparator. burettes and stand.
If a ppt does form within the 5 minutes. TOTAL HARDNESS DETERMINATION .5). titrate until just colourless. Rinse and fill a burette with standard EDTA solution and record the initial burette reading. 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. Add a full dropper of bromphenol blue indicator and titrate until the colour changes from yellow to blue ("MO" Acidity). Add the last few drops at 3-5 second intervals so that you do not "overshoot" the end-point. Add stir-bar. add 90% of the titrant to the sample before adjusting the pH with buffer.EDTA TITRIMETRIC METHOD 1. Measure 100 ml of sample (with minimum agitation or exposure to air) using a graduated cylinder and transfer carefully to an Erlenmeyer flask. Complete the titration within 5 minutes to minimize the tendency toward CaCO 3 precipitation. (If the solution is already blue after indicator.. Titrate to the first uniform pink colour (colour stays). Record burette reading (P). III. Apply dilution factor to obtain final result. Record the volume of titrant required in the following table. Measure 100 ml of water sample into a clean. 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. Rinse and fill a labelled burette with standard acid solution. add 1 drop of 0. 2. (BG or MO) 6.. 3. 2. Record the burette reading in the following table.?) 4.II. Pour into a 250 ml Erlenmeyer flask. 40 September 2007 CIVL 407 Lab M anual . IV. DETERMINATION OF ACIDITY 1. The buffer elevates the pH to 10. until the reddish tinge disappears from the solution and a pure blue remains. DETERMINATION OF ALKALINITY 1. it may be necessary to repeat the titration with the sample diluted 1 to 1 (again) with distilled water.. Add a full dropper of phenolphthalein (P) indicator and if the solution stays pink or red. Measure out a new sample and add 1 drop of sodium thiosulphate and a full dropper of phenolphthalein. 3..1 N sodium thiosulphate to the sample. In order to remove any free residual chlorine (which interferes with the indicator colour response).think what that means!) 4. 2. Add a stir-bar. 5. A pH much above 10 promotes CaCO 3 precipitate. Alternatively. Add 1 drop of sodium thiosulphate solution. 4. sample-rinsed graduated cylinder and transfer to an Erlenmeyer flask. while stirring. Titrate slowly with EDTA. if the approximate hardness is known (by unsuccessful attempts). White paper placed under the flask will facilitate this determination. 3. If sample is highly alkaline (titration goes over one burette full) dilute the sample and titrate again. Rinse and fill another burette with standard base solution and label it. (If solution is clear after addition of P.. Measure out 25 mls of sample using a graduated cylinder and dilute to 50 ml with distilled water.
HACH Spectrophotometer. 2. Use the technique outlined in Part A. 3.. Values are reported as APHA Colour Units (eqv. to determine the apparent colour (measures the influence of turbidity on the reading). Filter about 20 mls of sample using a filter funnel and stand and the pre-fluted filter paper provided 2. Press: I/O. CALCIUM HARDNESS DETERMINATION . Cap the cell. V. with continuous stirring to a pure blue end-point. 4. Add a stir-bar and stir to mix.TRUE AND APPARENT PART A: TRUE COLOUR . Close the lid. 4. 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. Fill a sample cell to the line (about 15 mL).BY HACH 2100P Turbidity Meter. The instrument may suggest diluting if over range. Pour the supernatant into one of the cells (to the etched mark) and fill another with distilled water. 2. Record the final burette reading in the following table. Select automatic range by pressing the RANGE key. The instrument will turn on but will turn off automatically after a short time so you should be ready. except on an unfiltered sample. 3.EDTA TITRIMETRIC METHOD 1. The display will show AUTO RNG when the instrument is in automatic range.0 ml of 1 N NaOH solution (use 3 droppers full). Place the cell containing water into the spectrophotometer and press Zero. COLOUR . lint-free cloth to remove water spots and fingerprints. Record all colour data in the following table. Demonstration of Jackson Candle and Hellige PART A: Hach Turbidimeter 1. 4. Measure out 50 ml of sample into an Erlenmeyer flask and add 2. VII. or a volume sufficient to produce a pH of 13-14 (designed to precipitate the magnesium as a hydroxide). TURBIDITY .. Refill the burette with EDTA titrant and note the initial reading. taking care to handle the sample cell by the top. Record the final burette reading in the following table. 5. 2. Add one aliquot of the Calcium Hardness Indicator and titrate with EDTA slowly. The display will show SIG AVG when the instrument is using signal averaging. 3. Use signal average mode if 41 September 2007 CIVL 407 Lab M anual . The reading might be higher so if you had to dilute in Part A dilute for Part B. to mg/L platinum as chloroplatinate) PART B: APPARENT COLOUR 1. VI. Wipe the cell with a soft.Enter the stored program number for true colour=120 1. Place the cell containing sample in it and press Read.. .5. Select signal averaging mode by pressing the SIGNAL AVERAGE key.
PART C: Jackson Candle Turbidity .. that the filter selector shows "none" and that bulb B is in use. DO NOT PLACE AN EMPTY TUBE IN THE TUBE HOLDER WITH THE CANDLE LIT . Read the scale on the dial. Wipe the outside of the tube to dry it and place it in the turbidimeter (in the circular groove of the mirror unit). 42 September 2007 CIVL 407 Lab M anual .NTU.. Carefully fill the 20 mm viewing depth tube to the mark with sample. 16th edition. then the turbidity in NTU.T. T = Total alkalinity.demonstration only. Note that there are several different scales on the side of the tube. record the number and refer to the appropriate graph to determine the turbidity as mg/L SiO 2.. Pour sample into the tube in small increments until you can no longer distinguish the shape of the candle flame. If you take too long. (This allows you to make a more gradual and accurate approach to the end-point. Close the door and switch on the light.) 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.Demonstration only 1..U.the sample causes a noisy signal (display changes constantly). 2. 3. Try to be quick and not burn the candles more than a few minutes at a time. Record the turbidity after the lamp symbol turns off. the turbidity may start to settle and condense on the bottom of the tube. Be sure that the calibrated tubes are clean before adding samples to the tubes. Note that the rectangular door mirror is closed. Press: READ The display will show .s. Lower the plunger into the liquid making sure no bubbles are trapped under the plunger and the top is dry. page 272-273. PART B: Hellige Turbidimeter . 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.adding liquid to a hot tube will cause it to shatter. Take the reading where the dark central part first merges with the surrounding field.. obscuring vision. See Standard Methods. 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. 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. 2. 3. 1. 6. Record value in following table as J.
O.O. Alkalinity titre (mls) Net "M.O.Sample volume "M." Acidity end-point reading (mls)__________ TOTAL Hardness Sample volume (mls)__________ Calcium hardness as mg CaCO 3/L __________ 43 September 2007 CIVL 407 Lab M anual ." Alkalinity end-point (mls) Initial burette reading Net P." Alkalinity titre (mls) ACIDITY.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.Initial pH by test paper Initial pH by pH meter ALKALINITY.Sample volume Initial burette reading P.O. Acidity end-point reading (mls) Initial burette reading Net "M. Alkalinity end-point reading (mls) __________ "M.
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 .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 1.00 ml EDTA titrant at the calcium indicator endpoint = 1.
Hach .T.ion (mg/L) CO 3 -2 Alkalinity as CO 3 -2 ion (mg/L) HCO 3.ion (mg/L) "M.Alkalinity as HCO 3 .Alkalinity as CaCO 3 (mg/L) CO 3 -2 Alkalinity as CaCO 3 (mg/L) HCO 3." Alkalinity as CaCO 3 (mg/L) OH . Alkalinity as CaCO 3 (mg/L) "M.O.Alkalinity as CaCO 3 (mg/L) OH .RESULTS (sample) P.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 .O.APHA Colour Units: True Apparent Turbidity .U.Units= N. ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 45 September 2007 CIVL 407 Lab M anual .
7 x 10 -11 and K w = 10 -14. calculate the carbonate alkalinity in mg/L as CaCO 3 for the water sample analyzed during this lab. 46 September 2007 CIVL 407 Lab M anual . 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. Is this water suitable for a domestic water supply? Why? 2. given K 2 = 4. A water has the following analysis: Na + K+ Ca +2 15 mg/L 33 mg/L 12 mg/L ClSO 4-2 HCO 3 70 mg/L Mg+2 18 mg/L 42 mg/L 8 mg/L What is the carbonate. What errors can occur in the calcium hardness determination? Why? 3. From equations (17-12) and (17-13) in Sawyer and McCarty.Lab 6 Questions 1. Does this agree with the results from Part II of this lab? Explain. What sanitary significance does colour have? 4. non-carbonate and total hardness of this water in mg/L as CaCO 3 ? in meq/L? Is the analysis complete? Why? 6.
and all of the materials from Lab. The optimum alum dosage for coagulation of the sample will then be chosen.for the next session (b). This is a two-step experiment. You should have with you the part of Exercise 7 that gives instructions for the various tests. alum solution (Al2 (SO 4 )3 @18H 2 O). soda ash solution (Na 2CO 3) and lime (Ca(OH)2) or sodium hydroxide (NaOH). MATERIALS: -Jar Test Apparatus. 47 September 2007 CIVL 407 Lab M anual . 7 (except Jackson Candle and Hellige Turbidimeter). 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. No. The first step will be to analyse the raw water sample and the supernate from a number of alum dosages (to be announced in the class) using the techniques learned in Lab. Several lime and soda ash dosages will also be selected for determining the efficiency of water softening. Please try to coordinate your work with the rest of the class. In the second lab you will use the previously determined dosages to coagulate and soften the sample and then re-analyse the resultant water. to be conducted over two lab periods. OBJECT: -to illustrate the principles of coagulation and water softening. 1000 ml beakers.
hardness (total and calcium). and clarity of supernatant liquid. clear. Carefully decant into a graduated cylinder 800 mls of the supernatant liquid and pour into fresh beakers. 2. 2. After the floc has settled (allow 15 min). Determine which dosages were most appropriate for this sample. not a beaker ) and transfer to 1000 ml beakers. Analyze the supernates for pH. Part B: WATER SOFTENING PROCEDURE (to be done the following week) again work as teams of 2 or 3. Stop stirrer and allow floc to settle. Analyze these supernates plus a sample of raw water for pH. position them under stirrers. Lift paddles out of beakers and secure. Reduce the stirring rate to about 20 rpm for 10 minutes. moderate. Measure out six 1000 ml aliquots of sample (using graduated cylinder to measure. times of appearance and descriptions and record. 3. 6. alkalinity. Stir at 100 rpm for 1 minute. Discard the floc and clean beakers in preparation for a second decanting in Step 4. true colour and turbidity (Hach). 6. medium. hardness (total and calcium). Pour six 1000 ml aliquots of sample into 1000 ml beakers and place on the jar test stirrer apparatus. Stop stirrer. Add the assigned alum dosages as quickly as possible and then start the stirrer. A pH meter could be used for the alkalinity and acidity determinations. if time permits. but because of equipment limitations. clarity of supernatant liquid (very clear. 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. slow). alkalinity. Stir at 100 rpm for 1 minute and at 20 rpm for 10 minutes. use indicators as specified in Lab 6. In the lecture. hazy. and fill in the table on the board. 3. floc volume. drop of 0. Add the designated lime (or NaOH) and soda ash additions as quickly as possible to the decanted samples. Observe and make notes describing floc formation and note the time of appearance. COAGULATION USING ALUM . decant the supernatant liquid carefully into another set of beakers (try not to stir up what has settled). Add the appropriate alum dosage and immediately start stirring. Make sure it is centred. decant the supernatant liquid carefully into a clean set of beaker (try not to stir up what has settled) and discard the floc. Brief Methods Summary for Analyses Alkalinity: @ 100 ml of sample. acidity. 4. 5. and settling characteristics of the floc (rapid. 7. 4. turbidity and colour. Place beakers on the jar tester apparatus. acidity.work in 2 or 3 teams to assess a total of 6 dosages for each team. small. Record observations on settling rate. 5. Make relative estimates of floc sizes. you will discuss the data and select the appropriate alum dosage to be used by all groups in the water softening step next week.Part A:. 1.1 N sodium thiosulphate 48 September 2007 CIVL 407 Lab M anual . cloudy). After the flocs have settled (allow 15 min). large).5 to 1 cm off the bottom of the beaker. Record relative floc sizes (pin-point. 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). You may need to use blocks to position the paddle 0. Complete the coagulation summary sheet during the lab period.. if available. (NB dosage now based on 800 ml sample size). 1. Stir at 100 rpm for 1 minute.
Compare removal of hardness components under the different doses .pH 3. show by calculation the theoretical amount of lime and soda ash required for excess lime/soda ash softening. Analyze the response of colour and turbidity removal as a function of alum dose.use graphical presentation. 2.000/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 .7 ("methyl orange" acidity) and with phenolphthalein (using a fresh sample) to pink .3 ("phenolphthalein" or total acidity) @ calculation .pH 8. 2.3 and with bromcresol green (continuing with the same sample) to yellow .1 N sodium thiosulphate @ titrate with 0. Based on your chemical analysis of the raw water.acidity (mg/L CaCO 3)= mls titre x N base x 50.alkalinity (mg/L CaCO 3)= mls titre x N acid x 50. @ calculation: hardness (mg/L CaCO 3 )= mls titre x 1000/mls sample Calcium Hardness: @ 50 ml sample @ add 1 ml 1 N NaOH (or to pH 13) and Ca Hardness Indicator (Hydroxy Naphthol Blue) @ titrate quickly with EDTA to blue end-point (no pink/purple tinge . 3. 4.02 N base with bromphenol blue to blue .02 N acid with phenolphthalein to colourless . How does the consumption of alkalinity by alum compare with theoretical calculation based on balanced equations? How much extra soda ash should you add to compensate for the alkalinity consumption by 70 mg/L alum? B. Coagulation 1. @ calculation: Ca hardness (mg/L CaCO 3 )= mls titre x 1000/mls sample Lab 7 Questions A.5.pure blue). @ calculation .@ titrate with 0.pH 4. Compare your experimental softening results to the theoretical results based on solubility equations or mass balance equations. What treatment would you recommend to prepare this water for human consumption? Why? 49 September 2007 CIVL 407 Lab M anual .000/mls sample Acidity: @ 100 ml of sample. drop of 0. Softening 1.pure blue).pH 8.
sample vol Net titre Notes: 50 September 2007 CIVL 407 Lab M anual .5 “TOT” Total Hardness sample vol Net titre Ca Hardness .Coagulation Raw Data Sheet DATA Raw Sample Alum mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L pH Alkalinity-sample vol ml Net titre to pH 8.5 “TOT” Acidity .5 “P” Net titre to pH 4.7 “MO” Net titre to pH 8.sample vol ml Net titre to pH 3.
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.A.) unfiltered Notes: 51 September 2007 CIVL 407 Lab M anual .T.) True Colour (A.U.) (filtered) Turbidity (N.H.P.A.H.P.
5 Total Hardness sample vol Net titre Ca Hardness .7 Net (Total) titre pH 8.sample vol Net titre Notes: 52 September 2007 CIVL 407 Lab M anual .5 Net (Total) titre pH 4.5 Acidity .Coagulation and Softening Raw Data D ATA Lime/NaOH mg/L Soda Ash mg/L Alum mg/L pH Alkalinity-sample vol ml Net titre to pH 8.sample vol ml Net titre to pH 3.
Coagulation and Softening Summary Sheet Data Lime/NaOH mg/L Soda Ash mg/L Alum mg/L pH P.Alkalinity -mg/L CaCO 3 *Total Alkalinity-mg/L CaCO 3 “MO” Acidity -mg/L CaCO 3 *Total Acidity -mg/L CaCO 3 Total Hardness -mg/L CaCO 3 Ca Hardness -mg/L CaCO 3 Mg Hardness -mg/L CaCO 3 **Carbonate Hard.A. -mg/L CaCO 3 Apparent Colour (A.T. Calculation 53 September 2007 CIVL 407 Lab M anual ** By .P.) True Colour (A.H.U.P.) (Filtered) Turbidity (N.H.A. -mg/L CaCO 3 **Non-Carb Hard -mg/L CaCO 3 **Excess Alk.) Note:* Total Alkalinity must include Phenolphthalein Alkalinity just as Total Acidity must include “MO” Acidity.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 18. . . . . . . . . . . . . . . . Alkalinity Species as a Function of pH . . . . . . . . . . . . . . . . . . 117 29. . . . . . DO. . . . . . . . . . . . 82 15. . . . . . . . . . . . . . . . . . . . . Bacteriology and Water-related diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . Extra tables for Chlorine Lab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . COD . . . . . . . . . . . . . Normal Solutions and Equivalent Weights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4. . Reading and Reference Material . . . . . . . . . 71 10. . . . . . . . 74 13. . . . 73 12. . . . . . . . Chlorine . . . . . .COD . . . . . . 112 28. . . . . . . . . Typical Problems for Acid-Base and General Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 3. . . Concepts and Definitions . . . . . . 69 8. . Balancing an Oxidation Reduction Equation . . . . . . . . . . . . . . . . .pdf 1. . . . . . . . . . . . 104 24. . . . . . . . . . . . . . . . . . . . Colour. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Solids Removal in Wastewater Treatment . Titrations . . . . Hach Absorption Method . . . . . . . . . 70 9. . . 67 6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 25. . . Turbidity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alkalinity. . . . . Bacteriological Examination . . . . . . . . . . . . . Standard Solutions. . . . Known Addition Technique . . . . . . . . . . . . . . 84 17. . . . . . . . . 64 5. . . . . . . . . . . . . . . . . . . . . . . . . . . 83 16. . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 27. . . . . . . . . . . . . . Periodic table . . . . . . . . . . . Chloride Analytical Methods . . . . . . . . . . . Instructions for Laboratory Report Writing . . . . . . . . . . . . . . . . . .Chlorine Demand . . . . . . . . . . . . . . . . . . . . . Water Softening . . . . . . . . . . Chemical Requirements for Water Softening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Faecal Coliforms . . . . . . . . Milliequivalent and mg/l as CaCO 3 . 80 14. . . . . . . . . . . 96 21. . . . . . . . . . . . . . . . . . . pH . . . . . . . . . . . . . . . Equilibrium Relationships of Chlorine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Additional Notes . . . . . . . . . . . . . . . . . . . . . . Introduction . .Levels and Water Use . . . . . . . . . . . 118 54 September 2007 CIVL 407 Lab M anual . Hardness Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bar Diagram Method for Water Softening Problems . . . . . . . 92 19. . . . . Acidity. . . . . 55 2. . . . . 72 11. . . . . . . 68 7. . . . . . . . . . . . . . 100 22. . . . . . . . Coagulation . . . . . . . . Hardness. . . . . . . . . . . . . . . 94 20. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 Appendices see: CIVL407Manual_CourseNotes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BOD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 23. . . . . . . . . . . . . . . . . . . . . . . . Residues . . . . . . . . . 107 26. . . . . . . . . . . . . . . . . . . . . .
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