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Prepared for Dept. Of Civil Engineering by Susan C.M. Harper September 2007
Table of Contents
Course Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Station/Drawer Supply List (if supplies are missing, please report) . . . . . 2 First Session: Laboratory Safety and Orientation . . . . . . . . . . . . . . . . . . . . 3
L ABORATORY R EPORT W RITE U P G UIDELINES . . . . . . . . . . . 5
Laboratory Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Laboratory 1: Solids Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Laboratory 2: COD, DO and BOD 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Laboratory 3: Bacteriological Examination of Sewage . . . . . . . . . . . . . . . 17 Laboratory 4: Chloride Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Laboratory 5: Chlorine and Chlorine Demand . . . . . . . . . . . . . . . . . . . . . 34 Laboratory 6: Alkalinity, Acidity, pH, Hardness, Colour, Turbidity . . . . 39 Laboratory 7a: Coagulation and 7b: Coagulation & Softening . . . . . . . . . 47
Appendices see: CIVL407Manual_CourseNotes.pdf . . . . . . . . . . . . . . . . . 54
(Environmental Laboratory Analysis - 1-3-0, 0-0-0) Testing procedures used in water quality studies and in the operation of water and wastewater treatment plants. Prerequisite Chem 151. Sc h e d u le Lecture CEME1204: Laboratory CEME1301: Wednesday 1:00 pm 1) Tuesday (time to be arranged by consensus) 2) Wednesday 2:00 to 5:00 pm 3) Thursday (time to be arranged by consensus) 4) Friday (time to be arranged by consensus) (Note: Lab Session must have at least 4 students to run)
Subject No Lab No Lab Laboratory Safety & Orientation (~1 hr) #1 Solids determination #2 Dissolved oxygen, COD, BOD No lab - Thanksgiving week - catch up #3 Bacteriological Examination of waste water #4 Chloride #5 Chlorine demand #6 Acidity, alkalinity, hardness, colour, turbidity No lab - Remembrance day - catch up #7a Coagulation #7b Coagulation and Softening
Date 2007 Sep. 4 - 7 Sep. 11 - 14 Sep. 19 Sep. 25 - 28 Oct 2 - 5 Oct 9 - 12 Oct. 16 - 19 Oct. 23 - 26 Oct. 30 - Nov.2 Nov. 6 - 9 Nov. 13 - 16 Nov. 20- 23 Nov. 27 - 30
Lecturer: Lab instructor: T.A.s: Lab: Marker:
Victor Lo, email: Susan Harper, email: Margaret Parsotan, email: Kazi Parvez Fattah, email:
email@example.com firstname.lastname@example.org email@example.com firstname.lastname@example.org
(604) 822-4880 (604) 822-4397
September 2007 CIVL 407 Lab M anual
Sta tio n /D ra w e r Su p p ly Lis t (if s u p p lie s a re m is s in g , p le a s e re p o rt)
The following items will be maintained in each stations supply drawer or at station on bench when required: 2 pair safety glasses 2 permanent marker pens 2 stir bars and a bar retriever 1 stir plate 2 buret funnels 4 30 ml beakers 4 50 ml beakers 2 100 ml beakers 2 150 ml beakers 2 250 ml beakers 2 1 L plastic beakers 2 600 ml plastic beakers 2 400 ml plastic beakers 2 10 ml grad cylinders 2 25 ml grad cylinders 2 50 ml grad cylinders 2 100 ml grad cylinders 1 buret stand 1 double buret clamp 2 50 ml burets 2 25 ml burets 2 10 ml burets 0-14 pH strips Thermometer 2 Stir stick or spatula Filter funnel (Nalgene magnetic) Tweezers (Millipore or Gelman for membrane filters)) On benches each row: Latex gloves- small, medium and large Non-latex gloves (keep on reserve for latex sensitive persons only) Filter racks with funnels #4 Whatman filter paper or equivalent Vacuum flasks and hoses 8 1 L grad cylinders 8 3 L plastic beakers
September 2007 CIVL 407 Lab M anual
0. gloves as required. Clean-up: You must leave your work station as you found it . eye protection. Safety Shower. 1. Pipettes must be placed with tips pointing up into the pipet basket supplied. 15. with contaminated gloves will contaminate those items.0. no matter how minor. calculators. 25. Please ask for assistance if chemicals are spilled. if there is room. Do not put lids and small items onto racks that may slip through to the filthy drip tray. They are calibrated by weighing the volume of distilled water that will flow from them by gravity. Putting pens in your mouth that may have been on the bench or handled with gloves could be hazardous. Pipettes/ graduated cylinders/ beakers/ flasks: 5) 6) 7) 8) 9) 10) Volumetric pipettes are the most accurate way to measure volume. 20. 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. Spills: Wipe up all spills immediately. 50 and 100 ml. Please take your labcoat away in a plastic bag. If you cut yourself.clean and dry. A small amount of liquid always remains in the tip and must not be blown out. with the tip against the side of the receiving vessel. No sandals or open-toe or heel shoes... DO NOT PUT BROKEN GLASS INTO THE REGULAR GARBAGE CONTAINER. MSDS: is a supplier information sheet that must be available for each product and describe the hazards and necessary handling requisites. Remember to empty and rinse burets as well. wash down and dry the bench. Sanitation: antibiotic soap for hand washing. Some pipettes may be “to 3 September 2007 CIVL 407 Lab M anual .2 First Session: Laboratory Safety and Orientation 1) 2) 3) 4) NO Food or drink. WHMIS: Workplace Hazardous Materials Information System is a government regulation. Hygiene: At times you will be working with sewage. Be aware that handling your pens. These should be used whenever standard solutions are measured or when upmost accuracy is required. disinfectant for bench surfaces.up to 10. etc. but if you do please ask for assistance.5. Personal Protective Equipment: Lab coat (long). They are available in 0. Most pipettes are calibrated “to deliver” or TD and should never be blown out. 2. packs. please get attendant help. You must be informed of the hazards of a substance before using it. Safety Equipment: Eye Wash Station. Broken Glass: Be careful not to break glass. Wearing lab coats out of the lab is forbidden.0 ml.
do not use a 1 L graduated cylinder. Some are blow-out. They are useful for measuring samples 20 mls . Pipettes are in drawers indicated. 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.1000 mls.merely approximations. 250 mls. 12) Cup sinks: Please do not use these sinks for the disposal or delivery of liquids. 50 mls. especially at smaller volumes. 25 mls.deliver with Blow-Out”. Graduated cylinders: Not as accurate as pipettes. please rinse out immediately and set aside to dry.pay attention to the markings. Undetected gas leaks can be deadly. 500 mls. They are containers best suited for “swirling” liquid ie to mix reagents with samples in a colourimetric determination (Lab 4 or 6). 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. 4 September 2007 CIVL 407 Lab M anual . not wastefully as quantities are limited. 100 mls.. Erle n m e y e r or conical flask markings are not accurate. 11) Gas/air/vacuum taps: Do not touch these unless instructed to do so. Make sure that you label beakers and flasks to avoid confusion and waste. Used to hold/transfer approximate volumes of samples. If liquid should enter a bulb or pump. some are not meant to drain completely . Do not squish bulbs in the direction of another person. 1. Even the force of air from the air taps can be dangerous because it can contain grit or water. They are calibrated the same way as TD except that the remaining drop is blown into the receiving vessel. Chemical residues can be splashed in you face. 13) Stir bars: Please remove from containers before pouring contents down the drain. 10 mls. They are usually identified by a double etched or coloured band at the top of the pipette. Flasks: Vo lu m e tric flasks are very accurate and should be used when diluting standards (together with volumetric pipettes). Ie If you want to measure 20 mls. YOU WILL USUALLY NEED THE INFORMATION GIVEN TO COMPLETE YOUR LAB WRITE-UPS. NEVER mouth pipette. chemicals or for titrations. Cylinders are available: 5 mls. 14) Glassware/supplies: Each station has a drawer where some glassware and other useful tools will be kept. They can be unpredictable in force and are close to eye level. LISTEN AND MAKE NOTE OF VERBAL AND CHALK BOARD INSTRUCTIONS. Chemicals will be put out as required for each lab and should be used sparingly. A demonstration will be given. markings are not accurate at all . Additional supplies are available in the supply room (around the corner). Pipette bulbs or pumps must be used and great care must be taken not to contaminate bulbs.
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. State that the procedures were in accordance with CIVL 407 Lab Manual and note any deviations to the manual’s procedure. 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. Please note that the marking outline is intended only as a guideline to assist in report write-up and that marks would also be allocated for overall presentation and clarity of the report. R EPORT E LEMENTS Title page Objective Materials and Method S HORT R EPORT L ABS 1 TO 6 Required Required Details are not required. Required  Required  Required  Required  Required  Required  Required   5 September 2007 CIVL 407 Lab M anual .L ABORATORY R EPORT W RITE U P G UIDELINES Guidelines for laboratory report write up are also given in the CIVL407 Manual Course Notes and summarized here. The short report is intended to be a concise yet complete laboratory report whereas the long report is more elaborate.
If large pockets of liquid form between the particles of settled matter. 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. A. Mix the samples thoroughly and fill individually marked Imhoff cones to the 1 litre mark. keep hands and pencils away from your mouth and wash hands frequently. Use pipette bulbs. evaporating dishes. settle for 15 minutes longer. Do not include any surface floating material as settled solids. 6 September 2007 CIVL 407 Lab M anual . tin dishes. to measure sewage treatment plant efficiency in removing residue. and record the volume of settleable solids. MATERIALS: -Imhoff cones. A 1 L graduated cylinder may be used in place of the Imhoff cone for the sludge sample. distilled water bottles. desiccators. oven muffle furnace. graduated cylinders. SETTLEABLE SOLIDS 1. Alternatively a place will be provided in the lab to store your coat. sludge. subtract an estimated volume from the measured volume of matter. Do not wear lab coats outside of lab and do not take away from lab unless stored in a plastic bag. balance. if necessary. gently dislodge any solids that have clung to the sides using a stirring rod. 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. samples of raw sewage. final effluent. Settle for 45 minutes. wipe up all spills and wash with disinfectant.
Assemble filtering apparatus. 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. 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 . rinse the cylinder. Subtract the tare weight to obtain the total solids weight and calculate the mg/L value. Record the total volume filtered. The furnace will then be allowed to cool significantly before dishes are removed to a desiccator.so be quick.done for you.] Using very small amounts of distilled water. position the filter and begin suction (vacuum tap). pre-fired and desiccated . Once dishes are at room temperature they can be re-weighed. the solids remaining represent the fixed solids. Make sure that you have recorded all the pertinent details: Dish #. Conversely. Pinch sides of dish in a bit to protect the filter from oven drafts. 2. Wet filter with a small volume of distilled water to seat it. stir the beaker contents and then rapidly (so that it doesn't settle) measure a volume of sample into the appropriate graduated cylinder.[Note: Sludge Volume Index is the volume occupied by 1 gram of sludge after 30 minutes of settling. Suggested portions: 100-200 ml of final effluent. After drying overnight.] Rinse the graduated cylinder with small amounts of distilled water and add to filter. dried. The dish containing the dried total solids can now be placed in a muffle furnace or. adding the rinsings to the dish. Using the sample poured out for Part B (for consistency). rapidly pour sample into a 50 ml graduated cylinder to approximately equal 35-40 mls (if using 50 ml dishes). 3. pour about 500 mls (for Part B & Part C) into a beaker and then. if instructed. 5. 2. tare weight. SVI= settled sludge volume (ml/L) x 1000/suspended solids (mg/L)] B. sample source and sample volume. on a cart in preparation for staff to do so when all are ready. prior to session). immediately after stirring the beaker. 6. This is a different test to the one we are doing using Imhoff cones and should not be confused.[Note: it may be harder to wash out solids if you allow them to settle before pouring into the dish . the dish will be transferred to a desiccator. Mix the sample in the carboy thoroughly. Obtain the tare weight of a properly prepared porcelain evaporating dish (ie one that has been washed. Carefully remove filter from filtration apparatus and transfer it back to the aluminum dish. The samples will be fired at 550° C for one hour.]. Calculate the mg/L volatile and fixed solids. 10 mls for Raw & 5 mls for sludge) of well-mixed sample and filter entire portion before pouring another portion. Put the dish in the 103-105 °C oven for drying. . Do not write on crucibles as high temperatures during the next steps will burn writing off. TOTAL SOLIDS and TOTAL VOLATILE AND FIXED SOLIDS AT 550o C 1. Obtain the tare weights of three aluminum dishes each containing a glass fibre filter (already pre-washed. 3.TOTAL AND VOLATILE 1. C.not beakers. 4. SUSPENDED SOLIDS . [Notes: to allow for rinsing and to avoid spillage when transferring dish to oven don't pour more than 40 mls]. The loss in weight represents the volatile solids lost from the originally measured sample volume. dried and fired). [Hint: pour out small portions (say 25 mls at a time for Effl. Obtain the gross weight of the dish [Note: it should be room temperature before weighing. Quickly record the exact volume and pour the sample into the dish. when filtering slows significantly do not add more.
What. overnight). Lab personnel will perform the timed-firing after the class. 6. What major types of solids are removed in primary treatment? in secondary treatment? 2. Enter all data in the tables on following page. If all of the volatile solids reduced is given off as gas and if the specific gravity of the volatile solids is 1. The aluminum dish and filter (from #4) must now be fired in a muffle furnace at 550°C for at least 30 minutes (or until a constant weight is achieved).e. They must be allowed to cool completely before re-weighing them to determine the loss on ignition or volatile suspended solids. A raw sewage sludge goes through an anaerobic digestion process.) a) a bacterium b) sodium chloride c) sugar d) clay 8 September 2007 CIVL 407 Lab M anual . Please return tomorrow to obtain final weight. DO NOT DISCARD! The gain in weight represents the total suspended solids of the sample. After firing the dishes will be moved to a desiccator. Calculate as the mg/L total suspended solids. fixed (use more than one adjective to describe each substance. what is the percentage reduction in solids. Calculate the percent solids reduction through the treatment plant. volatile. Calculate the mg/L total volatile solids and total fixed suspended solids. if any. Your hands may have moisture and natural and/or applied oil/lotion on them.50. What two techniques can be used to overcome or correct for the loss of material from filter pads when firing at 550 oC? 4. From the above determinations it will now be possible to obtain a value for the dissolved solids present in the sample. 6. dissolved. 7.3 and fixed solids 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? 5.5. Transfer dish to a desiccator. where the volatile solids are reduced from 65% to 40%. cool and weigh. suspended. place the dishes on a cart designated by the instructor. 8. If sludge is used in Part A. compute the sludge volume index (SVI). For the time being. Discuss the meaning and significance of this index. solids _________ % reduction fixed suspended solids _________ % reduction dissolved solids _________ Lab 1 Questions 1. % reduction in settleable matter _________ % reduction in total solids _________ % reduction in volatile solids _________ % reduction in fixed solids _________ % reduction in suspended solids _________ % reduction volatile susp. Classify each of the following into its proper solids category(s) i. 3.
fired (G) Loss on ignition (G) Volatile solids (mg/L) Fixed solids (mg/L) Percentage fixed residue C.use permanent mark on dish) Sample volume (mL) Gross weight .DATA AND RESULTS Raw Sewage Aerobic Sludge A.use embossed mark) Sample volume (ml) Gross weight . Total Solids Dish marking (do not use ink . Settleable Matter After 1 hour (mL/L) B.oven dry (G) Dish tare weight (G) Total solids (G) Total solids (mg/L) Gross weight .fired (G) Loss on ignition (G) Volatile suspended solids (mg/L) Fixed suspended solids (mg/L) Dissolved solids (mg/L) 9 September 2007 CIVL 407 Lab M anual Final Effluent .oven dry (G) Tin dish with filter tare weight (G) Total suspended solids (G) Total suspended solids (mg/L) Gross weight . Suspended Solids Tin dish marking (do not use ink .
Wash hands frequently with soap. Wipe up all spills immediately. COD . OBJECT: -to familiarize you with the commonest tests run for assessing treatment plant efficiency and pollution loading to receiving waters. potassium hydrogen phthalate standards: 800. 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. Keep fingers. I. as well as handling samples probably containing pathogenic bacteria. wash/rinse bench tops with water/disinfectant. Always use and store pipettes with the top higher than the tip. etc. homogenization of samples containing suspended solids may be necessary in order to get a representative sample and to improve reproducibility. Materials: Raw sewage and final effluent samples. spectrophotometer.La b o ra to ry 2: CO D . NEVER pour water into acid. 400. pipettes. etc out of your mouth. Hach block digester. vials with pre-measured reagents. and to demonstrate the use of dissolved oxygen probes. to illustrate the shortcomings of these tests. otherwise reagents will run to the bulb end and make pipetting difficult and contamination likely. The closed reflux method is more economical but because a small sample volume (2 mls) is used. 200. pencils. Digests from either method can be titrated or read colourimetrically. 10 September 2007 CIVL 407 Lab M anual . 100 and 50 mg/L as COD.Closed Reflux.
Note: You are going to measure DO on four different liquids using two different methods. aerated dilution water. a smaller aliquot (making volume up to 2 mls with distilled water) or 2 mls of a pre-diluted sample can be used. If less than two mls of sample or diluted sample was used. into the appropriate tubes and screw cap on snugly. Raw Sewage (Infl) and/or Effluent (Effl) in the upper 1/4 of the tube using permanent markers. Standards have been digested for you. 4. 2. Allow samples to digest for 30 minutes (normally this would be 2 hours). which is set at 600 nm to read absorbance of light by the sample. BOD bottles. 11 September 2007 CIVL 407 Lab M anual . If sample is known to be outside the standard range (above 800 mg/L). 100. [Note: it should look homogenous and be careful it will be very hot!] 3. Read your samples and the set of COD standards: 800. Prepare calibration curve. alkaline-iodide-azide reagent. 50 and 0 COD as mg O 2 /L. 200. The four liquids are cold tap water. use wide mouth graduated pipettes) in duplicate. before the lab. Put identifying labels on each tube ie your initials.Procedure: 1. concentrated sulphuric acid. Remove the vials and allow them to cool to room temperature (use cold water to speed up if necessary). Take vials to fumehood and place in block digester . manganous sulfate. a volumetric correction must be made to the value obtained from the curve. 400. DISSOLVED OXYGEN (DO) Materials: Raw sewage and final effluent samples. except without mercuric sulfate). burette stand with burettes. 500-ml Erlenmeyer flasks. II.025 N sodium thiosulphate. and are at the spectrophotometer. At your station you will find four vials with pre-measured solutions containing potassium dichromate and sulfuric acid (prepared according to standard methods. Pipette 2 mls of sample (using volumetric pipettes unless particulate is present.the azide modification of the iodometric method) and a dissolved oxygen probe and meter. The instructor will demonstrate the use of the spectrophotometer. dilution water. thermometers. well-mixed samples in the 25-position digester. starch indicator. 6. in which case. DO NOT ADJUST THE SETTINGS ON THE METER. You will use a chemical titration (Winkler . 250-ml graduated cylinder. 0. The probe has been previously calibrated.noting the location of your well-labelled. raw sewage and final effluent. 10-ml graduated pipettes. The vials should be clean and dry on the outside. 5. Make sure sample is well mixed with the acid contents of the vial. absorbance versus concentration and calculate the COD in samples.
) You can save these bottles for B. aerated dilution water. . Allow the floc to settle to about 1/3 the bottle volume. to thoroughly mix contents. (Note: DO meter temperature is usually not accurate. 2. (Hint: if you measured very low DO level with the meter. 4. Do not mix droppers and do not allow tap water into the reagent bottles. 1. Neglect any reappearance of the blue colour. To the same bottles. Fill four BOD bottles with the four samples (cold tap water. add 1 ml of alkaline-iodide-azide solution the same way. (Fill carefully. 8. 9. Instructions on the use of the probe will be given in the lab. Record the volume of titre.A. WINKLER TITRATION (Azide modification) NOTE: Use only the droppers attached to the reagent bottles for dispensing into samples. right to the top and place stopper on bottle. raw sewage or influent and final effluent). B. 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. Set back in sink. The chemical floc should completely dissolve. Note: if solution already seems pale yellow add starch right away. Fill a BOD bottle with each of the four samples (or use ones saved from A ).025 N Na 2 S2 O 3 until only a faint yellow or "straw" colour remains.) Transfer 201 ml of each bottle (do one at a time) to a 500 ml conical flask. Determine the percent saturation for each sample. again quickly replacing the lid. 1. then the same sample will require little or no titre and may not turn blue when starch is added . taking care not to entrain additional air. Titrate this volume with 0. until the first complete disappearance of the blue colour. Record the temperature of each sample using the DO meter or a thermometer. 7. remove the stopper. add 1 ml of concentrated H 2 SO 4 and quickly restopper. For the cold tap water sample. Stopper the bottles without trapping any air bubbles. Add enough starch solution to give a strong blue colour (one dropper full) and continue to titrate. Take each stoppered bottle. When the floc has settled the second time to about 1/3 bottle volume. as demonstrated. 2. for each of the four samples. Place the bottles in the sink and one at a time remove the stopper. 5. tip the liquid out of the space around the stopper (caution -it may be corrosive) and invert several times. 12 September 2007 CIVL 407 Lab M anual 3.think about it!) Complete all four titrations. The DO concentration in mg/L is equal to the number of ml of titre as long as 201 ml is titrated. dropwise. Use the specially marked volumetric flask to dispense the 201 ml. invert several times again and allow to settle again. Determine DO. 6. submerge the hose in the bottle and allow the water to overflow for 15-30 seconds). DISSOLVED OXYGEN PROBE NOTE: DO NOT add any reagents to the samples used for the probe method. using the DO probe and meter. Tip out the liquid from the area around the stopper and invert several times to mix thoroughly. (Note: biological solids originally present in the sample will not dissolve.
Draw a straight line between the water temperature and the mg/l of dissolved oxygen. Raw Sewage Final Effluent Tap Water Dil’n Water 13 September 2007 CIVL 407 Lab M anual . Pair up the mg/l of dissolved oxygen you measured and the temperature of the water in degrees C. (mg/L) DO conc. use the saturation chart above. (Probe) Temp (oC) (thermometer) Saturation concentration Percent saturation DETERMINING PERCENT SATURATION THE "QUICK AND EASY" METHOD For a quick and easy determination of the percent saturation value for dissolved oxygen at a given temperature.Dissolved Oxygen Data Sample/Data Bottle # Sample Volume (mls) Initial Buret reading Final Buret reading Net Titre (mls) DO conc. 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. The percent saturation is the value where the line intercepts the saturation scale.
These caps are designed to prevent the evaporation of the liquid around the stopper. groups). Materials: Raw sewage and final effluent samples. it may indicate that probe may not have been calibrated properly or the dilution water had been contaminated. 3. be careful not to contaminate it with sample. 1. The blank is used to check that the dilution water has negligible BOD. dilution water. Stopper the BOD bottles. In addition.if not add a some dilution water (or distilled water) to the well. preferably using the same meter and probe used to get IDO reading. 5. thereby maintaining a water seal for each bottle. There should be negligible BOD in the Dilution water. graduated cylinders. After five (or six)days. 6. 10-ml graduated pipettes. 4. to bring the levels above the ground glass neck of the bottle. Note: Each group should have 9 BOD bottles in the incubator. BOD bottles. ie Rinse the filling tube if it has been in the sample. BIOCHEMICAL OXYGEN DEMAND Note: Because of the logistics of organization some groups may have 6 day BOD 5 instead of the usual 5 day. Dissolved oxygen meter and probe. showing sample calculations. All bottles should have liquid in the well around the stopper . being sure to exclude any air bubbles that might be trapped under the stopper by adding 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). 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. Be sure to note this in your lab write-up. If the dilution water BOD is greater than 1 ppm. Be sure to record the bottle numbers and sample volumes on the following table . Calculate the 5-day (or 6-day if unavoidable) BOD. 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. remove bottles and measure the DO. Measure the Initial DO of the blank using the calibrated probe and meter.do not write on the bottles and do not use the numbers on the lids. Because you want to be sure that the dilution water has no BOD. carefully measure the waste water samples into BOD bottles. 14 September 2007 CIVL 407 Lab M anual .III. Carefully top up the BOD bottles with DILUTION WATER. fill one BOD bottle with DILUTION WATER alone. Using the volumes and samples provided by the instructor. Place plastic caps over all bottles. but if there is a significant BOD then it will have to be subtracted before the dilution factor is applied in the calculation.
= = = = decimal volumetric fraction of sample used DO of seed control before incubation. mg/L DO of seed control after incubation. mg/L = DO of diluted sample after 5 d incubation at 20 oC. and ratio of seed water in diluted sample to seed in seed control= (%seed in diluted sample) / (%seed in seed control). 15 September 2007 CIVL 407 Lab M anual . 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.
Diln. Both wastes have equal BOD 5 temperature and volume. Calculate theoretical oxygen demand for 300 mg/L of methyl alcohol. If an industrial waste has a COD of 450 mg/L. 2) 3) 4) Show on a graph the approximate dissolved oxygen sag curves in a river receiving two wastes. Diln. 6) 7) 8) 16 September 2007 CIVL 407 Lab M anual .) Why is your COD result different from the BOD result? 5. 2 Final Effluent. One waste has a K value of 0. 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. 1 Final Effluent. titrimetric method. 1 Raw Sewage. open reflux.17. 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.25.Summary Raw Sewage. what would you expect its I) BOD 5 and ii) BOD L to be. Diln. if at all possible. Diln.
. Keep hands. lab coats. Heterotrophic plate count: dilution tubes. DO NOT MOUTH PIPETTE. plates. 35oC incubator. Get raw data from another group and include in your report. F iv e d if f e re n t p ro c e d u re s w ill b e s tu d ie d . Students will do only sections marked ? MATERIALS: Multiple tube method: dilution tubes. LES Endo agar plates. Demonstration only SAMPLE: You will do either a raw sewage sample or a final effluent sample. sterile pipettes.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. Wash hands with soap before leaving. T h e re f o re . filtering apparatus. etc. sterile dilution water. sterile pipets. eye protection. EC tubes and/or Hach A1 tubes. Demonstration only.leave it here or take away in a plastic bag to launder. Brilliant Green Lactose Broth tubes. pencils. 35 oC incubator. lauryl tryptose broth tubes. Quebec colony counter. wipe up all spills and disinfect the area of the spill. Membrane filter 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 . not both. OBJECT: -to demonstrate different methods for the determination of the coliform group of organisms and to acquaint you with bacteriological procedures. Do not wear lab coat out of lab . agar tubes. away from your mouth. membrane filters. Wipe lab benches with disinfectant before and after each lab. 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 .
COLI in 4 to 24 hours. 2. Use Table 2 on page 27. EC or A-1 tubes positives: 1. as demonstrated. 1. 3. Brilliant green lactose bile (BGLB) tubes are inoculated and incubated at 35oC for 48 hours (confirmed test for total coliforms) and EC tubes are inoculated and incubated at 44.7oC for 24 hours (confirmed test for faecal coliforms). E. Demonstration. All tubes have been examined for gas production. 18 September 2007 CIVL 407 Lab M anual . Using aseptic technique. Gas formation indicates the possible presence of coliform organisms. using a ready-to-use P/A Broth with MUG. 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.Pour Plate Method: Serial dilutions of samples mixed with agar and incubated for 48 hrs at 35oC. Results will be provided for the demonstration set. D. (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. ? B. Calculate the MPN value from the number of positives using the Table 1. Another series of tubes are inoculated from these positive lauryl tryptose tubes. 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. Express as Total coliforms. Membrane Filter Technique: Serial dilutions of sample are collected on membrane filters and placed on M-Endo media. ? C. 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. The number of colonies counted directly.A. is USEPA-accepted for testing non-potable waters. MUG produces a fluorogenic product when hydrolyzed by an enzyme specific to E.coli. Gas-positive Hach A-1 tubes indicate the presence of faecal coliform. Streak plates in a manner to insure presence of some discrete colonies. Hach’s Most Probable Number Method 8368. streak one LES Endo agar plate from each tube of BGLB broth showing gas from the highest dilution and the second highest dilution. It is used as a faster alternative to the LTB/EC procedure for enumerating faecal coliforms in water. Presence/Absence Testing: HACH technique to simultaneously screen for total coliform and E. Parallel positive BGLB broth cultures with negative EC broth cultures indicate the presence of nonfaecal coliforms. 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).coli. Microscope Slides (Completed Phase of Multiple-Tube Fermentation): Single colonies arising from single cells are prepared and stained for microscope examination. 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. A-1 Medium. Consider positive EC broths incubated at the elevated temperature (44.) Methods: ? Recording BGLB.5 oC) as a positive completed test response for both Total and faecal coliform. Bacterial numbers are calculated from the numbers of positive tubes.
Prepare Gram Stain slides for examination under a microscope. transfer 1 ml of sample from dilution tube # 6 (most dilute for Raw sewage) or #5 (for Effluent) to an empty (large) Petrie dish. then immediately replace the cover. ?B. Verification of both types of colonies is advisable as some typical sheen colonies may be noncoliform and some atypical may be coliform. 4.2. Using sterile forceps. Incubate plates (inverted) at 35 oC for 24 ± 2 hr. red. Membrane Filter Technique -Total Coliform 1. Heterotrophic Plate Count . Verification is usually done by a test for lactose fermentation. 2. Be sure dishes are labelled with your name and dilutions. 3. choose appropriate dilutions for counting. -Note: “typical” colonies count as coliforms in this procedure. prepare one set of six dilution tubes following the instructions given under Multiple Tube Fermentation. coliform bacteria are Gram-negative (pink) rods (sometimes coccus-like). removing the cover just enough to insert pipette tip and dispense sample. Do not push down on the filter except on the outside edge and use only sterile forceps . Repeat for the rest of the dilution tubes to # 2. 2. open the valve slowly and allow all the liquid to be pulled through. If you haven’t already. with a motion that excludes air as it comes in contact with the agar. Use dishes with 20 to 80 coliform colonies and not more than 200 of all types. Prepare one set of six dilution tubes as described in Section A..1. place a sterile membrane filter (grid side up) onto the filtration apparatus. In general. ?C. Atypical coliform colonies can be dark red or nucleated without sheen. Incubate inverted Petrie dishes for 20-22 hr at 35 oC. A high count of non-coliform colonies may interfere with the maximum development of coliforms. Flame loop between second and third quadrants to improve colony isolation. Dilution tubes. The typical coliform colony has a pink to dark-red colour with a golden green metallic surface sheen. Colonies that lack sheen may be pink. 5. Gram staining technique can be also be used. When filtering is complete and the valve is closed. 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. therefore the results table should specify #coliform colonies/100 ml. The sheen area may vary in size from a small pinhead to complete coverage of the colony surface. Inoculating. Dilution tubes. or colourless and are considered to be non-coliforms. After 20-22 hr. The filtration apparatus has been sterilized by autoclaving. from one edge. The sheen area may vary in size from a small pinhead to complete coverage of the colony surface. Mount apparatus on suction flask with valve sideways to block suction. remove filter using flamesterilized forceps (allow forceps to cool or they will damage the filter).Pour Plate Method 1. Close valve and rinse the funnel with three aliquots (portions) of sterile dilution water opening and closing valve after each aliquot. Place the filter on a small LES Endo agar plate carefully.applying gentle pressure. Using a sterile pipette. white. 19 September 2007 CIVL 407 Lab M anual . Pipette about 10 ml of sterile dilution water over the surface of the filter with the suction valve still closed. Counting. Pour the full volume of dilution tube # 6 onto the filter. Filtering.
marking (on edge of dish so as not to impede counting) and incubating for 48 hr at 35 oC. a. Do this for a typical and an atypical colony. Wash slide in tap water. Work quickly or the agar will solidify in the tube before you get a chance to pour it. ?4. The aim of using several dilutions is to have at least one dilution giving colony counts between these limits. 2. Take 5 tubes of melted agar from the incubator. f. on the bench. refer to Standard Methods for more instruction. Microscope Slides: 1. Do not report as “too numerous to count” if all plates exceed 300 colonies.coli). count four representative squares. Multiply the number by 5 to compute estimated colonies per plate (66 cm 2 ). Compute bacterial density and report as “colony-forming units” (CFU) per ml. Slide staining. After 48 hr incubation.too hot for a bacterium. if fewer than 10/cm 2. Ideally. Resterilise the loop and remove one colony from the surface of the Les Endo streaked plate (prepared previously). 4. count colonies in 13 squares (of the colony counter) of the most dilute plate having representative colony distribution. Preferably select seven consecutive squares horizontally and six consecutive squares vertically. average and multiply by 57 (for plastic Petrie dish). Allow iodine to act for 30 seconds. e. use the plate(s) that will give from 30 to 300 colonies/plate.d e m o n s tra tio n -s ta rte d a h e a d o f la b 1. If more than 10/cm 2. Test temperature on your wrist . c. A hand counter is also available. Counting. 6. Examine for yellow colour (total coliforms) and fluorescence (E. D. Flood slide with crystal violet solution and allow to act for 30 seconds. Gently swirl the dish.too hot for a baby . 5. Let them cool down from the 60C that they are kept at but not enough to solidify. Spread the colony in the drop of water so that you get a uniform dispersion over an area about the size of a dime. Air dry the slide high above the Bunsen burner and fix by passing slide briefly through a flame. Flood the slide with fresh alcohol-acetone solution to act for 30 seconds. select the appropriate dilution to count. Instead. Slide the cover of a Petrie dish back just far enough to pour the agar. Slide preparation: With a flame sterilized inoculating loop. d. Allow the agar to solidify in the dish before inverting.3. Record results of previously prepared test. Drain off excess iodine and wash freely with water and then alcohol-acetone decolourising solution. pour and replace cover. Wash off stain with water and then with iodine solution (Lugol’s). Repeat for the rest of the dilutions. Repeat with dilutions 5 to 2 (for Raw) and 4-1 (for Effluent). E. Presence/Absence Testing . 20 September 2007 CIVL 407 Lab M anual . 2. b. Incubate at 35 oC for 24-48 hours. Use the Quebec colony counter to aid in counting and a grease pencil to mark off colonies counted. place 1 loopful of sterile dilution water in the centre of a slide. Label the slide using a grease pencil (T or A). to mix the sample with the agar. Add 100 mls of sample directly to pre-measured medium provided. If necessary. ?3.
Microscopic examination. . or in short chains.Come in and count Heterotrophic Plate colonies after 48 hours. .Membrane filter prepared dilutions. . Cells occur singly. in pairs.3 µm in size. . . place on LES Endo agar dishes and incubate.counts will be provided on the board. and on the slide: typical or atypical. smooth. .g. sometimes coccus-like and 0. Examine the prepared slides . Note: in the staining process: before decolourisation all organisms appear Gram positive (blue).5 . h. In general. Day 3.Examine prepared slides. Apply fuchsin or safranin (counter)stain for 30 seconds.Prepare dilutions for Heterotrophic Plate Count and Membrane Filter technique. Description of bacteria: Describe what you see on the plates: colonial morphology of each type of colony seen ie surface (shiny. odour. cell groupings (single.Prepare and incubate Heterotrophic (Pour) Plates using dilutions above. after decolourisation Gram negative organisms are no longer visible. .Record positives in all Multiple Tube Fermentation inoculations: LTB. a. pairs chains). Wash in water. coliform are Gram-negative (pink). Schedule Summary Day 1.do not turn knobs except the positioning ones. . concave). b. cocci or rods. Plates ready on a weekend will be put in cold room until Monday. gram positive or negative. usually motile. 21 September 2007 CIVL 407 Lab M anual . After application of the counterstain. nonsporing. blot and then dry in air high above the Bunsen burner (so as not to burn away your efforts). Gram negative organisms are visualized (pink). metallic. plump rods. short. ?3.Record Presence/absence testing results of Hach Prepared Media bottles. colour. BGLB and EC and AI tubes . transparency. c. length to width ratio.Come in and count colonies on LES Endo Membrane Filter dishes. Day 2. rough. dull.
35 oC/48 hr Tube # 1 2 3 Mls of inoculum Infl Effl Plate count Infl Effl *CFU/ml Infl Effl *CFU= colony-forming units Membrane Filter Technique Tube # Dilution (mls) Infl Effl Coliform Colonies Infl Effl #Colonies/100 mls Infl Effl 1 2 3 4 5 6 4 5 6 22 September 2007 CIVL 407 Lab M anual . 1 2 3 4 5 6 Heterotrophic Plate Count .pour plate method.Bacteriological Examination Data Multiple Tube Fermentation : indicate the number of positives for each dilution Tube # Mls of sample/diln tube LTB 24 hr (+ or -) BGLB 48 hr (+ or -) EC 24 hr (+ or -) AI 24 hr (+ or -) Infl Effl Infl Effl Infl Effl Infl Effl Infl Effl From MPN Index table find MPN Index per 100 ml.. 1/ 10 and 1/100 are used. Multiply the MPN index by the dilution factor from the series used when dilutions other than 1/1.
In the examples given below.Discussion: -sources of error? -compare MPN.coli. A long-wave UV light is required to detect fluorescence. To select the three dilutions to be used in determining the MPN index. If zero total coliform is the maximum contamination goal. Do they agree? Why/why not? -compare MPN/MF with Heterotrophic Plate Count. 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.01 ml 2/5 2/5 0/5 0.1 ml 5/5 4/5 1/5 0. the total tubes planted. MUG (4methylumbelliferyl-$-D-glucuronide) can be used to detect E. Use the results at these three volumes in computing the MPN index. the significant dilution results are in boldface. With MUG reagent added to P/A Broth. Estimation of Bacterial Density . you can simultaneously detect total coliforms and E. The number in the numerator represents positive tubes. it is not necessary to enumerate.Most Probably Number (MPN) When more than three dilutions are used in a decimal series of dilutions. Both media meet USEPA guidelines for testing of total coliforms in drinking water. MF results. 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.coli because it produces a fluorogenic product when hydrolyzed by glucuronidase.coli. They are in concentrated form and merely require the addition of 100 ml of sample (or diluted sample) to the media and incubation. an enzyme specific to E. E.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 will fluoresce as early as 4-24 hours. the combination of positives simply represents the total number of positive tubes per dilution: Example a b c 1 ml 5/5 5/5 0/5 0. use the results from only three of these in computing the MPN. that in the denominator.
0 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 .Table 1 .1 mL) Comb. 1. 0.MPN Index and 95% Confidence Limits for Various Combinations of Positive Results when 5 Tubes are used per Dilution (10 mL. 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.
Hach .MPN Table 2 25 September 2007 CIVL 407 Lab M anual .
What are the important pathogens transmitted by water? How is quality control achieved in microbiological analysis.com/ ¸Information Central/Learning Library/Microbiological Testing ASTM: (STP635) D.microbelibrary.D.htm Lab 5 Questions: 1) Compare the advantages and disadvantages of the multiple tube fermentation and membrane filter techniques. -The Use of Indicator Organisms to Assess Public Water Safety .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.http://www. 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 .org/microbelibrary/files/ccImages/Articleimages/keen/Gram stainkeen.hach.
Hint: optimum ranges will coincide with the standards provided or will be indicated within the pertinent section. If chemicals are spilled alert instructors. spectrophotometer. graduated cylinders. semi-log graph paper. beakers. buffer. chloride specific ion electrode. standard Hg(NO 3)2. 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. standard AgNO 3 solution. That means you will probably have to dilute the sample by the most accurate means available. standard chloride solution (0. indicator-acidifier reagent. MATERIALS: -millivolt meter. burettes.5 mg/L). known addition and calibration techniques and colourimetric and potentiometric titrations. OBJECT: -to acquaint you with the use of specific ion electrodes. to illustrate the differences in operation and accuracy between instrumental methods and wet chemical methods.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. Mercuric thiocyanate is very toxic. pH meter with double-junction electrode and silver billet electrode. volumetric pipettes. indicator. conc. beakers. Use personal protective equipment: gloves. Note and consider carefully: Different methods of analysis have different optimum ranges. reference electrode (double junction). HNO 3. immediately. 27 September 2007 CIVL 407 Lab M anual . standards. NaHCO 3. goggles or glasses and lab coats. 125 ml Erlenmeyer flasks. magnetic stirrer. sample.
SPECIFIC ION ELECTRODE Part One: Preparation and reading standards and samples 1. 2. 2 minutes) and use it for all standards and samples.e. labelled beakers.in sample = mg/L Cl. Using semi-log paper or software equivalent. Repeat for rest of standards. 100 and 10 ppm (mg/L) have been prepared for you and are at the meter. Add 2 mls of IONIC STRENGTH ADJUSTMENT (ISA) buffer to each using the plastic dropper (filled to the mark). Standards of 1000. Turn on stirrer and while stirring record the millivolt (mv) reading when the reading appears to be stable. Unless already done by a previous group (values written on the board). As probe tends to drift for some time it may be expedient to select a “standard time to read” (i. Measure 100 mls of sample.= ____________________ 28 September 2007 CIVL 407 Lab M anual .I. 6. 4. plot the millivolt readings (linear axis) against the concentration of the standards (log axis) to produce a calibration curve. 3. pour out 100 mls of each standard into separate. place the lowest standard on the stirrer and lower the electrodes into it.added to the sample "slope" = change in potential over 10 fold change in concentration of standard (use data from Step 2). added 2 mls of ISA. stir and obtain a reading for it. where: E1 = E2 = E = Vo = Vs = A = S = potential of sample (mV) potential of sample plus stock addition potential change = E 2 . Determine the concentration of chloride in the sample. rinsing and blotting dry the electrodes between each. Calculate the chloride concentration. Unless already done by a previous group.E 1 volume of sample volume of stock solution added mg Cl. Method of Standard Addition :Add 5 mls of the stock (4 mg/ml) chloride solution (V s) to the sample measured in Step 3 (E 1) and take a new reading after value is stable (E 2 ). 5. mg Cl. CHLORIDE .
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 . it should be rerun from the start after diluting. Near the end-point.II.0 and 8.1. (Light green indicates a pH of less than 2. 2.+ Hg(SCN)2 º HgCl2 + 2SCN SCN . pure blue. III. Using a 25 ml graduated cylinder. zero instrument on distilled water and obtain an absorbance value for the blank. 3. 6. and Hg ++ (excess) + DPC º violet colour 3.º HgCl2 1. fill. the excess mercuric ions combine with diphenylcarbazone (DPC) to produce a violet colour (the end-point). Wait two or three minutes and then add the reagent to the first (lowest) standard. Pour 100 ml of sample (or a portion diluted such that the concentration is less than 100 mg/L) into a 250 ml beaker. (Use extreme caution as this is a corrosive & toxic chemical. measure 25 mls of “zero” standard or “blank” (halide-free distilled water). 4. Determine the chloride concentration in the sample from the calibration curve. 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. add 5 ml of the “combined reagent” (containing ferric alum and mercuric thiocyanate solutions) to the blank first. If sample is above the highest standard. the 1 ml of the indicator-acidifier reagent will adjust the pH to 2.. which then must be subtracted from the standards and sample. Marking the time (T=0). 4.0 ppm). MERCURIC NITRATE METHOD FOR CHLORIDE DETERMINATION The principle of this method is as follows: Cl. using the dispenser provided. [Note: this delay between additions is to allow you time to measure absorbance of each at the 10 minute time required . Hg ++ + 2Cl.ions have been bound. After all Cl. The colour of the solution should be green-blue at this point.] After 10 minutes has passed (since the combined reagent was added to the blank). the three standards (2. (Alternatively. 5. use a plastic dropper to transfer blank into a spectrophotometer cuvette (do not fill more than 3/4 ). and the sample (diluted if necessary) into separate and labelled 125 ml Erlenmeyer flasks (5 in total).8. CHLORIDE . Add 1 ml (use pipette to measure accurately) of indicator-acidifier reagent to sample.) Mix thoroughly by gently swirling the flask.they can’t all be at 10 minutes at once.5 ± 0. For most samples. 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. insert in the spectrometer and record the absorbance displayed on the meter. after a few more minutes.ions in the sample combine with added mercuric ions forming a soluble but virtually undissociate-able complex. Titrate with 0.0.) Prepare a calibration curve by plotting the absorbance readings versus the concentration of chloride in the standards.) Every two or three minutes thereafter you will rinse the cuvette with the next standard or sample.BY COLOURIMETRIC METHOD The principle of this method is based on the following formula: 2Cl.+ Fe+++ º Fe(SCN) ++ (Reddish brown) 1.014N Hg(NO 3) 2 to a definite purple end-point. discard and refill at least twice to rinse cuvette. 2. a pH of greater than 3. (No further adjustment is required after the blank or distilled water has been set to zero.
. Don’t forget to add the indicator. smaller increments (0. The first group will do the standard (part one) in duplicate or triplicate depending on the size of the group and the other will do the sample (part two) in duplicate (or triplicate). as the end-point of the reaction is approached (ª mV/ml increases). with standard AgNO 3. recording the volume and millivolt reading for each increment. 2. Continue the titration about 5 ml past the end-point. 3. At the start. Calculate the chloride concentration: 5.. Take care that stirrer is not hitting the electrodes. Begin titrating.if one is necessary. in increments. A reference set of data will be provided for comparison. CHLORIDE BY POTENTIOMETRIC TITRATION Note: For this part form two groups comprised of 1 member from each station. Place 10.1 to 0. Immerse stir bar. Calculate the Normality of the AgNO 3 using the following equation: 30 September 2007 CIVL 407 Lab M anual .0 ml (use volumetric pipette) of standard chloride solution in a 250 ml beaker. 4. Rotate the work within the group so that different pairs are doing the titrating and recording of numbers. 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 . where: A = ml titrant for sample B = ml titrant for blank N = normality of titrant IV. 5. This way each station will have data for each part.0 ml of concentrated HNO 3 (use plastic dropper). Start the stirrer and set the millivolt reading to zero or record initial mv reading [Note: Some meters can not be set to zero]. Plot a differential titration curve to determine the exact end-point. then. place beaker on stir plate and lower electrodes into the solution. and add 2. until past the endpoint when larger increments can be used again (ª mV/ml decreases). drops of the purple end-point. 6.2 ml or drop-wise) should be added. The titre from this step must be subtracted from the titre for the sample. The end-point occurs at the greatest mV change per unit addition of AgNO 3. Part one: Standard 1.4. large increments (1-2 mls) may be added. Nitric acid is in the sink and is very corrosive. dilute to about 100 ml with distilled water.
Millivolts per ml per ml versus ml of titrant. in this case. 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 . b. 2. Millivolt reading versus ml of titrant. (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.) Plot three curves for this titration: a. which are merely first and second derivatives. Millivolts per ml versus ml of titrant.Part Two: Sample 1. Measure 100 ml of sample or portion made up to 100 ml. 4. Add 2 ml of HNO 3 and proceed as described in Part One. It is not. Calculate the chloride concentration in the sample using the following equation: 3. be careful to use the average value of ml of titrant on the abscissa. if dilution is necessary into a 250 ml beaker (use a graduated cylinder to measure). c. In plotting b and c.
Potentiometric Titration RAW DATA A Volume AgNO 3 Added (Vol) (ml) B Meter Reading (E) (mV) C FIRST DERIVATIVE D SECOND DERIVATIVE F G H ª (Vol) (ml) ª (E) (mV) E ave (AgNO 3 ) Volume (ml) ª (E)/ ª (Vol) (mV/ml) ª ( ª E/ ª Vol) (mV/ml) ª [Ave(Vol)] (ml) I Ave of Ave(Vol) (ml) J ª( ª E/ ª Vol) /ml (mV/ml/ml ) Extra pages are at the back of this lab manual in appendix. 32 September 2007 CIVL 407 Lab M anual .Chloride .
# High levels of ions which form very insoluble salts of silver may deposit a layer of salt on the electrode membrane. This is a complex. # In practice. can you see any advantage in suing the first or second derivative curve? Discuss. SPECIFIC ION ELECTRODE # All halides interfere (the electrode is a halide electrode). dichromate. # Mercury must be absent from samples. requires knowledge of the compounds in the sample which are likely to interfere. ferric ion. fluoride. # Environmental samples usually require complex pre-treatment. # Environmental samples usually require pretreatment. chromate. MERCURIC NITRATE TITRATION METHOD # Iodide. It can only correct for substances that enhance or suppress the test response proportional to the amount of chloride ion present. 3. in itself. fluoride and bromide titrate as chloride. cyanide. In the Mohr method for chlorides (described in Sawyer and McCarty) what would the equilibrium concentration of silver ions be (in mg/L) when the chloride concentration has been reduced to 0. strongly reducing solutions may form a surface layer of silver. # Colour may obscure or interfere with end-point determination. # Other interferences: ferricyanide. and sulfite ions interfere when concentration greater than 10 mg/L. specific ion electrodes are rarely usable in environmental samples. timeconsuming process requiring a sound knowledge of chemistry and extensive experience working with environmental samples. hydrazine.INTERFERENCES IN CHLORIDE DETERMINATIONS POTENTIOMETRIC METHOD # Iodide. PRETREATMENT TECHNIQUES # There are no "standard" pretreatment techniques. iodide. KNOWN (STANDARD) ADDITION TECHNIQUE # The known addition techniques will only correct for certain types of interferences. known addition cannot correct for any ions that test as chloride ion. Each sample type requires development of an appropriate method which. Also. ferric. 33 September 2007 CIVL 407 Lab M anual . and nitrite interfere. # Sample pH may need adjustment beyond the capacity of indicator-acidifier reagent. # Chromate. COLOURIMETRIC METHOD # Bromide. # Pretreatment of environmental samples usually required. causing electrode malfunction. 2. # Colour in the sample may interfere in the absorbance measurement. thiosulphate. Lab 3 Questions 1. fluoride and bromide titrate as chloride.3 mg/L? Outline the advantages and disadvantages of all the methods used in this lab to measure chloride concentration. From your chloride results. In the above tests.
025 N sodium thiosulphate solution (use a volumetric pipette). Personal protective equipment required at all times when working in the lab are: eye protection. Mix well (on stir plate with stir bar). about 50 ml of chlorine-free water (distilled water will do). potassium iodide (KI) crystals. Rinse a burette and fill it with this stock chlorine solution. 1995. REFERENCES: Standard Methods 19th ed. Titrate rapidly with the stock chlorine solution (in burette) until the appearance of a constant 34 September 2007 CIVL 407 Lab M anual . 0. N. PREPARATION OF STOCK CHLORINE SOLUTION (Iodometric method . 250 ml volumetric flasks. Wipe up all liquid spills immediately. allow to mix again. Make up to the 250 ml and invert flask several times to mix.. standard ferrous ammonium sulphate (FAS) solution. starch indicator. about 5 ml of glacial acetic acid (use a graduated cylinder). 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. and exactly 10 ml of 0. to illustrate the concepts of free and combined chlorine residuals. 3. Take a 250 ml Erlenmeyer flask and add about 1 gram of potassium iodide. pipettes. bleach or hypochlorite solution. Materials: -600 ml beakers. phosphate buffer solution. 2. chlorine demand and break-point chlorination. add 1 ml of starch solution. 100 ml graduated cylinder. pages 4-56 to 4-57 or older/newer editions. chlorine free water. OBJECT: -to acquaint you with disinfection of water supplies and to demonstrate techniques for the determination of chlorine residuals. When weighing chemicals at the balance.025 N sodium thiosulphate.total chlorine residual) 1. 250 ml Erlenmeyer flasks. gloves and lab coats.La b o ra to ry 5: Ch lo rin e a n d Ch lo rin e D e m a n d WARNING Glacial acetic acid is very corrosive. Bleach/chlorine solutions are strong oxidants.N-diethyl-p-phenylenediamine (DPD) indicator solution. burettes and stands. I. (concentrated) glacial acetic acid. clean up all spills with a brush into a container and wash crystals down the drain.
IIb. d) Alternatively: (Not to be done in this lab.purplish-blue colour. Total chlorine (Free plus Combined) = Reading C Free residual chlorine = Reading A Combined residual chlorine (dichloramine plus monochloramine) = Reading C . add full amount of KI at the start (ie 2 drops KI solution and 1 g of KI crystals) to a fresh sample. 35 September 2007 CIVL 407 Lab M anual . For dichloramine concentrations greater than 1 mg/L. (*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. pp 4-43. etc. Allow at least 5 minutes of time to elapse between applications of chlorine doses (use the buret containing diluted chlorine solution from Part I above) to successive beakers in order to allow enough time for titrating the free and combined chlorine residuals. Determination of Free and Combined Chlorine by the DPD Method 1. c) Dichloramine: Add about 1 g KI (to the same sample) and mix to dissolve. let stand for two minutes and titrate until the red colour (if present) is discharged. a) Free chlorine: titrate rapidly with standard FAS (Ferrous ammonium sulfate) titrant until the red colour is discharged. Method 4500-Cl F. 18th edition. Go to c.). 3. let stand 2 min more if colour drifts back indicating incomplete reaction. After 15 minutes of contact time. 4.) Free and combined chlorine or total chlorine: To obtain total chlorine in one reading. Record the burette reading (mls) = Reading A. Place 5 ml of phosphate buffer solution and 5 ml of DPD solution in a 250 ml conical (Erlenmeyer) flask and mix. These 6 flasks can be prepared at once to make them ready for the next step. It is likely that the highest dose will require dilution. 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. 2. b) Monochloramine: Add two drops of KI solution (to the same sample) and mix. refilling burette. 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). Record the burette reading (Reading C). Let stir for 2 min and then continue titrating until red colour (if any) is discharged (Reading C). From the amount of chlorine solution used. as there is lots to do . titrate to colourless again.Breakpoint Chlorination using Free and Combined Chlorine Residuals Values 1. and again after 1 hour of contact time determine the free and combined chlorine (see below) in 100 ml samples taken from each of the beakers at the appropriate time spacing (15 minutes and 60 minutes after the addition of the chlorine dose to each beaker). 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). calculate the chlorine concentration in the stock solution (See Data and Results section for formula). Add 100 ml of sample or diluted* sample using a 100 ml graduated cylinder and mix again. Let stand 2 minutes more.A Monochloramine= B-A Dichloramine= C-B For interferences. Continue titrating until red colour (if any) is discharged once again (Reading B). mix to dissolve. 2.COORDINATE! IIa. Work in groups of three or four for this part. please refer to Standard Methods. if colour drifts back (indicating an incomplete reaction).
Under what conditions is the practice of break point chlorination necessary? 4. Given: HOCl W H + + OClK eqv = 2. Make a separate graph for the 15 minute and 1 hour contact time. II. Guaranteed analysis is normally printed on the container and is usually 5-6%.e. FAS titrant concentration is 1 ml = 100 µg Cl as Cl2 a. Discuss your results as a function of the residual forms of chlorine present at different chlorine dosages and at different contact times. The rate of kill of bacteria by chlorination follows first-order reaction kinetics. If this is true.5 mg/L if 50% are killed in 1½ minutes at this concentration? b. How would you expect the forms of residual chlorine and their speed of formation to change as a function of 1) temperature and 2) pH? Lab 5 Questions 1. Determine the concentration of total. 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. how many coliforms would remain after a chlorine contact time of 10 minutes? 20 minutes? 36 September 2007 CIVL 407 Lab M anual .DATA AND RESULTS I. free and combined residual chlorine for each applied chlorine dose. 250) then convert mg/L to g/100 mls = %. c. d. what percentage of bacteria would be killed in 10 minutes at a chlorine residual of 0.7 x 10-8 at 25 o C % HOCl = 27 What is the pH of the solution? 3. b. Why is it important to determine chlorine residuals in domestic water supplies? 2. a. If a sewage sample contains 10 x 10 6 coliforms/100 ml. Make a plot of the three residual chlorine components against the applied chlorine dosage.
(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 .60 MINUTE CONTACT TIME BEAKER NUMBER Vol. of Cl Soln. of FAS to endpoint B (mls) (includes A) Vol. (mls) (Stock=_____mg/L Cl as Cl2) Applied Cl Dosage (mg/L) Vol. of FAS to endpoint A (mls) Vol.
hardness indicators. 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. colour comparator. Ac id ity . standard acid. Calibrate the pH meter by following the procedure outlined on the instruction sheet beside the meter unless told otherwise. (Take care not to contaminate paper in case. beakers. OBJECT: -to familiarize you with the most common water treatment tests.Lab o rato ry 6: Alkalin ity . place a beaker containing the sample and stir-bar on the stir-plate. lower probe. 39 September 2007 CIVL 407 Lab M anual . bromphenol blue.(Do not allow the stir bar to hit the probe. 4. metres and calibrated glassware are available in limited quantities and are very expensive to replace. if above 7. MATERIALS: -pH meter. Always wear personal protective equipment. buffer solution. to acquaint you with the use of pH metres. phenolphthalein. burettes and stand. I. graduated cylinders. metacresol purple. magnetic stirrer. then use buffers 7 and 10. Co lo u r. then use buffers 4 and 7 to calibrate. pH test paper and other water quality assessment equipment. particularly those labelled with specific hazards. 3. turn on stirrer and obtain a stable reading. turbidimeters. Rinse probe when you are finished and leave immersed in pH 7 buffer for storage. T u rb id ity WARNING Please use caution when handling all chemicals. Normally. the two buffers that are chosen for calibration will bracket your sample ie if sample is less than 7. pH test paper. standard base.) Record value in following table. 1 N NaOH. rinse & dry probe. flasks. bromcresol green. Probes. p H. Hard n e s s . EDTA. After calibration is complete. Use care when using lab equipment. DETERMINATION OF pH 1. water samples. 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.) 2.
it may be necessary to repeat the titration with the sample diluted 1 to 1 (again) with distilled water. If sample is highly alkaline (titration goes over one burette full) dilute the sample and titrate again. Rinse and fill a burette with standard EDTA solution and record the initial burette reading. Titrate to the first uniform pink colour (colour stays). Add a stir-bar. 3. TOTAL HARDNESS DETERMINATION . Add 1-2 ml of buffer solution using dropper bottle (3 full droppers) and one aliquot of Total Hardness Indicator using the special dispenser on the chemical bottle. until the reddish tinge disappears from the solution and a pure blue remains. Add a full dropper of phenolphthalein (P) indicator and if the solution stays pink or red. A pH much above 10 promotes CaCO 3 precipitate. (BG or MO) 6. add 1 drop of 0. Pour into a 250 ml Erlenmeyer flask. Measure out a new sample and add 1 drop of sodium thiosulphate and a full dropper of phenolphthalein.. Record burette reading (P). In order to remove any free residual chlorine (which interferes with the indicator colour response).?) 4. (If the solution is already blue after indicator.. DETERMINATION OF ACIDITY 1.. titrate until just colourless.II. Apply dilution factor to obtain final result. Add a full dropper of bromphenol blue indicator and titrate until the colour changes from yellow to blue ("MO" Acidity).5). Complete the titration within 5 minutes to minimize the tendency toward CaCO 3 precipitation. IV. If a ppt does form within the 5 minutes. if the approximate hardness is known (by unsuccessful attempts). Measure out 25 mls of sample using a graduated cylinder and dilute to 50 ml with distilled water. Record the volume of titrant required in the following table.1 N sodium thiosulphate to the sample. 2. sample-rinsed graduated cylinder and transfer to an Erlenmeyer flask. 3. (If solution is clear after addition of P. Rinse and fill another burette with standard base solution and label it. III. Add the last few drops at 3-5 second intervals so that you do not "overshoot" the end-point. 2. Add stir-bar... Rinse and fill a labelled burette with standard acid solution. 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. Alternatively. 3. DETERMINATION OF ALKALINITY 1. while stirring. Titrate slowly with EDTA. The buffer elevates the pH to 10.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. Measure 100 ml of water sample into a clean. 4. White paper placed under the flask will facilitate this determination. Add 1 drop of sodium thiosulphate solution. Record the burette reading in the following table. 40 September 2007 CIVL 407 Lab M anual . add 90% of the titrant to the sample before adjusting the pH with buffer. 2. 5.think what that means!) 4.
The display will show SIG AVG when the instrument is using signal averaging.EDTA TITRIMETRIC METHOD 1. Press: I/O. COLOUR .. Demonstration of Jackson Candle and Hellige PART A: Hach Turbidimeter 1. or a volume sufficient to produce a pH of 13-14 (designed to precipitate the magnesium as a hydroxide). 2. 4. 3. 4. to determine the apparent colour (measures the influence of turbidity on the reading). Pour the supernatant into one of the cells (to the etched mark) and fill another with distilled water. Refill the burette with EDTA titrant and note the initial reading.BY HACH 2100P Turbidity Meter.5. VI. CALCIUM HARDNESS DETERMINATION . Add one aliquot of the Calcium Hardness Indicator and titrate with EDTA slowly. Use the technique outlined in Part A. 3. Values are reported as APHA Colour Units (eqv.0 ml of 1 N NaOH solution (use 3 droppers full). 2. The display will show AUTO RNG when the instrument is in automatic range. 5. Select automatic range by pressing the RANGE key. Close the lid. Record the final burette reading in the following table. Record the final burette reading in the following table. V. Record all colour data in the following table. Insert the sample cell in the instrument cell compartment so the diamond or orientation mark aligns with the raised orientation mark in front of the cell compartment. 4. TURBIDITY . except on an unfiltered sample. The instrument will turn on but will turn off automatically after a short time so you should be ready. with continuous stirring to a pure blue end-point. 3. Place the cell containing sample in it and press Read. Measure out 50 ml of sample into an Erlenmeyer flask and add 2. Select signal averaging mode by pressing the SIGNAL AVERAGE key. The reading might be higher so if you had to dilute in Part A dilute for Part B. 2. The instrument may suggest diluting if over range. lint-free cloth to remove water spots and fingerprints.Enter the stored program number for true colour=120 1. to mg/L platinum as chloroplatinate) PART B: APPARENT COLOUR 1. . Cap the cell. Filter about 20 mls of sample using a filter funnel and stand and the pre-fluted filter paper provided 2. Wipe the cell with a soft.HACH Spectrophotometer. Use signal average mode if 41 September 2007 CIVL 407 Lab M anual . VII. taking care to handle the sample cell by the top. Fill a sample cell to the line (about 15 mL).. Place the cell containing water into the spectrophotometer and press Zero.TRUE AND APPARENT PART A: TRUE COLOUR . Add a stir-bar and stir to mix..
Lower the plunger into the liquid making sure no bubbles are trapped under the plunger and the top is dry. Wipe the outside of the tube to dry it and place it in the turbidimeter (in the circular groove of the mirror unit). record the number and refer to the appropriate graph to determine the turbidity as mg/L SiO 2.NTU.adding liquid to a hot tube will cause it to shatter. Pour sample into the tube in small increments until you can no longer distinguish the shape of the candle flame. 3.. Note that there are several different scales on the side of the tube. Try to be quick and not burn the candles more than a few minutes at a time.Demonstration only 1. Read the scale on the dial.. DO NOT PLACE AN EMPTY TUBE IN THE TUBE HOLDER WITH THE CANDLE LIT . 2. PART C: Jackson Candle Turbidity . Record the turbidity after the lamp symbol turns off. Carefully fill the 20 mm viewing depth tube to the mark with sample. Record value in following table as J. Pinch all excess charred wick with fingers before lighting or soot may be deposited on the glass tubes.) Remove the glass tube from the holder and read the turbidity at the meniscus of the sample. that the filter selector shows "none" and that bulb B is in use. then the turbidity in NTU. page 272-273. 3. Immediately balance the light intensity of the central spot with the surrounding observation field by turning the dial on the right-hand side of the instrument.T. Be sure that the calibrated tubes are clean before adding samples to the tubes. 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. the turbidity may start to settle and condense on the bottom of the tube. 16th edition.. 6. 1. PART B: Hellige Turbidimeter . T = Total alkalinity. 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. Close the door and switch on the light..demonstration only. 42 September 2007 CIVL 407 Lab M anual .s.U. (This allows you to make a more gradual and accurate approach to the end-point. Take the reading where the dark central part first merges with the surrounding field. Press: READ The display will show . If you take too long. 2. Note that the rectangular door mirror is closed. obscuring vision..the sample causes a noisy signal (display changes constantly).
O.Initial pH by test paper Initial pH by pH meter ALKALINITY. Alkalinity end-point reading (mls) __________ "M.O.O.Sample volume Initial burette reading P." Alkalinity end-point (mls) Initial burette reading Net P." Acidity end-point reading (mls)__________ TOTAL Hardness Sample volume (mls)__________ Calcium hardness as mg CaCO 3/L __________ 43 September 2007 CIVL 407 Lab M anual . Acidity end-point reading (mls) Initial burette reading Net "M." 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. Alkalinity titre (mls) Net "M." Alkalinity titre (mls) ACIDITY.O.Sample volume "M.DATA Sample 1 pH .
0 Total Hardness (EDTA): where: A= B= ml titrant for sample mg CaCO 3 equivalent to 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 = 1.0 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.
Alkalinity as HCO 3 .ion (mg/L) "M.U.Alkalinity as CaCO 3 (mg/L) OH .Hach . ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 45 September 2007 CIVL 407 Lab M anual . Alkalinity as CaCO 3 (mg/L) "M.O." Alkalinity as CaCO 3 (mg/L) OH .Units= N.ion (mg/L) CO 3 -2 Alkalinity as CO 3 -2 ion (mg/L) HCO 3.Alkalinity as CaCO 3 (mg/L) CO 3 -2 Alkalinity as CaCO 3 (mg/L) HCO 3.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 .RESULTS (sample) P.O.T.APHA Colour Units: True Apparent Turbidity .
Does this agree with the results from Part II of this lab? Explain.Lab 6 Questions 1. Is this water suitable for a domestic water supply? Why? 2. calculate the carbonate alkalinity in mg/L as CaCO 3 for the water sample analyzed during this lab. From equations (17-12) and (17-13) in Sawyer and McCarty. given K 2 = 4.7 x 10 -11 and K w = 10 -14. non-carbonate and total hardness of this water in mg/L as CaCO 3 ? in meq/L? Is the analysis complete? Why? 6. 46 September 2007 CIVL 407 Lab M anual . What errors can occur in the calcium hardness determination? Why? 3. A water has the following analysis: Na + K+ Ca +2 15 mg/L 33 mg/L 12 mg/L ClSO 4-2 HCO 3 70 mg/L Mg+2 18 mg/L 42 mg/L 8 mg/L What is the carbonate. Note: You should bring a copy of this lab to the next two sessions as you will need the same instructions to perform the same tests in the Coagulation and Softening Labs. What sanitary significance does colour have? 4.
and all of the materials from Lab. No. 47 September 2007 CIVL 407 Lab M anual .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. soda ash solution (Na 2CO 3) and lime (Ca(OH)2) or sodium hydroxide (NaOH). Please try to coordinate your work with the rest of the class. to be conducted over two lab periods. 7 (except Jackson Candle and Hellige Turbidimeter). 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. In the second lab you will use the previously determined dosages to coagulate and soften the sample and then re-analyse the resultant water. alum solution (Al2 (SO 4 )3 @18H 2 O). OBJECT: -to illustrate the principles of coagulation and water softening. MATERIALS: -Jar Test Apparatus. You should have with you the part of Exercise 7 that gives instructions for the various tests. No. 1000 ml beakers. The optimum alum dosage for coagulation of the sample will then be chosen.6. Several lime and soda ash dosages will also be selected for determining the efficiency of water softening.for the next session (b). This is a two-step experiment.
Stop stirrer. 5. moderate. Lift paddles out of beakers and secure. Part B: WATER SOFTENING PROCEDURE (to be done the following week) again work as teams of 2 or 3. floc volume. times of appearance and descriptions and record. Stop stirrer and allow floc to settle. turbidity and colour. Complete the coagulation summary sheet during the lab period. 4. Record observations on settling rate. In the lecture. After the floc has settled (allow 15 min). 7. You may need to use blocks to position the paddle 0. decant the supernatant liquid carefully into a clean set of beaker (try not to stir up what has settled) and discard the floc. and fill in the table on the board. alkalinity. acidity. Place beakers on the jar tester apparatus. 3. and clarity of supernatant liquid. if available. use indicators as specified in Lab 6. hardness (total and calcium). Add the designated lime (or NaOH) and soda ash additions as quickly as possible to the decanted samples. 4. 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). COAGULATION USING ALUM . After the flocs have settled (allow 15 min). hazy. Make relative estimates of floc sizes. acidity. 2. if time permits. clarity of supernatant liquid (very clear. Measure out six 1000 ml aliquots of sample (using graduated cylinder to measure. small. 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.work in 2 or 3 teams to assess a total of 6 dosages for each team. decant the supernatant liquid carefully into another set of beakers (try not to stir up what has settled). 5. Analyze these supernates plus a sample of raw water for pH. Analyze the supernates for pH. 6. Stir at 100 rpm for 1 minute. 3. medium. Stir at 100 rpm for 1 minute and at 20 rpm for 10 minutes. Add the appropriate alum dosage and immediately start stirring. clear. Stir at 100 rpm for 1 minute. Stop stirring. you will discuss the data and select the appropriate alum dosage to be used by all groups in the water softening step next week. Discard the floc and clean beakers in preparation for a second decanting in Step 4. Record relative floc sizes (pin-point. Reduce the stirring rate to about 20 rpm for 10 minutes. cloudy).1 N sodium thiosulphate 48 September 2007 CIVL 407 Lab M anual . 1. position them under stirrers. Observe and make notes describing floc formation and note the time of appearance. large).5 to 1 cm off the bottom of the beaker. alkalinity. Pour six 1000 ml aliquots of sample into 1000 ml beakers and place on the jar test stirrer apparatus. hardness (total and calcium). Carefully decant into a graduated cylinder 800 mls of the supernatant liquid and pour into fresh beakers. true colour and turbidity (Hach).. 1. drop of 0. Add the assigned alum dosages as quickly as possible and then start the stirrer. slow). A pH meter could be used for the alkalinity and acidity determinations. 2.Part A:. Determine which dosages were most appropriate for this sample. and settling characteristics of the floc (rapid. Make sure it is centred. Brief Methods Summary for Analyses Alkalinity: @ 100 ml of sample. (NB dosage now based on 800 ml sample size). not a beaker ) and transfer to 1000 ml beakers. 6. but because of equipment limitations.
3 ("phenolphthalein" or total acidity) @ calculation . 2.3 and with bromcresol green (continuing with the same sample) to yellow . Analyze the response of colour and turbidity removal as a function of alum dose. What treatment would you recommend to prepare this water for human consumption? Why? 49 September 2007 CIVL 407 Lab M anual . 2. drop of 0.pH 4. show by calculation the theoretical amount of lime and soda ash required for excess lime/soda ash softening. @ calculation: Ca hardness (mg/L CaCO 3 )= mls titre x 1000/mls sample Lab 7 Questions A.000/mls of sample Total Hardness: @ 25 ml sample diluted to 50 ml @ add 1 ml buffer and Total Hardness Indicator (Calmagite) @ titrate quickly with EDTA to blue end-point (no pink/purple tinge . 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.use graphical presentation.pure blue).pH 8.000/mls sample Acidity: @ 100 ml of sample.pH 3. Based on your chemical analysis of the raw water. Compare removal of hardness components under the different doses . Softening 1.pure blue).7 ("methyl orange" acidity) and with phenolphthalein (using a fresh sample) to pink .acidity (mg/L CaCO 3)= mls titre x N base x 50.1 N sodium thiosulphate @ titrate with 0. Compare your experimental softening results to the theoretical results based on solubility equations or mass balance equations.pH 8. Coagulation 1.02 N base with bromphenol blue to blue .02 N acid with phenolphthalein to colourless .5. @ 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 .alkalinity (mg/L CaCO 3)= mls titre x N acid x 50. 3. 4.@ titrate with 0. @ calculation .
sample vol ml Net titre to pH 3.7 “MO” Net titre to pH 8.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” Total Hardness sample vol Net titre Ca Hardness .5 “TOT” Acidity .5 “P” Net titre to pH 4.sample vol Net titre Notes: 50 September 2007 CIVL 407 Lab M anual .
U.T.A.H.A.Coagulation Summary Sheet DATA Raw Sample Alum mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L pH Floc size Clarity Floc settling rate Alkalinity (mg/L CaCO 3 ) “P” Alkalinity (mg/L CaCO 3 ) “TOT” Acidity (mg/L CaCO 3 ) “MO” Acidity (mg/L CaCO 3 ) “TOT” Total Hardness (mg/L CaCO 3 ) Ca Hardness (mg/L CaCO 3 ) Apparent Colour (A.P.H.) (filtered) Turbidity (N.P.) unfiltered Notes: 51 September 2007 CIVL 407 Lab M anual .) True Colour (A.
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 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 3.5 Net (Total) titre pH 4.
-mg/L CaCO 3 **Non-Carb Hard -mg/L CaCO 3 **Excess Alk. -mg/L CaCO 3 Apparent Colour (A.Coagulation and Softening Summary Sheet Data Lime/NaOH mg/L Soda Ash mg/L Alum mg/L pH P.U.H. Calculation 53 September 2007 CIVL 407 Lab M anual ** By .P.P.) Note:* Total Alkalinity must include Phenolphthalein Alkalinity just as Total Acidity must include “MO” Acidity.) True Colour (A.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.) (Filtered) Turbidity (N.H.T.A.
. . . . . . . . . . . . . . . . . . Hardness. . . . 71 10. . . . . . . . . . . 104 24. . . . . . 69 8. COD . . . . . . . Periodic table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .COD . . . . . . . . . . . . pH . . . . . . . Introduction . . . . . . . . . . . . . . . . 74 13. . . . . . . . . . . . . . . . . . . . 68 7. . . . . . . . . . . . . . . . . . . . . . . 84 17. . . . . . . . . . . . . . . . . . . . . . . . . . . . Instructions for Laboratory Report Writing . .Levels and Water Use . . . . . . . . . . . Residues . . . . . . . . . . . . . . . . . Chemical Requirements for Water Softening . . . . . . . . . . . 105 25. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Chlorine Demand . . . . . . . . . . . . . . . . . . . . . . . 80 14. . . . . . . . . . Titrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 18. . . . . Water Softening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 9. . . . . . . . . . . . 110 27. . . . . . . . . . . . . . . . . . . . . . . . 55 2. . . . . . . . . . . . . . . . . . Alkalinity. . . . . . DO. . Coagulation . . . . . . . . . . . . . . . . . 72 11. . . . . . . . . . . . . . . . . . 112 28. . . . . . . 57 3. . . . . . . . . . . . . 64 5. . . . . . . . . . . . . . 117 29. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 20. . Turbidity. . . . . . . . . . . . . . . Chloride Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . Milliequivalent and mg/l as CaCO 3 . . BOD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acidity. . . . . . . . . . . . . 82 15. . 83 16. 60 4. . . . . . . . . . . . . . . 67 6. . . . . . . . . . . . . . . . . . Equilibrium Relationships of Chlorine . . . . . . . . . . . Typical Problems for Acid-Base and General Chemistry . . . . . . . . . . . . . . . . . . . . .pdf 1. . . . . Colour. . . . . . . . Bacteriological Examination . . . . . . . Balancing an Oxidation Reduction Equation . . . . . . . . . . . Faecal Coliforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 12. Extra tables for Chlorine Lab . . . Normal Solutions and Equivalent Weights . Solids Removal in Wastewater Treatment . . . . . . . . . . . 96 21. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hach Absorption Method . . . . . . . .4 Appendices see: CIVL407Manual_CourseNotes. . . . . . . . . . . . . . . . . 118 54 September 2007 CIVL 407 Lab M anual . . . . . . . . . . . . . 107 26. . . . . . . . . . . . . . . . . Concepts and Definitions . . . . . . . . . Reading and Reference Material . . . . . . . . Chlorine . . . . Bar Diagram Method for Water Softening Problems . . . . . . . . . . . . . . 100 22. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Known Addition Technique . . . . . . . . . . Alkalinity Species as a Function of pH . . . . . . . . . . Bacteriology and Water-related diseases . . . . . . . . . . . . . . . . . . . . . . . . 92 19. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hardness Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Additional Notes . . . . . 102 23. . . . . Standard Solutions. . . . . . . . . . . . .
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