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Prepared for Dept. Of Civil Engineering by Susan C.M. Harper September 2007
Table of Contents
Course Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Station/Drawer Supply List (if supplies are missing, please report) . . . . . 2 First Session: Laboratory Safety and Orientation . . . . . . . . . . . . . . . . . . . . 3
L ABORATORY R EPORT W RITE U P G UIDELINES . . . . . . . . . . . 5
Laboratory Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Laboratory 1: Solids Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Laboratory 2: COD, DO and BOD 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Laboratory 3: Bacteriological Examination of Sewage . . . . . . . . . . . . . . . 17 Laboratory 4: Chloride Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Laboratory 5: Chlorine and Chlorine Demand . . . . . . . . . . . . . . . . . . . . . 34 Laboratory 6: Alkalinity, Acidity, pH, Hardness, Colour, Turbidity . . . . 39 Laboratory 7a: Coagulation and 7b: Coagulation & Softening . . . . . . . . . 47
Appendices see: CIVL407Manual_CourseNotes.pdf . . . . . . . . . . . . . . . . . 54
(Environmental Laboratory Analysis - 1-3-0, 0-0-0) Testing procedures used in water quality studies and in the operation of water and wastewater treatment plants. Prerequisite Chem 151. Sc h e d u le Lecture CEME1204: Laboratory CEME1301: Wednesday 1:00 pm 1) Tuesday (time to be arranged by consensus) 2) Wednesday 2:00 to 5:00 pm 3) Thursday (time to be arranged by consensus) 4) Friday (time to be arranged by consensus) (Note: Lab Session must have at least 4 students to run)
Subject No Lab No Lab Laboratory Safety & Orientation (~1 hr) #1 Solids determination #2 Dissolved oxygen, COD, BOD No lab - Thanksgiving week - catch up #3 Bacteriological Examination of waste water #4 Chloride #5 Chlorine demand #6 Acidity, alkalinity, hardness, colour, turbidity No lab - Remembrance day - catch up #7a Coagulation #7b Coagulation and Softening
Date 2007 Sep. 4 - 7 Sep. 11 - 14 Sep. 19 Sep. 25 - 28 Oct 2 - 5 Oct 9 - 12 Oct. 16 - 19 Oct. 23 - 26 Oct. 30 - Nov.2 Nov. 6 - 9 Nov. 13 - 16 Nov. 20- 23 Nov. 27 - 30
Lecturer: Lab instructor: T.A.s: Lab: Marker:
Victor Lo, email: Susan Harper, email: Margaret Parsotan, email: Kazi Parvez Fattah, email:
firstname.lastname@example.org email@example.com firstname.lastname@example.org email@example.com
(604) 822-4880 (604) 822-4397
September 2007 CIVL 407 Lab M anual
Sta tio n /D ra w e r Su p p ly Lis t (if s u p p lie s a re m is s in g , p le a s e re p o rt)
The following items will be maintained in each stations supply drawer or at station on bench when required: 2 pair safety glasses 2 permanent marker pens 2 stir bars and a bar retriever 1 stir plate 2 buret funnels 4 30 ml beakers 4 50 ml beakers 2 100 ml beakers 2 150 ml beakers 2 250 ml beakers 2 1 L plastic beakers 2 600 ml plastic beakers 2 400 ml plastic beakers 2 10 ml grad cylinders 2 25 ml grad cylinders 2 50 ml grad cylinders 2 100 ml grad cylinders 1 buret stand 1 double buret clamp 2 50 ml burets 2 25 ml burets 2 10 ml burets 0-14 pH strips Thermometer 2 Stir stick or spatula Filter funnel (Nalgene magnetic) Tweezers (Millipore or Gelman for membrane filters)) On benches each row: Latex gloves- small, medium and large Non-latex gloves (keep on reserve for latex sensitive persons only) Filter racks with funnels #4 Whatman filter paper or equivalent Vacuum flasks and hoses 8 1 L grad cylinders 8 3 L plastic beakers
September 2007 CIVL 407 Lab M anual
up to 10. Safety Equipment: Eye Wash Station. WHMIS: Workplace Hazardous Materials Information System is a government regulation.. 1.0 ml. Putting pens in your mouth that may have been on the bench or handled with gloves could be hazardous. packs. They are available in 0. MSDS: is a supplier information sheet that must be available for each product and describe the hazards and necessary handling requisites. 50 and 100 ml. 15. Most pipettes are calibrated “to deliver” or TD and should never be blown out.5. etc. Pipettes must be placed with tips pointing up into the pipet basket supplied. Broken Glass: Be careful not to break glass. Wearing lab coats out of the lab is forbidden. Some pipettes may be “to 3 September 2007 CIVL 407 Lab M anual . if there is room. Hygiene: At times you will be working with sewage. Sanitation: antibiotic soap for hand washing. Personal Protective Equipment: Lab coat (long). A small amount of liquid always remains in the tip and must not be blown out. They are calibrated by weighing the volume of distilled water that will flow from them by gravity. Remember to empty and rinse burets as well. 20. gloves as required. but if you do please ask for assistance. eye protection. Safety Shower. with contaminated gloves will contaminate those items. You must be informed of the hazards of a substance before using it.clean and dry. If you cut yourself. with the tip against the side of the receiving vessel. Do not put lids and small items onto racks that may slip through to the filthy drip tray. 2. Pipettes/ graduated cylinders/ beakers/ flasks: 5) 6) 7) 8) 9) 10) Volumetric pipettes are the most accurate way to measure volume. please get attendant help.2 First Session: Laboratory Safety and Orientation 1) 2) 3) 4) NO Food or drink. 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. disinfectant for bench surfaces. 25. No sandals or open-toe or heel shoes. DO NOT PUT BROKEN GLASS INTO THE REGULAR GARBAGE CONTAINER. Please ask for assistance if chemicals are spilled. These should be used whenever standard solutions are measured or when upmost accuracy is required. Spills: Wipe up all spills immediately. no matter how minor. wash down and dry the bench.0. Be aware that handling your pens. Clean-up: You must leave your work station as you found it . Please take your labcoat away in a plastic bag. calculators..0.
especially at smaller volumes.1000 mls. Ie If you want to measure 20 mls. They can be unpredictable in force and are close to eye level. markings are not accurate at all . Make sure that you label beakers and flasks to avoid confusion and waste. Chemical residues can be splashed in you face. They are containers best suited for “swirling” liquid ie to mix reagents with samples in a colourimetric determination (Lab 4 or 6). not wastefully as quantities are limited. 4 September 2007 CIVL 407 Lab M anual . A demonstration will be given.merely approximations. Some are blow-out. please rinse out immediately and set aside to dry. Erle n m e y e r or conical flask markings are not accurate. 1. 13) Stir bars: Please remove from containers before pouring contents down the drain. Graduated cylinders: Not as accurate as pipettes. 100 mls. Do not squish bulbs in the direction of another person.deliver with Blow-Out”. 10 mls. YOU WILL USUALLY NEED THE INFORMATION GIVEN TO COMPLETE YOUR LAB WRITE-UPS. NEVER mouth pipette. Additional supplies are available in the supply room (around the corner). Use the size of cylinder CLOSEST TO THE VOLUME YOU WISH to measure for best accuracy. chemicals or for titrations. They are useful for measuring samples 20 mls . Flasks: Vo lu m e tric flasks are very accurate and should be used when diluting standards (together with volumetric pipettes). 500 mls. If liquid should enter a bulb or pump. 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. Pipette bulbs or pumps must be used and great care must be taken not to contaminate bulbs. Pipettes are in drawers indicated. 250 mls. Undetected gas leaks can be deadly. LISTEN AND MAKE NOTE OF VERBAL AND CHALK BOARD INSTRUCTIONS. 50 mls. some are not meant to drain completely . 11) Gas/air/vacuum taps: Do not touch these unless instructed to do so. They are calibrated the same way as TD except that the remaining drop is blown into the receiving vessel.pay attention to the markings. do not use a 1 L graduated cylinder. 14) Glassware/supplies: Each station has a drawer where some glassware and other useful tools will be kept. Even the force of air from the air taps can be dangerous because it can contain grit or water. Used to hold/transfer approximate volumes of samples. Chemicals will be put out as required for each lab and should be used sparingly. Cylinders are available: 5 mls. 2 & 4 L Beakers: are cylindrical in shape with straight sides and flat bottoms. 12) Cup sinks: Please do not use these sinks for the disposal or delivery of liquids. 25 mls. They are usually identified by a double etched or coloured band at the top of the pipette..
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. Total Mark L ONG R EPORT L AB 7 Required Required Required Include a brief description of the procedure.L ABORATORY R EPORT W RITE U P G UIDELINES Guidelines for laboratory report write up are also given in the CIVL407 Manual Course Notes and summarized here. Sufficient detail should be provided so that what was done could be understood and the experiment could be repeated from the procedure reported. 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. State that the procedures were in accordance with CIVL 407 Lab Manual and note any deviations to the manual’s procedure. R EPORT E LEMENTS Title page Objective Materials and Method S HORT R EPORT L ABS 1 TO 6 Required Required Details are not required. The mark allocation for some sections for both reporting format is indicated in parenthesis in the table below. The short report is intended to be a concise yet complete laboratory report whereas the long report is more elaborate. Required  Required  Required  Required  Required  Required  Required   5 September 2007 CIVL 407 Lab M anual .
2. A 1 L graduated cylinder may be used in place of the Imhoff cone for the sludge sample. desiccators. samples of raw sewage.. Use pipette bulbs. 6 September 2007 CIVL 407 Lab M anual . Settle for 45 minutes. Mix the samples thoroughly and fill individually marked Imhoff cones to the 1 litre mark. gently dislodge any solids that have clung to the sides using a stirring rod. MATERIALS: -Imhoff cones. Alternatively a place will be provided in the lab to store your coat. if necessary. sludge. distilled water bottles. Do not wear lab coats outside of lab and do not take away from lab unless stored in a plastic bag. to measure sewage treatment plant efficiency in removing residue. graduated cylinders. and record the volume of settleable solids. keep hands and pencils away from your mouth and wash hands frequently. settle for 15 minutes longer. subtract an estimated volume from the measured volume of matter. Do not include any surface floating material as settled solids. A. 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. tin dishes. SETTLEABLE SOLIDS 1. balance. wipe up all spills and wash with disinfectant. oven muffle furnace. evaporating dishes.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. If large pockets of liquid form between the particles of settled matter. final effluent.
]. the solids remaining represent the fixed solids. This is a different test to the one we are doing using Imhoff cones and should not be confused. Obtain the tare weights of three aluminum dishes each containing a glass fibre filter (already pre-washed. TOTAL SOLIDS and TOTAL VOLATILE AND FIXED SOLIDS AT 550o C 1. if instructed. 3. sample source and sample volume. Place the aluminum dish into the 103°C oven to dry for at least one hour (or leave drying 7 September 2007 CIVL 407 Lab M anual 4. 6. Using the sample poured out for Part B (for consistency).TOTAL AND VOLATILE 1. prior to session). Obtain the gross weight of the dish [Note: it should be room temperature before weighing. 3. pre-fired and desiccated . 10 mls for Raw & 5 mls for sludge) of well-mixed sample and filter entire portion before pouring another portion. 4. dried and fired).] Using very small amounts of distilled water. Carefully remove filter from filtration apparatus and transfer it back to the aluminum dish. rapidly pour sample into a 50 ml graduated cylinder to approximately equal 35-40 mls (if using 50 ml dishes). stir the beaker contents and then rapidly (so that it doesn't settle) measure a volume of sample into the appropriate graduated cylinder.] Rinse the graduated cylinder with small amounts of distilled water and add to filter. Wet filter with a small volume of distilled water to seat it. [Hint: pour out small portions (say 25 mls at a time for Effl. The samples will be fired at 550° C for one hour. Make sure that you have recorded all the pertinent details: Dish #. dried. tare weight. Mix the sample in the carboy thoroughly. Obtain the tare weight of a properly prepared porcelain evaporating dish (ie one that has been washed. The furnace will then be allowed to cool significantly before dishes are removed to a desiccator. rinse the cylinder. when filtering slows significantly do not add more. .not beakers. Assemble filtering apparatus. Once dishes are at room temperature they can be re-weighed. 2. Suggested portions: 100-200 ml of final effluent. 5.so be quick. the dish will be transferred to a desiccator. SVI= settled sludge volume (ml/L) x 1000/suspended solids (mg/L)] B. Put the dish in the 103-105 °C oven for drying. 2. Calculate the mg/L volatile and fixed solids. The dish containing the dried total solids can now be placed in a muffle furnace or. C. adding the rinsings to the dish. SUSPENDED SOLIDS . Do not write on crucibles as high temperatures during the next steps will burn writing off. [Notes: to allow for rinsing and to avoid spillage when transferring dish to oven don't pour more than 40 mls]. pour about 500 mls (for Part B & Part C) into a beaker and then. The loss in weight represents the volatile solids lost from the originally measured sample volume. 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 . Subtract the tare weight to obtain the total solids weight and calculate the mg/L value. Record the total volume filtered. Conversely.done for you. position the filter and begin suction (vacuum tap). After drying overnight.[Note: Sludge Volume Index is the volume occupied by 1 gram of sludge after 30 minutes of settling. immediately after stirring the beaker. Pinch sides of dish in a bit to protect the filter from oven drafts. Quickly record the exact volume and pour the sample into the dish.[Note: it may be harder to wash out solids if you allow them to settle before pouring into the dish . on a cart in preparation for staff to do so when all are ready.
What. solids _________ % reduction fixed suspended solids _________ % reduction dissolved solids _________ Lab 1 Questions 1. They must be allowed to cool completely before re-weighing them to determine the loss on ignition or volatile suspended solids.50. Calculate the mg/L total volatile solids and total fixed suspended solids. place the dishes on a cart designated by the instructor. For the time being. compute the sludge volume index (SVI). suspended. A raw sewage sludge goes through an anaerobic digestion process. DO NOT DISCARD! The gain in weight represents the total suspended solids of the sample. 3. 8.3 and fixed solids 2. volatile. From the above determinations it will now be possible to obtain a value for the dissolved solids present in the sample. fixed (use more than one adjective to describe each substance. % reduction in settleable matter _________ % reduction in total solids _________ % reduction in volatile solids _________ % reduction in fixed solids _________ % reduction in suspended solids _________ % reduction volatile susp. cool and weigh. What two techniques can be used to overcome or correct for the loss of material from filter pads when firing at 550 oC? 4.) a) a bacterium b) sodium chloride c) sugar d) clay 8 September 2007 CIVL 407 Lab M anual . Enter all data in the tables on following page. The aluminum dish and filter (from #4) must now be fired in a muffle furnace at 550°C for at least 30 minutes (or until a constant weight is achieved). Calculate as the mg/L total suspended solids. 6. where the volatile solids are reduced from 65% to 40%. What major types of solids are removed in primary treatment? in secondary treatment? 2. affect would there be to the TSS and TVSS results if your bare hands were used to handle a tin dish a) prior to obtaining the initial tare weight and b) after obtaining the initial tare weight? 5. 7.5. Calculate the percent solids reduction through the treatment plant.e. if any. After firing the dishes will be moved to a desiccator. If sludge is used in Part A. Classify each of the following into its proper solids category(s) i. If all of the volatile solids reduced is given off as gas and if the specific gravity of the volatile solids is 1. Please return tomorrow to obtain final weight. Discuss the meaning and significance of this index. what is the percentage reduction in solids. Lab personnel will perform the timed-firing after the class. Your hands may have moisture and natural and/or applied oil/lotion on them. overnight). Transfer dish to a desiccator. dissolved. 6.
use embossed mark) Sample volume (ml) Gross weight . Settleable Matter After 1 hour (mL/L) B.oven dry (G) Tin dish with filter tare weight (G) Total suspended solids (G) Total suspended solids (mg/L) Gross weight .fired (G) Loss on ignition (G) Volatile suspended solids (mg/L) Fixed suspended solids (mg/L) Dissolved solids (mg/L) 9 September 2007 CIVL 407 Lab M anual Final Effluent .DATA AND RESULTS Raw Sewage Aerobic Sludge A. Total Solids Dish marking (do not use ink . Suspended Solids Tin dish marking (do not use ink .use permanent mark on dish) Sample volume (mL) Gross weight .fired (G) Loss on ignition (G) Volatile solids (mg/L) Fixed solids (mg/L) Percentage fixed residue C.oven dry (G) Dish tare weight (G) Total solids (G) Total solids (mg/L) Gross weight .
COD . spectrophotometer. Keep fingers. NEVER pour water into acid. The closed reflux method is more economical but because a small sample volume (2 mls) is used. Always use and store pipettes with the top higher than the tip.La b o ra to ry 2: CO D . etc. as well as handling samples probably containing pathogenic bacteria. etc out of your mouth. otherwise reagents will run to the bulb end and make pipetting difficult and contamination likely. and to demonstrate the use of dissolved oxygen probes.Closed Reflux. Digests from either method can be titrated or read colourimetrically. 10 September 2007 CIVL 407 Lab M anual . vials with pre-measured reagents. Wipe up all spills immediately. wash/rinse bench tops with water/disinfectant. 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). 200. I. 100 and 50 mg/L as COD. Materials: Raw sewage and final effluent samples. Wash hands frequently with soap. pencils. D O a n d B O D 5 WARNING You will be using concentrated acid and other corrosive and toxic chemicals. 400. homogenization of samples containing suspended solids may be necessary in order to get a representative sample and to improve reproducibility. pipettes. OBJECT: -to familiarize you with the commonest tests run for assessing treatment plant efficiency and pollution loading to receiving waters. to illustrate the shortcomings of these tests. Hach block digester.
II. 50 and 0 COD as mg O 2 /L. Pipette 2 mls of sample (using volumetric pipettes unless particulate is present. well-mixed samples in the 25-position digester. manganous sulfate. Note: You are going to measure DO on four different liquids using two different methods. 4. 400. Read your samples and the set of COD standards: 800. BOD bottles. 11 September 2007 CIVL 407 Lab M anual . aerated dilution water. Put identifying labels on each tube ie your initials. 6. and are at the spectrophotometer. Prepare calibration curve. Make sure sample is well mixed with the acid contents of the vial. If less than two mls of sample or diluted sample was used. a volumetric correction must be made to the value obtained from the curve. The vials should be clean and dry on the outside.Procedure: 1. 0. 200. Raw Sewage (Infl) and/or Effluent (Effl) in the upper 1/4 of the tube using permanent markers. 2. except without mercuric sulfate). 10-ml graduated pipettes. Allow samples to digest for 30 minutes (normally this would be 2 hours). burette stand with burettes. 500-ml Erlenmeyer flasks.noting the location of your well-labelled. 100. a smaller aliquot (making volume up to 2 mls with distilled water) or 2 mls of a pre-diluted sample can be used. The four liquids are cold tap water.the azide modification of the iodometric method) and a dissolved oxygen probe and meter. Remove the vials and allow them to cool to room temperature (use cold water to speed up if necessary).025 N sodium thiosulphate. use wide mouth graduated pipettes) in duplicate. thermometers. starch indicator. 250-ml graduated cylinder. The instructor will demonstrate the use of the spectrophotometer. The probe has been previously calibrated. before the lab. in which case. into the appropriate tubes and screw cap on snugly. You will use a chemical titration (Winkler . absorbance versus concentration and calculate the COD in samples. DISSOLVED OXYGEN (DO) Materials: Raw sewage and final effluent samples. [Note: it should look homogenous and be careful it will be very hot!] 3. which is set at 600 nm to read absorbance of light by the sample. alkaline-iodide-azide reagent. Standards have been digested for you. 5. DO NOT ADJUST THE SETTINGS ON THE METER. At your station you will find four vials with pre-measured solutions containing potassium dichromate and sulfuric acid (prepared according to standard methods. Take vials to fumehood and place in block digester . dilution water. If sample is known to be outside the standard range (above 800 mg/L). concentrated sulphuric acid. raw sewage and final effluent.
Record the temperature of each sample using the DO meter or a thermometer. 4. dropwise. Instructions on the use of the probe will be given in the lab. add 1 ml of concentrated H 2 SO 4 and quickly restopper. invert several times again and allow to settle again. Titrate this volume with 0. then the same sample will require little or no titre and may not turn blue when starch is added . To the same bottles. until the first complete disappearance of the blue colour. Fill four BOD bottles with the four samples (cold tap water. Add enough starch solution to give a strong blue colour (one dropper full) and continue to titrate. Note: if solution already seems pale yellow add starch right away. For the cold tap water sample. tip the liquid out of the space around the stopper (caution -it may be corrosive) and invert several times. Determine DO. 1.) Transfer 201 ml of each bottle (do one at a time) to a 500 ml conical flask. . add 1 ml of alkaline-iodide-azide solution the same way. Fill a BOD bottle with each of the four samples (or use ones saved from A ).think about it!) Complete all four titrations. WINKLER TITRATION (Azide modification) NOTE: Use only the droppers attached to the reagent bottles for dispensing into samples. The DO concentration in mg/L is equal to the number of ml of titre as long as 201 ml is titrated. Determine the percent saturation for each sample. Record the volume of titre. B. 7. Take each stoppered bottle. (Note: biological solids originally present in the sample will not dissolve. 1. (Fill carefully. Use the specially marked volumetric flask to dispense the 201 ml. (Note: DO meter temperature is usually not accurate. DISSOLVED OXYGEN PROBE NOTE: DO NOT add any reagents to the samples used for the probe method. When the floc has settled the second time to about 1/3 bottle volume. remove the stopper. for each of the four samples.) You can save these bottles for 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. The chemical floc should completely dissolve. taking care not to entrain additional air. aerated dilution water. Stopper the bottles without trapping any air bubbles. Do not mix droppers and do not allow tap water into the reagent bottles. to thoroughly mix contents. 2. using the DO probe and meter. as demonstrated. Neglect any reappearance of the blue colour. right to the top and place stopper on bottle. raw sewage or influent and final effluent). 8. Allow the floc to settle to about 1/3 the bottle volume. 2.025 N Na 2 S2 O 3 until only a faint yellow or "straw" colour remains. Tip out the liquid from the area around the stopper and invert several times to mix thoroughly. Place the bottles in the sink and one at a time remove the stopper. 12 September 2007 CIVL 407 Lab M anual 3. 5. submerge the hose in the bottle and allow the water to overflow for 15-30 seconds).A. 9. again quickly replacing the lid. Set back in sink. 6. (Hint: if you measured very low DO level with the meter.
Pair up the mg/l of dissolved oxygen you measured and the temperature of the water in degrees C. Draw a straight line between the water temperature and the mg/l of dissolved oxygen. (mg/L) DO conc. Note that this nomogram assumes that the water is at sea level The saturation value can also vary slightly depending on barometric pressure with lower values expected when a storm front moves through as compared to bright and sunny "high pressure" days. use the saturation chart above.Dissolved Oxygen Data Sample/Data Bottle # Sample Volume (mls) Initial Buret reading Final Buret reading Net Titre (mls) DO conc. Raw Sewage Final Effluent Tap Water Dil’n Water 13 September 2007 CIVL 407 Lab M anual . (Probe) Temp (oC) (thermometer) Saturation concentration Percent saturation DETERMINING PERCENT SATURATION THE "QUICK AND EASY" METHOD For a quick and easy determination of the percent saturation value for dissolved oxygen at a given temperature. The percent saturation is the value where the line intercepts the saturation scale.
5. In addition. Materials: Raw sewage and final effluent samples. Because you want to be sure that the dilution water has no BOD. showing sample calculations. 6.do not write on the bottles and do not use the numbers on the lids. dilution water. The blank is used to check that the dilution water has negligible BOD. Stopper the BOD bottles. 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. 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). All bottles should have liquid in the well around the stopper . Dissolved oxygen meter and probe. Measure the Initial DO using a calibrated probe and meter. Using the volumes and samples provided by the instructor. Place plastic caps over all bottles. Be sure to note this in your lab write-up. Carefully top up the BOD bottles with DILUTION WATER. 1. There should be negligible BOD in the Dilution water. groups). BIOCHEMICAL OXYGEN DEMAND Note: Because of the logistics of organization some groups may have 6 day BOD 5 instead of the usual 5 day. Calculate the 5-day (or 6-day if unavoidable) BOD. BOD bottles. If the dilution water BOD is greater than 1 ppm. graduated cylinders. fill one BOD bottle with DILUTION WATER alone. Measure the Initial DO of the blank using the calibrated probe and meter. 10-ml graduated pipettes. Note: Each group should have 9 BOD bottles in the incubator. After five (or six)days. but if there is a significant BOD then it will have to be subtracted before the dilution factor is applied in the calculation. Be sure to record the bottle numbers and sample volumes on the following table . it may indicate that probe may not have been calibrated properly or the dilution water had been contaminated. thereby maintaining a water seal for each bottle. ie Rinse the filling tube if it has been in the sample. 4. carefully measure the waste water samples into BOD bottles. preferably using the same meter and probe used to get IDO reading. 3. These caps are designed to prevent the evaporation of the liquid around the stopper. be careful not to contaminate it with sample. being sure to exclude any air bubbles that might be trapped under the stopper by adding dilution water. Place all bottle sets in the 20 o C incubator for five days (or 6 for Tues.III. to bring the levels above the ground glass neck of the bottle.if not add a some dilution water (or distilled water) to the well. 14 September 2007 CIVL 407 Lab M anual . remove bottles and measure the DO.
mg/L DO of seed control after incubation. mg/L. = = = = decimal volumetric fraction of sample used DO of seed control before incubation. 15 September 2007 CIVL 407 Lab M anual .BIOCHEMICAL OXYGEN DEMAND (BOD) DATA Sample source: ________________________________ Raw Sewage Bottle number (not lid #) Sample volume (ml) Initial DO (mg/L) Final DO (mg/L) Final Effluent Bottle number (not lid #) Sample volume (ml) Initial DO (mg/L) Final DO (mg/L) Dilution water Bottle number (not lid #) Sample volume (ml) Initial DO (mg/L) Final DO (mg/L) _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ BOD Calculation When dilution water is not seeded (our water is not seeded for this exercise): When seeded water is used: where: D 1 = D2 P B1 B2 f mg/L. DO of diluted sample immediately after preparation. and ratio of seed water in diluted sample to seed in seed control= (%seed in diluted sample) / (%seed in seed control). mg/L = DO of diluted sample after 5 d incubation at 20 oC.
Calculate theoretical oxygen demand for 300 mg/L of methyl alcohol.Summary Raw Sewage.) Why is your COD result different from the BOD result? 5. Diln. Diln. 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. the other 0.17. 2 Final Effluent. titrimetric method. if at all possible. 6) 7) 8) 16 September 2007 CIVL 407 Lab M anual . Both wastes have equal BOD 5 temperature and volume.25. 2) 3) 4) Show on a graph the approximate dissolved oxygen sag curves in a river receiving two wastes. 1 Final Effluent. 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 Raw Sewage. what would you expect its I) BOD 5 and ii) BOD L to be. open reflux. If an industrial waste has a COD of 450 mg/L. Diln. One waste has a K value of 0. Diln.
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 . OBJECT: -to demonstrate different methods for the determination of the coliform group of organisms and to acquaint you with bacteriological procedures. EC tubes and/or Hach A1 tubes. Wipe lab benches with disinfectant before and after each lab. HACH P/A Broth with MUG. eye protection. membrane filters.La b o ra to ry 3: B a c te rio lo g ic a l Exa m in a tio n o f Se w a g e WARNING As you are handling and pipetting samples probably containing pathogenic organisms please observe the following rules: Wear gloves. sterile dilution water. Demonstration only SAMPLE: You will do either a raw sewage sample or a final effluent sample. Wash hands with soap before leaving. away from your mouth. pencils. Brilliant Green Lactose Broth tubes. Keep hands. sterile pipets. Heterotrophic plate count: dilution tubes.. 35oC incubator. 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 .leave it here or take away in a plastic bag to launder. DO NOT MOUTH PIPETTE. 35 oC incubator. Demonstration only. sterile pipettes. Get raw data from another group and include in your report. T h e re f o re . Students will do only sections marked ? MATERIALS: Multiple tube method: dilution tubes. Membrane filter method: dilution tubes. plates. not both. etc. filtering apparatus. Quebec colony counter. LES Endo agar plates. Do not wear lab coat out of lab . agar tubes. lab coats. lauryl tryptose broth 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 . wipe up all spills and disinfect the area of the spill.
Membrane Filter Technique: Serial dilutions of sample are collected on membrane filters and placed on M-Endo media. Demonstration. D.) Methods: ? Recording BGLB. Completed Phase LES Endo agar Plates: A p la te w ill b e a v a ila b le to lo o k a t.7oC for 24 hours (confirmed test for faecal coliforms). as demonstrated. 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.coli. EC or A-1 tubes positives: 1. Hach’s Most Probable Number Method 8368.A.5 oC) as a positive completed test response for both Total and faecal coliform. Consider positive EC broths incubated at the elevated temperature (44. 1. Using aseptic technique. E. It is used as a faster alternative to the LTB/EC procedure for enumerating faecal coliforms in water. Heterotrophic Plate Count . Another series of tubes are inoculated from these positive lauryl tryptose tubes. ? B.Pour Plate Method: Serial dilutions of samples mixed with agar and incubated for 48 hrs at 35oC. Presence/Absence Testing: HACH technique to simultaneously screen for total coliform and E. The broth will turn yellow indicating total coliform and fluoresce in the presence of E. 3. Use Table 2 on page 27. MUG produces a fluorogenic product when hydrolyzed by an enzyme specific to E. Calculate the MPN value from the number of positives using the Table 1. is USEPA-accepted for testing non-potable waters. The number of colonies counted directly. 18 September 2007 CIVL 407 Lab M anual . 2. 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. Express as Total coliforms.COLI in 4 to 24 hours. Microscope Slides (Completed Phase of Multiple-Tube Fermentation): Single colonies arising from single cells are prepared and stained for microscope examination. (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. using a ready-to-use P/A Broth with MUG.coli. All tubes have been examined for gas production. ? C. Parallel positive BGLB broth cultures with negative EC broth cultures indicate the presence of nonfaecal coliforms. Gas-positive Hach A-1 tubes indicate the presence of faecal coliform. Bacterial numbers are calculated from the numbers of positive tubes. Streak plates in a manner to insure presence of some discrete colonies. Colonies that are pink to dark red with a golden green metallic sheen are counted after incubation 20-22 hrs at 35oC. Gas formation indicates the possible presence of coliform organisms. Note: these methods will be demonstrated but data must be recorded. streak one LES Endo agar plate from each tube of BGLB broth showing gas from the highest dilution and the second highest dilution. 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). Results will be provided for the demonstration set. A-1 Medium.
The filtration apparatus has been sterilized by autoclaving. Filtering..1. Incubate plates (inverted) at 35 oC for 24 ± 2 hr. or colourless and are considered to be non-coliforms. -Note: “typical” colonies count as coliforms in this procedure. Do not push down on the filter except on the outside edge and use only sterile forceps . Inoculating. Be sure dishes are labelled with your name and dilutions. 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. Mount apparatus on suction flask with valve sideways to block suction. prepare one set of six dilution tubes following the instructions given under Multiple Tube Fermentation. When filtering is complete and the valve is closed. Close valve and rinse the funnel with three aliquots (portions) of sterile dilution water opening and closing valve after each aliquot. If you haven’t already. Verification is usually done by a test for lactose fermentation. The typical coliform colony has a pink to dark-red colour with a golden green metallic surface sheen.2. 5. Pour the full volume of dilution tube # 6 onto the filter. therefore the results table should specify #coliform colonies/100 ml. Repeat for the rest of the dilution tubes to # 2. coliform bacteria are Gram-negative (pink) rods (sometimes coccus-like). Place the filter on a small LES Endo agar plate carefully. Atypical coliform colonies can be dark red or nucleated without sheen. Incubate inverted Petrie dishes for 20-22 hr at 35 oC. with a motion that excludes air as it comes in contact with the agar. Heterotrophic Plate Count . Using a sterile pipette. Dilution tubes. removing the cover just enough to insert pipette tip and dispense sample. The sheen area may vary in size from a small pinhead to complete coverage of the colony surface. 4. remove filter using flamesterilized forceps (allow forceps to cool or they will damage the filter). Flame loop between second and third quadrants to improve colony isolation. Pipette about 10 ml of sterile dilution water over the surface of the filter with the suction valve still closed. Verification of both types of colonies is advisable as some typical sheen colonies may be noncoliform and some atypical may be coliform. Dilution tubes. 2.applying gentle pressure. ?C. Gram staining technique can be also be used. 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. After 20-22 hr. choose appropriate dilutions for counting.Pour Plate Method 1. Use dishes with 20 to 80 coliform colonies and not more than 200 of all types. ?B. white. Prepare one set of six dilution tubes as described in Section A. 3. place a sterile membrane filter (grid side up) onto the filtration apparatus. Using sterile forceps. A high count of non-coliform colonies may interfere with the maximum development of coliforms. In general. from one edge. Counting. Prepare Gram Stain slides for examination under a microscope. Colonies that lack sheen may be pink. Membrane Filter Technique -Total Coliform 1. open the valve slowly and allow all the liquid to be pulled through. The sheen area may vary in size from a small pinhead to complete coverage of the colony surface. 19 September 2007 CIVL 407 Lab M anual . 2. red.
Incubate at 35 oC for 24-48 hours. Do not report as “too numerous to count” if all plates exceed 300 colonies. if fewer than 10/cm 2. Repeat with dilutions 5 to 2 (for Raw) and 4-1 (for Effluent). D. Examine for yellow colour (total coliforms) and fluorescence (E. Add 100 mls of sample directly to pre-measured medium provided. Microscope Slides: 1. Slide preparation: With a flame sterilized inoculating loop. 20 September 2007 CIVL 407 Lab M anual . Take 5 tubes of melted agar from the incubator. Flood the slide with fresh alcohol-acetone solution to act for 30 seconds. c. Counting.coli). After 48 hr incubation. Let them cool down from the 60C that they are kept at but not enough to solidify. Allow iodine to act for 30 seconds. b. marking (on edge of dish so as not to impede counting) and incubating for 48 hr at 35 oC. 2. If necessary. pour and replace cover. refer to Standard Methods for more instruction. Drain off excess iodine and wash freely with water and then alcohol-acetone decolourising solution. Instead. count four representative squares. Flood slide with crystal violet solution and allow to act for 30 seconds.too hot for a baby . ?4. Ideally. If more than 10/cm 2. place 1 loopful of sterile dilution water in the centre of a slide.3. Presence/Absence Testing . Preferably select seven consecutive squares horizontally and six consecutive squares vertically. count colonies in 13 squares (of the colony counter) of the most dilute plate having representative colony distribution. to mix the sample with the agar.too hot for a bacterium. Record results of previously prepared test. 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. 2. average and multiply by 57 (for plastic Petrie dish). select the appropriate dilution to count. Label the slide using a grease pencil (T or A). Spread the colony in the drop of water so that you get a uniform dispersion over an area about the size of a dime. Use the Quebec colony counter to aid in counting and a grease pencil to mark off colonies counted. Wash off stain with water and then with iodine solution (Lugol’s). Work quickly or the agar will solidify in the tube before you get a chance to pour it. e. 5. d. Air dry the slide high above the Bunsen burner and fix by passing slide briefly through a flame. Repeat for the rest of the dilutions. a.d e m o n s tra tio n -s ta rte d a h e a d o f la b 1. The aim of using several dilutions is to have at least one dilution giving colony counts between these limits. Slide the cover of a Petrie dish back just far enough to pour the agar. use the plate(s) that will give from 30 to 300 colonies/plate. 4. Resterilise the loop and remove one colony from the surface of the Les Endo streaked plate (prepared previously). 6. ?3. Allow the agar to solidify in the dish before inverting. A hand counter is also available. Slide staining. Do this for a typical and an atypical colony. Wash slide in tap water. on the bench. E. f. Test temperature on your wrist . Gently swirl the dish.
transparency. length to width ratio. dull. a. Examine the prepared slides .Come in and count Heterotrophic Plate colonies after 48 hours.Examine prepared slides. . short. . odour. .5 . rough. sometimes coccus-like and 0. plump rods. concave). nonsporing.do not turn knobs except the positioning ones. Note: in the staining process: before decolourisation all organisms appear Gram positive (blue).Prepare dilutions for Heterotrophic Plate Count and Membrane Filter technique. cocci or rods. h. Gram negative organisms are visualized (pink).Record Presence/absence testing results of Hach Prepared Media bottles. and on the slide: typical or atypical. Wash in water. c. BGLB and EC and AI tubes .counts will be provided on the board. cell groupings (single. gram positive or negative. colour.Record positives in all Multiple Tube Fermentation inoculations: LTB. metallic. Day 2. Apply fuchsin or safranin (counter)stain for 30 seconds. blot and then dry in air high above the Bunsen burner (so as not to burn away your efforts). 21 September 2007 CIVL 407 Lab M anual . usually motile.Come in and count colonies on LES Endo Membrane Filter dishes. Day 3. . In general.g. in pairs. Microscopic examination.3 µm in size. Schedule Summary Day 1. pairs chains). . b. . place on LES Endo agar dishes and incubate. smooth. After application of the counterstain.Membrane filter prepared dilutions. Plates ready on a weekend will be put in cold room until Monday. Description of bacteria: Describe what you see on the plates: colonial morphology of each type of colony seen ie surface (shiny.Prepare and incubate Heterotrophic (Pour) Plates using dilutions above. ?3. . after decolourisation Gram negative organisms are no longer visible. coliform are Gram-negative (pink). . or in short chains. Cells occur singly.
1/ 10 and 1/100 are used. 35 oC/48 hr Tube # 1 2 3 Mls of inoculum Infl Effl Plate count Infl Effl *CFU/ml Infl Effl *CFU= colony-forming units Membrane Filter Technique Tube # Dilution (mls) Infl Effl Coliform Colonies Infl Effl #Colonies/100 mls Infl Effl 1 2 3 4 5 6 4 5 6 22 September 2007 CIVL 407 Lab M anual .pour plate method.. Multiply the MPN index by the dilution factor from the series used when dilutions other than 1/1.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 2 3 4 5 6 Heterotrophic Plate Count .
coli because it produces a fluorogenic product when hydrolyzed by glucuronidase. 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. an enzyme specific to E.1 ml 5/5 4/5 1/5 0. you can simultaneously detect total coliforms and E. that in the denominator. In the examples given below.Most Probably Number (MPN) When more than three dilutions are used in a decimal series of dilutions.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 . 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. With MUG reagent added to P/A Broth. use the results from only three of these in computing the MPN. If zero total coliform is the maximum contamination goal. To select the three dilutions to be used in determining the MPN index. Do they agree? Why/why not? -compare MPN/MF with Heterotrophic Plate Count. the total tubes planted.Discussion: -sources of error? -compare MPN. E. Both media meet USEPA guidelines for testing of total coliforms in drinking water. it is not necessary to enumerate. A long-wave UV light is required to detect fluorescence. Use the results at these three volumes in computing the MPN index. MUG (4methylumbelliferyl-$-D-glucuronide) can be used to detect E. The number in the numerator represents positive tubes. MF results.coli will fluoresce as early as 4-24 hours. the significant dilution results are in boldface.01 ml 2/5 2/5 0/5 0.coli. 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. They are in concentrated form and merely require the addition of 100 ml of sample (or diluted sample) to the media and incubation.coli. Estimation of Bacterial Density .
MPN Index and 95% Confidence Limits for Various Combinations of Positive Results when 5 Tubes are used per Dilution (10 mL. 1.1 mL) Comb.Table 1 . Of Positiv es 0-0-0 0-0-1 0-1-0 0-2-0 1-0-0 1-0-1 1-1-0 1-1-1 1-2-0 2-0-0 2-0-1 2-1-1 2-2-0 2-3-0 3-0-0 3-0-1 3-1-0 3-1-1 3-2-0 3-2-1 4-0-0 4-0-1 4-1-0 4-1-1 4-1-2 MPN Index/ 100 ml 95% Confidence Limits Lower 1 1 1 1 1 1 2 2 1 2 3 3 5 3 4 4 6 6 7 5 7 7 9 12 Upper Comb. 0.0 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 .
MPN Table 2 25 September 2007 CIVL 407 Lab M anual .Hach .
D. What are the important pathogens transmitted by water? How is quality control achieved in microbiological analysis. 2) 3) 26 September 2007 CIVL 407 Lab M anual .http://www.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/keen/Gram stainkeen. Mara: -Bacterial Indicators/ Health Hazards Associated with Water -Bacteriology for Sanitary Engineers Microbiology 321: -Manual of Microbiological Techniques Website: http://www.htm Lab 5 Questions: 1) Compare the advantages and disadvantages of the multiple tube fermentation and membrane filter techniques.Further information can be found in the laboratory library (do not remove without permission): Standard Methods: -Part 9000 Microbiological Examination HACH publications:-Catalogue listing testing methods with descriptions. -The Use of Indicator Organisms to Assess Public Water Safety .com/ ¸Information Central/Learning Library/Microbiological Testing ASTM: (STP635) D.hach.
goggles or glasses and lab coats.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. 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. That means you will probably have to dilute the sample by the most accurate means available. graduated cylinders. MATERIALS: -millivolt meter. Note and consider carefully: Different methods of analysis have different optimum ranges. magnetic stirrer. HNO 3. semi-log graph paper. indicator-acidifier reagent. to illustrate the differences in operation and accuracy between instrumental methods and wet chemical methods. reference electrode (double junction). pH meter with double-junction electrode and silver billet electrode. standards. volumetric pipettes. Mercuric thiocyanate is very toxic. beakers. If chemicals are spilled alert instructors. indicator. NaHCO 3. chloride specific ion electrode. spectrophotometer. Hint: optimum ranges will coincide with the standards provided or will be indicated within the pertinent section. known addition and calibration techniques and colourimetric and potentiometric titrations. standard Hg(NO 3)2. Use personal protective equipment: gloves. 27 September 2007 CIVL 407 Lab M anual . conc. burettes. sample. standard chloride solution (0. 125 ml Erlenmeyer flasks. OBJECT: -to acquaint you with the use of specific ion electrodes. beakers. immediately.5 mg/L). buffer. standard AgNO 3 solution.
added to the sample "slope" = change in potential over 10 fold change in concentration of standard (use data from Step 2). Determine the concentration of chloride in the sample. 2. Calculate the chloride concentration. 4. Using semi-log paper or software equivalent. added 2 mls of ISA.= ____________________ 28 September 2007 CIVL 407 Lab M anual . 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 ). As probe tends to drift for some time it may be expedient to select a “standard time to read” (i. 3. Standards of 1000.SPECIFIC ION ELECTRODE Part One: Preparation and reading standards and samples 1. Add 2 mls of IONIC STRENGTH ADJUSTMENT (ISA) buffer to each using the plastic dropper (filled to the mark). plot the millivolt readings (linear axis) against the concentration of the standards (log axis) to produce a calibration curve. labelled beakers. Unless already done by a previous group (values written on the board). Unless already done by a previous group. 6. pour out 100 mls of each standard into separate.in sample = mg/L Cl.e.E 1 volume of sample volume of stock solution added mg Cl. 2 minutes) and use it for all standards and samples. rinsing and blotting dry the electrodes between each. Repeat for rest of standards. Measure 100 mls of sample. mg Cl. 5. Turn on stirrer and while stirring record the millivolt (mv) reading when the reading appears to be stable.I. where: E1 = E2 = E = Vo = Vs = A = S = potential of sample (mV) potential of sample plus stock addition potential change = E 2 . place the lowest standard on the stirrer and lower the electrodes into it. CHLORIDE . 100 and 10 ppm (mg/L) have been prepared for you and are at the meter. stir and obtain a reading for it.
using the dispenser provided.) Mix thoroughly by gently swirling the flask.8.they can’t all be at 10 minutes at once. Hg ++ + 2Cl. Pour 100 ml of sample (or a portion diluted such that the concentration is less than 100 mg/L) into a 250 ml beaker.+ Hg(SCN)2 º HgCl2 + 2SCN SCN . after a few more minutes. add 5 ml of the “combined reagent” (containing ferric alum and mercuric thiocyanate solutions) to the blank first. 2.5 ± 0. (Alternatively.) Every two or three minutes thereafter you will rinse the cuvette with the next standard or sample. Using a 25 ml graduated cylinder..0. which then must be subtracted from the standards and sample. 4. MERCURIC NITRATE METHOD FOR CHLORIDE DETERMINATION The principle of this method is as follows: Cl.ions have been bound. it should be rerun from the start after diluting. insert in the spectrometer and record the absorbance displayed on the meter.+ Fe+++ º Fe(SCN) ++ (Reddish brown) 1.) Prepare a calibration curve by plotting the absorbance readings versus the concentration of chloride in the standards. measure 25 mls of “zero” standard or “blank” (halide-free distilled water). a pH of greater than 3. pure blue. use a plastic dropper to transfer blank into a spectrophotometer cuvette (do not fill more than 3/4 ). Near the end-point. III. 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.0. and the sample (diluted if necessary) into separate and labelled 125 ml Erlenmeyer flasks (5 in total). 2. 4. CHLORIDE . Determine the chloride concentration in the sample from the calibration curve. (Use extreme caution as this is a corrosive & toxic chemical. discard and refill at least twice to rinse cuvette. After all Cl.] After 10 minutes has passed (since the combined reagent was added to the blank).1. Wait two or three minutes and then add the reagent to the first (lowest) standard. 3.0 ppm).0 and 8.II. zero instrument on distilled water and obtain an absorbance value for the blank. For most samples. Add 1 ml (use pipette to measure accurately) of indicator-acidifier reagent to sample. [Note: this delay between additions is to allow you time to measure absorbance of each at the 10 minute time required . (Light green indicates a pH of less than 2. 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 . 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). 6.º HgCl2 1.BY COLOURIMETRIC METHOD The principle of this method is based on the following formula: 2Cl. (No further adjustment is required after the blank or distilled water has been set to zero. the three standards (2. The colour of the solution should be green-blue at this point. Marking the time (T=0). and Hg ++ (excess) + DPC º violet colour 3. Titrate with 0. the excess mercuric ions combine with diphenylcarbazone (DPC) to produce a violet colour (the end-point).ions in the sample combine with added mercuric ions forming a soluble but virtually undissociate-able complex. fill. 5. If sample is above the highest standard. the 1 ml of the indicator-acidifier reagent will adjust the pH to 2.014N Hg(NO 3) 2 to a definite purple end-point.
0 ml (use volumetric pipette) of standard chloride solution in a 250 ml beaker.if one is necessary. Rotate the work within the group so that different pairs are doing the titrating and recording of numbers. Begin titrating. Part one: Standard 1.0 ml of concentrated HNO 3 (use plastic dropper). The titre from this step must be subtracted from the titre for the sample. Start the stirrer and set the millivolt reading to zero or record initial mv reading [Note: Some meters can not be set to zero]. Calculate the chloride concentration: 5. until past the endpoint when larger increments can be used again (ª mV/ml decreases). as the end-point of the reaction is approached (ª mV/ml increases).2 ml or drop-wise) should be added. Nitric acid is in the sink and is very corrosive. CHLORIDE BY POTENTIOMETRIC TITRATION Note: For this part form two groups comprised of 1 member from each station. place beaker on stir plate and lower electrodes into the solution. dilute to about 100 ml with distilled water. Take care that stirrer is not hitting the electrodes. The end-point occurs at the greatest mV change per unit addition of AgNO 3. recording the volume and millivolt reading for each increment. Plot a differential titration curve to determine the exact end-point.4. drops of the purple end-point. Place 10. Continue the titration about 5 ml past the end-point. 6.. This way each station will have data for each part. smaller increments (0. Immerse stir bar. 4.. where: A = ml titrant for sample B = ml titrant for blank N = normality of titrant IV. in increments. Calculate the Normality of the AgNO 3 using the following equation: 30 September 2007 CIVL 407 Lab M anual . with standard AgNO 3. The first group will do the standard (part one) in duplicate or triplicate depending on the size of the group and the other will do the sample (part two) in duplicate (or triplicate). 2. then. At the start. and add 2. 5. large increments (1-2 mls) may be added. A reference set of data will be provided for comparison.1 to 0. Don’t forget to add the indicator. Determine the blank by titrating 100 ml of distilled water containing 10 mg of NaHCO 3 which serves to provide some alkalinity to the blank (to make it similar to the sample) and provides a correction for alkalinity and reagents . 3.
) Plot three curves for this titration: a. which are merely first and second derivatives. It is not. Where: A = ml AgNO 3 B = ml Blank N = normality of titrant (AgNO 3) D = ml of sample ml From graph a b c mg/L Cl- S))))))))Q S))))))))Q S))))))))Q S))))))))Q S))))))))Q S))))))))Q 31 September 2007 CIVL 407 Lab M anual . b. 2. 4. Millivolts per ml per ml versus ml of titrant. c. Add 2 ml of HNO 3 and proceed as described in Part One. if dilution is necessary into a 250 ml beaker (use a graduated cylinder to measure). Millivolt reading versus ml of titrant.Part Two: Sample 1. In plotting b and c. be careful to use the average value of ml of titrant on the abscissa. in this case. Measure 100 ml of sample or portion made up to 100 ml. Calculate the chloride concentration in the sample using the following equation: 3. (If pH of the sample were quite high it would be necessary to first adjust the pH to about 4 and then add 2 ml HNO 3. Millivolts per ml versus ml of titrant.
Potentiometric Titration RAW DATA A Volume AgNO 3 Added (Vol) (ml) B Meter Reading (E) (mV) C FIRST DERIVATIVE D SECOND DERIVATIVE F G H ª (Vol) (ml) ª (E) (mV) E ave (AgNO 3 ) Volume (ml) ª (E)/ ª (Vol) (mV/ml) ª ( ª E/ ª Vol) (mV/ml) ª [Ave(Vol)] (ml) I Ave of Ave(Vol) (ml) J ª( ª E/ ª Vol) /ml (mV/ml/ml ) Extra pages are at the back of this lab manual in appendix. 32 September 2007 CIVL 407 Lab M anual .Chloride .
From your chloride results. fluoride and bromide titrate as chloride. Each sample type requires development of an appropriate method which. COLOURIMETRIC METHOD # Bromide. fluoride and bromide titrate as chloride. This is a complex. thiosulphate. # Colour in the sample may interfere in the absorbance measurement. # High levels of ions which form very insoluble salts of silver may deposit a layer of salt on the electrode membrane. # Pretreatment of environmental samples usually required.3 mg/L? Outline the advantages and disadvantages of all the methods used in this lab to measure chloride concentration.INTERFERENCES IN CHLORIDE DETERMINATIONS POTENTIOMETRIC METHOD # Iodide. 33 September 2007 CIVL 407 Lab M anual . requires knowledge of the compounds in the sample which are likely to interfere. Also. 2. # Mercury must be absent from samples. # Colour may obscure or interfere with end-point determination. It can only correct for substances that enhance or suppress the test response proportional to the amount of chloride ion present. in itself. # Environmental samples usually require pretreatment. # Environmental samples usually require complex pre-treatment. # In practice. ferric ion. dichromate. MERCURIC NITRATE TITRATION METHOD # Iodide. # Sample pH may need adjustment beyond the capacity of indicator-acidifier reagent. and nitrite interfere. KNOWN (STANDARD) ADDITION TECHNIQUE # The known addition techniques will only correct for certain types of interferences. iodide. timeconsuming process requiring a sound knowledge of chemistry and extensive experience working with environmental samples. cyanide. causing electrode malfunction. hydrazine. and sulfite ions interfere when concentration greater than 10 mg/L. 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. PRETREATMENT TECHNIQUES # There are no "standard" pretreatment techniques. SPECIFIC ION ELECTRODE # All halides interfere (the electrode is a halide electrode). Lab 3 Questions 1. specific ion electrodes are rarely usable in environmental samples. fluoride. chromate. 3. In the above tests. can you see any advantage in suing the first or second derivative curve? Discuss. known addition cannot correct for any ions that test as chloride ion. ferric. # Other interferences: ferricyanide. strongly reducing solutions may form a surface layer of silver. # Chromate.
250 ml Erlenmeyer flasks.total chlorine residual) 1. 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. (concentrated) glacial acetic acid. chlorine demand and break-point chlorination. potassium iodide (KI) crystals.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. Rinse a burette and fill it with this stock chlorine solution. about 5 ml of glacial acetic acid (use a graduated cylinder). OBJECT: -to acquaint you with disinfection of water supplies and to demonstrate techniques for the determination of chlorine residuals.. pages 4-56 to 4-57 or older/newer editions. Wipe up all liquid spills immediately. Materials: -600 ml beakers. pipettes. starch indicator. clean up all spills with a brush into a container and wash crystals down the drain. about 50 ml of chlorine-free water (distilled water will do). gloves and lab coats. to illustrate the concepts of free and combined chlorine residuals. REFERENCES: Standard Methods 19th ed. add 1 ml of starch solution. 250 ml volumetric flasks.N-diethyl-p-phenylenediamine (DPD) indicator solution. 3. and exactly 10 ml of 0. Titrate rapidly with the stock chlorine solution (in burette) until the appearance of a constant 34 September 2007 CIVL 407 Lab M anual . allow to mix again.025 N sodium thiosulphate solution (use a volumetric pipette). I. 0. 1995. Take a 250 ml Erlenmeyer flask and add about 1 gram of potassium iodide. N. 2. Mix well (on stir plate with stir bar). chlorine free water. When weighing chemicals at the balance. Bleach/chlorine solutions are strong oxidants. Personal protective equipment required at all times when working in the lab are: eye protection. standard ferrous ammonium sulphate (FAS) solution. PREPARATION OF STOCK CHLORINE SOLUTION (Iodometric method . phosphate buffer solution. 100 ml graduated cylinder. Make up to the 250 ml and invert flask several times to mix.025 N sodium thiosulphate. bleach or hypochlorite solution. burettes and stands.
Work in groups of three or four for this part. Determination of Free and Combined Chlorine by the DPD Method 1. 4. Method 4500-Cl F. Let stand 2 minutes more. 18th edition. 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. 3. 35 September 2007 CIVL 407 Lab M anual . Record the burette reading (Reading C). Sequentially add the necessary volumes of chlorine stock solution to each beaker at the appropriate time spacing to provide the applied dosages given by the instructor (see board). a) Free chlorine: titrate rapidly with standard FAS (Ferrous ammonium sulfate) titrant until the red colour is discharged. c) Dichloramine: Add about 1 g KI (to the same sample) and mix to dissolve. After 15 minutes of contact time. calculate the chlorine concentration in the stock solution (See Data and Results section for formula).purplish-blue colour. Go to c. Place 5 ml of phosphate buffer solution and 5 ml of DPD solution in a 250 ml conical (Erlenmeyer) flask and mix. 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).) Free and combined chlorine or total chlorine: To obtain total chlorine in one reading.Breakpoint Chlorination using Free and Combined Chlorine Residuals Values 1. It is likely that the highest dose will require dilution. refilling burette.A Monochloramine= B-A Dichloramine= C-B For interferences. please refer to Standard Methods.). These 6 flasks can be prepared at once to make them ready for the next step. From the amount of chlorine solution used. Total chlorine (Free plus Combined) = Reading C Free residual chlorine = Reading A Combined residual chlorine (dichloramine plus monochloramine) = Reading C . etc. Continue titrating until red colour (if any) is discharged once again (Reading B). (*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. as there is lots to do . if colour drifts back (indicating an incomplete reaction). let stand for two minutes and titrate until the red colour (if present) is discharged. 2.COORDINATE! IIa. d) Alternatively: (Not to be done in this lab. mix to dissolve. let stand 2 min more if colour drifts back indicating incomplete reaction. b) Monochloramine: Add two drops of KI solution (to the same sample) and mix. 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). pp 4-43. titrate to colourless again. 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. add full amount of KI at the start (ie 2 drops KI solution and 1 g of KI crystals) to a fresh sample. Add 100 ml of sample or diluted* sample using a 100 ml graduated cylinder and mix again. Record the burette reading (mls) = Reading A. IIb. Let stir for 2 min and then continue titrating until red colour (if any) is discharged (Reading C). For dichloramine concentrations greater than 1 mg/L. 2.
Given: HOCl W H + + OClK eqv = 2. how many coliforms would remain after a chlorine contact time of 10 minutes? 20 minutes? 36 September 2007 CIVL 407 Lab M anual . If a sewage sample contains 10 x 10 6 coliforms/100 ml. 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. free and combined residual chlorine for each applied chlorine dose. If this is true. Make a separate graph for the 15 minute and 1 hour contact time.7 x 10-8 at 25 o C % HOCl = 27 What is the pH of the solution? 3. Guaranteed analysis is normally printed on the container and is usually 5-6%. 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. 250) then convert mg/L to g/100 mls = %. Under what conditions is the practice of break point chlorination necessary? 4. The rate of kill of bacteria by chlorination follows first-order reaction kinetics. c. Make a plot of the three residual chlorine components against the applied chlorine dosage. Discuss your results as a function of the residual forms of chlorine present at different chlorine dosages and at different contact times.e. what percentage of bacteria would be killed in 10 minutes at a chlorine residual of 0.DATA AND RESULTS I. d. Determine the concentration of total. b. a. Why is it important to determine chlorine residuals in domestic water supplies? 2. FAS titrant concentration is 1 ml = 100 µg Cl as Cl2 a.5 mg/L if 50% are killed in 1½ minutes at this concentration? b. II.
15 MINUTE CONTACT TIME BEAKER NUMBER Vol. (mls) (Stock=_____mg/L Cl as Cl2) Applied Cl dosage (mg/L) Volume of FAS to endpoint A Volume of FAS to endpoint B (includes A) Volume of FAS to endpoint C (includes A & B) Free chlorine (mg/L) (A) Monochloramine (mg/L) (B-A) Dichloramine (mg/L) (C-B) Combined residual chlorine (mg/L) (C-A) Total chlorine (mg/L) © Time of Cl addition Time of Residual measurement 1 2 3 4 5 6 37 September 2007 CIVL 407 Lab M anual . of Cl Soln.
of FAS to endpoint A (mls) Vol. (mls) (Stock=_____mg/L Cl as Cl2) Applied Cl Dosage (mg/L) Vol. of FAS to endpoint C (mls) Free chlorine (mg/L) (A) Monochloramine (mg/L) (B-A) Dichloramine (mg/L) (C-B) Combined residual chlorine (mg/L) (C-A) Total chlorine (mg/L) © Time of Cl addition Time of residual measurement 1 2 3 4 5 6 (includes A+B) 38 September 2007 CIVL 407 Lab M anual . of Cl Soln.60 MINUTE CONTACT TIME BEAKER NUMBER Vol. of FAS to endpoint B (mls) (includes A) Vol.
then use buffers 4 and 7 to calibrate. MATERIALS: -pH meter.(Do not allow the stir bar to hit the probe. 4. DETERMINATION OF pH 1. Probes. Always wear personal protective equipment. pH test paper and other water quality assessment equipment. standard base. bromcresol green. the two buffers that are chosen for calibration will bracket your sample ie if sample is less than 7. metacresol purple. standard acid. p H. bromphenol blue. 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. After calibration is complete. pH test paper. Use care when using lab equipment. phenolphthalein. magnetic stirrer. I. 39 September 2007 CIVL 407 Lab M anual . flasks. metres and calibrated glassware are available in limited quantities and are very expensive to replace.) Record value in following table. water samples. colour comparator. (Take care not to contaminate paper in case. lower probe. to acquaint you with the use of pH metres. turbidimeters. rinse & dry probe. Ac id ity . Rinse probe when you are finished and leave immersed in pH 7 buffer for storage. 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. hardness indicators. then use buffers 7 and 10. T u rb id ity WARNING Please use caution when handling all chemicals. Calibrate the pH meter by following the procedure outlined on the instruction sheet beside the meter unless told otherwise. Hard n e s s . if above 7. burettes and stand. 1 N NaOH. graduated cylinders. particularly those labelled with specific hazards. place a beaker containing the sample and stir-bar on the stir-plate. beakers. EDTA. Normally. Co lo u r. 3.) 2. OBJECT: -to familiarize you with the most common water treatment tests. buffer solution.Lab o rato ry 6: Alkalin ity . turn on stirrer and obtain a stable reading.
3.. sample-rinsed graduated cylinder and transfer to an Erlenmeyer flask. 2. The buffer elevates the pH to 10. Record burette reading (P). 3. 3. TOTAL HARDNESS DETERMINATION . Rinse and fill a labelled burette with standard acid solution. DETERMINATION OF ALKALINITY 1.. while stirring. Add a dropper full of bromcresol green (BG= MO) to this same water sample and continue the titration until the colour changes from blue to yellow (at pH 4. add 90% of the titrant to the sample before adjusting the pH with buffer. Add the last few drops at 3-5 second intervals so that you do not "overshoot" the end-point. Add a stir-bar. (If the solution is already blue after indicator. In order to remove any free residual chlorine (which interferes with the indicator colour response). Measure 100 ml of sample (with minimum agitation or exposure to air) using a graduated cylinder and transfer carefully to an Erlenmeyer flask. Rinse and fill another burette with standard base solution and label it. Titrate slowly with EDTA. Record the volume of titrant required in the following table. Pour into a 250 ml Erlenmeyer flask. 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. Rinse and fill a burette with standard EDTA solution and record the initial burette reading. 40 September 2007 CIVL 407 Lab M anual . Alternatively. Record the burette reading in the following table. add 1 drop of 0. IV. If a ppt does form within the 5 minutes.II. Measure out a new sample and add 1 drop of sodium thiosulphate and a full dropper of phenolphthalein. Measure 100 ml of water sample into a clean. 2. White paper placed under the flask will facilitate this determination. Add a full dropper of bromphenol blue indicator and titrate until the colour changes from yellow to blue ("MO" Acidity). (BG or MO) 6. 4. it may be necessary to repeat the titration with the sample diluted 1 to 1 (again) with distilled water.1 N sodium thiosulphate to the sample.. Titrate to the first uniform pink colour (colour stays).. Apply dilution factor to obtain final result. A pH much above 10 promotes CaCO 3 precipitate. DETERMINATION OF ACIDITY 1.think what that means!) 4. 5. Complete the titration within 5 minutes to minimize the tendency toward CaCO 3 precipitation. titrate until just colourless. Measure out 25 mls of sample using a graduated cylinder and dilute to 50 ml with distilled water. Add a full dropper of phenolphthalein (P) indicator and if the solution stays pink or red.?) 4.EDTA TITRIMETRIC METHOD 1. if the approximate hardness is known (by unsuccessful attempts). 2.. If sample is highly alkaline (titration goes over one burette full) dilute the sample and titrate again. Add 1 drop of sodium thiosulphate solution.5). Add stir-bar. (If solution is clear after addition of P. III.
Record the final burette reading in the following table. to mg/L platinum as chloroplatinate) PART B: APPARENT COLOUR 1. Use signal average mode if 41 September 2007 CIVL 407 Lab M anual . V.EDTA TITRIMETRIC METHOD 1. TURBIDITY . Add a stir-bar and stir to mix.5. CALCIUM HARDNESS DETERMINATION . 4. VI. 4.TRUE AND APPARENT PART A: TRUE COLOUR . 3. 3. The display will show AUTO RNG when the instrument is in automatic range. taking care to handle the sample cell by the top. lint-free cloth to remove water spots and fingerprints. Place the cell containing sample in it and press Read. Record all colour data in the following table. COLOUR . Select automatic range by pressing the RANGE key. 3. Filter about 20 mls of sample using a filter funnel and stand and the pre-fluted filter paper provided 2. The display will show SIG AVG when the instrument is using signal averaging. or a volume sufficient to produce a pH of 13-14 (designed to precipitate the magnesium as a hydroxide). 2.. Wipe the cell with a soft. 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. Demonstration of Jackson Candle and Hellige PART A: Hach Turbidimeter 1. VII.0 ml of 1 N NaOH solution (use 3 droppers full).BY HACH 2100P Turbidity Meter. . The reading might be higher so if you had to dilute in Part A dilute for Part B.. 5. Fill a sample cell to the line (about 15 mL). Close the lid. Record the final burette reading in the following table. 2. Press: I/O. except on an unfiltered sample. 4. Refill the burette with EDTA titrant and note the initial reading. Add one aliquot of the Calcium Hardness Indicator and titrate with EDTA slowly. 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. Use the technique outlined in Part A.HACH Spectrophotometer. Select signal averaging mode by pressing the SIGNAL AVERAGE key. Pour the supernatant into one of the cells (to the etched mark) and fill another with distilled water. Cap the cell. to determine the apparent colour (measures the influence of turbidity on the reading). Values are reported as APHA Colour Units (eqv. 2.. Place the cell containing water into the spectrophotometer and press Zero.Enter the stored program number for true colour=120 1. Measure out 50 ml of sample into an Erlenmeyer flask and add 2. The instrument may suggest diluting if over range.
T = Total alkalinity. (This allows you to make a more gradual and accurate approach to the end-point. Carefully fill the 20 mm viewing depth tube to the mark with sample. Wipe the outside of the tube to dry it and place it in the turbidimeter (in the circular groove of the mirror unit).T. 3.the sample causes a noisy signal (display changes constantly). the turbidity may start to settle and condense on the bottom of the tube. Try to be quick and not burn the candles more than a few minutes at a time. 1.. Note that there are several different scales on the side of the tube.Demonstration only 1.NTU. obscuring vision..U. Record value in following table as J. 2. Note that the rectangular door mirror is closed.s.. See Standard Methods. If you take too long.demonstration only.adding liquid to a hot tube will cause it to shatter.. Result of Titration Hydroxide Alkalinity as CaCO 3 Carbonate Alkalinity as CaCO 3 Bicarbonate Concentration as CaCO 3 T T-2P 0 0 0 P=0 0 0 P<1/2T 0 2P P=1/2T 0 2P P>1/2T 2P-T 2(T-P) P=T T 0 Where: P = Phenolphthalein alkalinity. Be sure that the calibrated tubes are clean before adding samples to the tubes. PART C: Jackson Candle Turbidity . Take the reading where the dark central part first merges with the surrounding field. Pinch all excess charred wick with fingers before lighting or soot may be deposited on the glass tubes. Lower the plunger into the liquid making sure no bubbles are trapped under the plunger and the top is dry. 2. DO NOT PLACE AN EMPTY TUBE IN THE TUBE HOLDER WITH THE CANDLE LIT . 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. Record the turbidity after the lamp symbol turns off. that the filter selector shows "none" and that bulb B is in use. Close the door and switch on the light.) Remove the glass tube from the holder and read the turbidity at the meniscus of the sample. then the turbidity in NTU.. 42 September 2007 CIVL 407 Lab M anual . page 272-273. Pour sample into the tube in small increments until you can no longer distinguish the shape of the candle flame. 16th edition. Press: READ The display will show . 3. Read the scale on the dial. record the number and refer to the appropriate graph to determine the turbidity as mg/L SiO 2. PART B: Hellige Turbidimeter . 6. 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.
Acidity end-point reading (mls) Initial burette reading Net "M.Initial pH by test paper Initial pH by pH meter ALKALINITY.O.Sample volume Initial burette reading P.DATA Sample 1 pH ." Alkalinity end-point (mls) Initial burette reading Net P. Alkalinity titre (mls) 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.O.O." Acidity end-point reading (mls)__________ TOTAL Hardness Sample volume (mls)__________ Calcium hardness as mg CaCO 3/L __________ 43 September 2007 CIVL 407 Lab M anual . Alkalinity end-point reading (mls) __________ "M.O." Alkalinity titre (mls) ACIDITY.Sample volume "M.
00 ml EDTA titrant at the calcium indicator endpoint = 1.00 ml EDTA titrant at the calcium indicator endpoint = 1.CALCULATIONS Calcium: Calcium Hardness: where: A= B= ml titrant for sample mg CaCO 3 equivalent to 1.0 Total Hardness (EDTA): where: A= B= ml titrant for sample mg CaCO 3 equivalent to 1.0 Alkalinity: where: A= N= ml standard acid used normality of standard acid Acidity: where:A = N= ml standard base used normality of standard base 44 September 2007 CIVL 407 Lab M anual .
Alkalinity as HCO 3 . ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ 45 September 2007 CIVL 407 Lab M anual .Units= N. Alkalinity as CaCO 3 (mg/L) "M.O.T.O.U." 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 .Alkalinity as OH .ion (mg/L) CO 3 -2 Alkalinity as CO 3 -2 ion (mg/L) HCO 3.Alkalinity as CaCO 3 (mg/L) OH .RESULTS (sample) P.Alkalinity as CaCO 3 (mg/L) CO 3 -2 Alkalinity as CaCO 3 (mg/L) HCO 3." Alkalinity as CaCO 3 (mg/L) OH .ion (mg/L) "M.Hach .APHA Colour Units: True Apparent Turbidity .
46 September 2007 CIVL 407 Lab M anual . From equations (17-12) and (17-13) in Sawyer and McCarty. non-carbonate and total hardness of this water in mg/L as CaCO 3 ? in meq/L? Is the analysis complete? Why? 6. Is this water suitable for a domestic water supply? Why? 2.Lab 6 Questions 1. What sanitary significance does colour have? 4. calculate the carbonate alkalinity in mg/L as CaCO 3 for the water sample analyzed during this lab. 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.7 x 10 -11 and K w = 10 -14. Note: You should bring a copy of this lab to the next two sessions as you will need the same instructions to perform the same tests in the Coagulation and Softening Labs. Does this agree with the results from Part II of this lab? Explain. given K 2 = 4. What errors can occur in the calcium hardness determination? Why? 3.
Please try to coordinate your work with the rest of the class.6. 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. 1000 ml beakers. No. No. This is a two-step experiment. 7 (except Jackson Candle and Hellige Turbidimeter). You should have with you the part of Exercise 7 that gives instructions for the various tests. In the second lab you will use the previously determined dosages to coagulate and soften the sample and then re-analyse the resultant water. to be conducted over two lab periods. and all of the materials from Lab.for the next session (b). OBJECT: -to illustrate the principles of coagulation and water softening. The optimum alum dosage for coagulation of the sample will then be chosen. soda ash solution (Na 2CO 3) and lime (Ca(OH)2) or sodium hydroxide (NaOH). alum solution (Al2 (SO 4 )3 @18H 2 O). The first step will be to analyse the raw water sample and the supernate from a number of alum dosages (to be announced in the class) using the techniques learned in Lab. Several lime and soda ash dosages will also be selected for determining the efficiency of water softening. MATERIALS: -Jar Test Apparatus.
7. Make relative estimates of floc sizes. clarity of supernatant liquid (very clear. acidity. hardness (total and calcium). alkalinity. you will discuss the data and select the appropriate alum dosage to be used by all groups in the water softening step next week. Record observations on settling rate. Analyze the supernates for pH. Reduce the stirring rate to about 20 rpm for 10 minutes.work in 2 or 3 teams to assess a total of 6 dosages for each team. Observe and make notes describing floc formation and note the time of appearance. COAGULATION USING ALUM . 6. decant the supernatant liquid carefully into a clean set of beaker (try not to stir up what has settled) and discard the floc. moderate. 1. medium. You may need to use blocks to position the paddle 0. Record relative floc sizes (pin-point.. Add the designated lime (or NaOH) and soda ash additions as quickly as possible to the decanted samples. Stir at 100 rpm for 1 minute. 1. 4. 6. 3. Stir at 100 rpm for 1 minute and at 20 rpm for 10 minutes. times of appearance and descriptions and record. floc volume. large). cloudy). 2. hardness (total and calcium). Carefully decant into a graduated cylinder 800 mls of the supernatant liquid and pour into fresh beakers. Complete the coagulation summary sheet during the lab period. Stop stirrer and allow floc to settle. Lift paddles out of beakers and secure. 2. and settling characteristics of the floc (rapid. 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. (NB dosage now based on 800 ml sample size). alkalinity. Part B: WATER SOFTENING PROCEDURE (to be done the following week) again work as teams of 2 or 3. 4.5 to 1 cm off the bottom of the beaker.1 N sodium thiosulphate 48 September 2007 CIVL 407 Lab M anual . Measure out six 1000 ml aliquots of sample (using graduated cylinder to measure. Discard the floc and clean beakers in preparation for a second decanting in Step 4. position them under stirrers. Stop stirrer. slow). Analyze these supernates plus a sample of raw water for pH. Pour six 1000 ml aliquots of sample into 1000 ml beakers and place on the jar test stirrer apparatus. and clarity of supernatant liquid.Part A:. A pH meter could be used for the alkalinity and acidity determinations. small. In the lecture. Add the appropriate alum dosage and immediately start stirring. if available. Brief Methods Summary for Analyses Alkalinity: @ 100 ml of sample. After the flocs have settled (allow 15 min). if time permits. decant the supernatant liquid carefully into another set of beakers (try not to stir up what has settled). not a beaker ) and transfer to 1000 ml beakers. Determine which dosages were most appropriate for this sample. 3. true colour and turbidity (Hach). turbidity and colour. Make sure it is centred. Add the assigned alum dosages as quickly as possible and then start the stirrer. clear. acidity. drop of 0. and fill in the table on the board. Stop stirring. 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). use indicators as specified in Lab 6. Place beakers on the jar tester apparatus. but because of equipment limitations. 5. hazy. 5. After the floc has settled (allow 15 min). Stir at 100 rpm for 1 minute.
pH 3. 2.3 ("phenolphthalein" or total acidity) @ calculation . 3. @ 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 .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 .1 N sodium thiosulphate @ titrate with 0. 2.pH 4. How does the consumption of alkalinity by alum compare with theoretical calculation based on balanced equations? How much extra soda ash should you add to compensate for the alkalinity consumption by 70 mg/L alum? B.pH 8.use graphical presentation. What treatment would you recommend to prepare this water for human consumption? Why? 49 September 2007 CIVL 407 Lab M anual . Based on your chemical analysis of the raw water.pure blue). Compare your experimental softening results to the theoretical results based on solubility equations or mass balance equations. Compare removal of hardness components under the different doses .acidity (mg/L CaCO 3)= mls titre x N base x 50.alkalinity (mg/L CaCO 3)= mls titre x N acid x 50. 4.5. Coagulation 1. Analyze the response of colour and turbidity removal as a function of alum dose.3 and with bromcresol green (continuing with the same sample) to yellow .7 ("methyl orange" acidity) and with phenolphthalein (using a fresh sample) to pink .pure blue).pH 8.@ titrate with 0.02 N acid with phenolphthalein to colourless . Softening 1. @ calculation: Ca hardness (mg/L CaCO 3 )= mls titre x 1000/mls sample Lab 7 Questions A. @ calculation . drop of 0.02 N base with bromphenol blue to blue .000/mls sample Acidity: @ 100 ml of sample. show by calculation the theoretical amount of lime and soda ash required for excess lime/soda ash softening.
5 “TOT” Total Hardness sample vol Net titre Ca Hardness .sample vol ml Net titre to pH 3.5 “P” Net titre to pH 4.5 “TOT” Acidity .7 “MO” Net titre to pH 8.sample vol Net titre Notes: 50 September 2007 CIVL 407 Lab M anual .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.
A.) (filtered) Turbidity (N.) True Colour (A.A.T.P.H.H.P.U.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.) unfiltered Notes: 51 September 2007 CIVL 407 Lab M anual .
sample vol Net titre Notes: 52 September 2007 CIVL 407 Lab M anual .5 Total Hardness sample vol Net titre Ca Hardness .5 Net (Total) titre pH 4.7 Net (Total) titre pH 8.sample vol ml Net titre to pH 3.Coagulation and Softening Raw Data D ATA Lime/NaOH mg/L Soda Ash mg/L Alum mg/L pH Alkalinity-sample vol ml Net titre to pH 8.5 Acidity .
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.H.H.U. Calculation 53 September 2007 CIVL 407 Lab M anual ** By .) (Filtered) Turbidity (N. -mg/L CaCO 3 **Non-Carb Hard -mg/L CaCO 3 **Excess Alk.P.) Note:* Total Alkalinity must include Phenolphthalein Alkalinity just as Total Acidity must include “MO” Acidity.) True Colour (A. -mg/L CaCO 3 Apparent Colour (A.T.A.P.Coagulation and Softening Summary Sheet Data Lime/NaOH mg/L Soda Ash mg/L Alum mg/L pH P.A.
. . . . . . . Reading and Reference Material . . . . . . . . . . . . . . . . . . . . . . . . . . . .Additional Notes . . . . . . . Faecal Coliforms . . . . . . . . . . Turbidity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 8. . . . . . . . . . . 110 27. . . . . . Equilibrium Relationships of Chlorine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BOD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Typical Problems for Acid-Base and General Chemistry . . . . . . .Chlorine Demand . . . 74 13. . . . . . . . . . . . . . Concepts and Definitions . . . . . . . . . Chemical Requirements for Water Softening . 112 28. . . . . . . . . . . . . . . . . . . . . . . . . . Hardness Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 22. . . Milliequivalent and mg/l as CaCO 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alkalinity. . . . . . . . Hach Absorption Method . . . Extra tables for Chlorine Lab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 26. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 54 September 2007 CIVL 407 Lab M anual . . 80 14. . . . . . . . . . . . Bacteriology and Water-related diseases . Colour. . . . . . . . . . . Bacteriological Examination . . . . . . . . . . . . . . . pH . . . . 71 10. . . . . . . . . . . . . . . . . . . . 67 6. . . . . 91 18. . . . . . . . . . . . . . . . . . . . . . . 64 5. . . . . . .pdf 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 11. . . . . . . . 104 24. . . . . . . . . 82 15. . . . . . . . . . . . . . . . . . 70 9. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4. . . . . . . . . . . . . . . . . . . 84 17. . 117 29. . . . . . . . . . . . Normal Solutions and Equivalent Weights . . . . . . . . . . . . . . . . . . . Acidity. . . . Solids Removal in Wastewater Treatment . . . . . . . . . . . 57 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chloride Analytical Methods . . . . . . . 73 12. . . . . . . . . . . . Residues . 92 19. . . . . . . . . . . . . . . . . . . . . .COD . . . . . . . 94 20. . . . . COD . . . . . . . . . . . . . . . . . . . Titrations . . . . . . . . . . . . . . Water Softening . . . . . . . . 96 21. . . . . . . . . . . . . . . . . . . . . . . . . . . . Coagulation . . . . . . . . . . . . . . . . . . . . . . . Alkalinity Species as a Function of pH . . . . . . . . . . . . Chlorine . . . . . 105 25. . . . . . . . . . . . . . . Introduction . . . . .Levels and Water Use . . . . . . . . . . Standard Solutions. . . 55 2. . . . . . . . . . 83 16. . . . . . . . . . . .4 Appendices see: CIVL407Manual_CourseNotes. 68 7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Instructions for Laboratory Report Writing . . . . . . . . . . . . . . . . . . Bar Diagram Method for Water Softening Problems . . . . . . . . . . . . . . . . . . . . . . . . . . 102 23. . Periodic table . . . . . . Balancing an Oxidation Reduction Equation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DO. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Known Addition Technique . . . . . . Hardness. . . . . . . . . . . . . . . . . . . .
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